Polyphenolic profile and in vitro biological activity of Serbian orange (skin fermented white) wines.

Orange wines are made from white grapes, but with prolonged skin contact during fermentation. Available data on their composition and potential health benefits are limited, so polyphenolic profile (HPLC analysis) and in vitro biological activities (enzyme inhibition, antioxidant, and anti-inflammatory) of 24 Serbian orange wines were analyzed, including the correlation between determined composition and bioactivities. The wines displayed distinct polyphenolic profiles, enabling partial differentiation based on overall polyphenol content, including dominant components (catechin, gallic and caffeic acids), along with occasional occurrences of anthocyanins. However, no discernible distinctions were noted based on grape varieties, vintage, or producer. All twenty-four orange wines showed a reasonable inhibition of digestive enzymes and lipid peroxidation, twenty-one samples reduced ROS generation in the cell-based assay, but only two suppressed both PGE2 and TXA2 production in U937 cells, implicating possible functional food properties. No significant correlation between polyphenolic profile and determined biological activities was noticed.

Elemental Profile and Health Risk of Fruska Gora Wines.

The elemental composition of wine is influenced by endogenous sources and interventions from winemakers. The ICP-MS analysis of Fruska Gora wines (113) from vintages spanning across a decade (2011-2020), produced by 30 wineries and representing 18 autochthonous and international wine varieties, allowed a comprehensive insight into their elemental composition. Based on the mean concentrations of 23 investigated elements, B, Fe, and Mn, which were determined in mg per L of wine regardless of its colour or origin, were the most abundant. Red and white wines showed significant concentration differences in the case of B, Mn, and Sr (higher in red) as well as Be, Al, V, As, Mo, and Pb (higher in white). The elements of the highest toxicological concern were found in all (Pb and As) or almost all of the samples (Cd and Hg). Pb levels (maximum 47.1, 61.6, and 73.2 µg/L in red, rose, and white, respectively) were well below the legal limit. The applied risk assessment approaches (hazard quotient and index, margin of exposure) revealed no health concerns associated with consumption of Fruska Gora wines, except for a slightly increased lifetime cancer risk in the case of high wine consumption, and thus supported the promotion of Fruska Gora wines in the highly competitive international market.

Could elderberry fruits processed by modern and conventional drying and extraction technology be considered a valuable source of health-promoting compounds?

The influence of drying and extraction processes on the phytochemical composition and biopotential of elderberry is challenging for the food industry. For this reason in this research two drying techniques (lyophilization and natural convection) and three extraction techniques (ultrasound (UAE), microwave (MAE), and conventional (maceration (MAC)) was applied using two"green" solvents (water and 50 % ethanol). Results of the research showed that lyophilization was a better way of drying than natural convection, while MAE extraction was the most efficient technique for the isolation of secondary metabolites. The most abundant phenolic compounds established by LC-MS/MS analysis, were chlorogenic acid and rutin, identified in extracts of lyophilized elderberries. Elderberry extracts achieved great antioxidant (CUPRAC: 2.30-5.13 mg TE/mL) and enzyme inhibitor potential (α-amylase: 0.51-8.34 mg ACAE/mL). The results suggest that elderberry is a rich natural source of bioactive compounds and could be used for the future development of dietary supplements and functional foods.

Anti-Inflammatory, Antioxidant and Enzyme Inhibition Activities in Correlation with Mycochemical Profile of Selected Indigenous Ganoderma spp. from Balkan Region (Serbia).

The aim of this research work was to study the bioactivity potentials (antioxidant, anti-inflammatory, and enzyme inhibitory) of ethanol (EtOH), water (H2 O) and chloroform (CHCl3 ) extracts of G. applanatum, G. lucidum, G. pfeifferi and G. resinaceum as well as their mycochemical profile: the total content of phenolics (TP) and sugars (TS) and LC/MS/MS detection of phenolics. LC/MS/MS profile showed that p-hydroxybenzoic and protocatechuic acids were mostly found. The highest ABTS and DPPH activities were detected in polar G. applanatum extracts (159.84±0.59 mg TE/g d.w., IC50 =0.85±0.30 µg/mL, respectively), while G. resinaceum CHCl3 extract was the most potent in NO assay (IC50 =41.21±0.18-81.89±0.81 µg/mL). The highest TP and TS were generally determined in G. applanatum EtOH extracts. Enzyme inhibitory effects were determined in H2 O extracts. Generally, CHCl3 extracts showed the most powerful anti-inflammatory potential. These results suggest that analyzed species are a promising source of bioactive compounds and may be considered as candidates for new food supplements or drug formulations.

Plantain (Plantago L.) species as modulators of prostaglandin E2 and thromboxane A2 production in inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Worldwide distributed plantains (genus Plantago L.) are extensively used in the traditional, but some of them are also accepted in the modern medicine. Wide range of usages is mainly connected to the inflammation processes. AIM OF THE STUDY: To support usage of renowned P. lanceolata L. and P. major L., underinvestigated P. altissima L., P. argentea Chaix, P. holosteum Scop. and P. media L. methanol extracts, and their typical constituents (aucubin, apigenin, apigenin-7-O-glucoside, luteolin, luteolin-7-O-glucoside, chlorogenic and ursolic acid) in treatment of inflammation disorders, we conducted study on plantain potential to inhibit production of inflammatory mediators, prostaglandin E2 (PGE2) and thromboxane A2 (TXA2). MATERIALS AND METHODS: LPS-stimulated monocytes (U937 cell line) were used as a model-system to examine anti-inflammatory potential of plantains and their constituents. Produced PGE2 and TXA2 were quantified by LC-MS/MS; qPCR was applied to examine related gene (PLA2, COX-1, COX-2, mPGES-1, mPGES-2, cPGES, TXAS) expression; LC-MS/MS and LC-UV/VIS techniques to analyze extracts composition. RESULTS: In general, examined plantain extracts showed comparable inhibition activity of PGE2 and TXA2 production as aspirin at low-dose concentration. Underinvestigated P. altissima can be pointed out, since it exerted the strongest effect on both PGE2 production and related gene expression. Notable suppression of TXA2 production by P. lanceolata and P. major was observed. But, PCA analysis showed no obvious grouping, implicating that different mechanisms of action are responsible for each sample activity. In most cases, positive correlation was found between content of apigenin and ursolic acid and extracts suppression of PGE2 and TXA2 production and related genes expression. CONCLUSIONS: P. altissima can be regarded as promising anti-inflammatory agent, while novel aspect of P. lanceolata and P. major application (anti-aggregation) can be suggested. P. argentea and P. media could be considered as a good source of ursolic acid.

Elderberry (Sambucus nigra L.) juice as a novel functional product rich in health-promoting compounds.

The medicinal herbs of the Balkan Peninsula are highly represented in traditional medicine. The connection between traditional and modern life and health is reflected in the creation of new food products with added value. In this study, the plant species Sambucus nigra L. was used to obtain freshly pressed juice, whose chemical composition and various biological activities were evaluated. The most abundant compounds were phenolic acids: protocatechuic and chlorogenic acid, as well as flavonoids: quercetin-3-O-hexoside, quercetin, and rutin. The analyzed juice was very rich in total phenolic compounds (1945 mg GAE per mL juice), and a significant anthocyanin concentration was observed (30.85 mg Cy-3-GE per mL juice). Bioactivity testing revealed that elderberry juice was an extremely potent agent in the process of neutralizing NO free radicals (53.06 g TE per L juice), while in reducing over-enzyme activity, the best result was achieved in the inhibition of tyrosinase enzyme (54.70 mg KAE per g of juice).

The Effects of Trifluoromethylated Derivatives on Prostaglandin E2 and Thromboxane A2 Production in Human Leukemic U937 Macrophages.

BACKGROUND: A convenient approach to modulation of the inflammation has an influence on the production of inflammatory mediators - icosanoids, generated in arachidonic acid (AA) metabolism. The common therapeutic activity of non-steroidal anti-inflammatory drugs (NSAID), such as aspirin, includes inhibition of two crucial enzymes of AA metabolism - cyclooxygenase- 1 and -2 (COX-1/2), with certain risk for gastrointestinal and renal intolerance. Ever since the enrolment of COX-2, particularly overabundance of its main products prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) in numerous pathological processes was recognized, it became a significant therapeutic target. OBJECTIVE: The aim of this study was to examine the effects of synthesized organo-fluorine compounds on PGE2 and TXA2 production in the inflammation process. METHODS: Trifluoromethyl compounds were synthesized from N-benzyl trifluoromethyl aldimine, commercially available 2-methyl or 2-phenyl α-bromo esters (ß-lactams trans-1 and trans-2 and trifluoromethyl ß-amino ester, respectively) and methyl 2-isocyanoacetate (2-imidazoline trans-4). The reactions proceeded with high geometric selectivity, furnishing the desired products in good yields. The influence of newly synthesized compounds on PGE2 and TXA2 production in human leukemic U937 macrophages on both enzyme activity and gene expression levels was observed. RESULTS: Among the tested trifluoromethyl compounds, methyl trans-1-benzyl-5-(trifluoromethyl)- 4,5-dihydro-1H-imidazole-4-carboxylate (trans-4) can be distinguished as the most powerful antiinflammatory agent, probably due to its trifluoromethyl-imidazoline moiety. CONCLUSION: Some further structural modifications in tested compounds and particularly in the synthesis of different trifluoromethyl imidazolines could contribute to the development of new COX-2 inhibitors and potent anti-inflammatory agents.

Activity profiling of Serbian and some other European Merlot wines in inflammation and oxidation processes.

Merlot are worldwide recognized red wines. Several studies show that red wines have health benefits, mainly due to their phenolic constituents. This study evaluates twelve Serbian and other five European (French, Italian, Macedonian, Slovenian, Spanish) Merlot wines in respect of their phenolic composition and biological activity. The latter was evaluated through a set of in vitro experiments related to common benefits of moderate red wine consumption in the prevention of cardiovascular diseases. Among the examined phenolics, the most abundant acid in all samples was the gallic acid (14.3-58.3 mg/L), catechin (9.1-49.3 mg/L) was the dominant flavonoid, malvidin-3-O-glucoside (2.63-66.5 mg/L) leading anthocyanin, whereas resveratrol was found in a usual concentration (0.18-4.67 mg/L). Differences determined in phenolic profiles, mainly in content of quercetin, rutin and p-coumaric acid, leaded to separation of Serbian from foreing Merlot wines. Results of standard antioxidant assays (DPPH•, ABTS•+ and •NO scavenger capacity reducing power (FRAP), lipid peroxidation) revealed French Merlot as the most potent, but also pointed out some Serbian samples. The correlation between the content of dominant phenolics and antioxidant activity was sporadic, but samples with the highest overall phenolic content, generally had higher antioxidant potential. Concentration of wines and number of cells in ant-inflammatory assay were chosen to mimic in vivo conditions. So, the potency of examined wines to decrease the production of macrophage-derived PGE2 and TXA2 (up to 65.5 and 47.9%, respectively), could be considered as in vitro evidence of positve health effect. Regarding the phenolic content and anti-inflammatory contribution of the most abundant compounds, no correlation was witnessed. In general, this study showed interesting potential of Serbian Merlot wines, comparable to health-promoting effects of renewed Eurepean ones.

Phytochemical composition and in vitro functional properties of three wild rose hips and their traditional preserves.

The aim of the present study was investigation of the phenolic profile, ascorbic acid content, antioxidant, anti-acetylcholinesterase, anti-inflammatory and cytotoxic activity of rose hips and the preserves (purée and jam) of three insufficiently examined Rosa species: Rosa dumalis Bechst., R. dumetorum Thuill. and R. sempervirens L. The liquid chromatography-tandem mass spectrometry analysis resulted in quantification of 14 of the 45 phenolic compounds examined, with ellagic acid as the most dominant. Notable antioxidant activity of all three species was confirmed through several assays. Moderate inhibition of acetylcholinesterase by extracts of all investigated Rosa species was observed. Several extracts of examined Rosa species demonstrated inhibition potency towards production of some monitored eicosanoids in cyclooxygenase-1 and 12-lipoxygenase pathways. Two R. sempervirens extracts exerted cytotoxic activity against HeLa and HT-29 cell lines, but were inactive towards MRC-5 and MCF7. The results support the potential of these rose hips as food with health-promoting properties.

Crataegus orientalis Leaves and Berries: Phenolic Profiles, Antioxidant and Anti-inflammatory Activity.

The present study was designed to define the phenolic content, antioxidant and anti-inflammatory activity of Crateagus orientalis Pall. ex M. Bieb., traditionally used by local people in southern parts of F.Y.R. Macedonia. The presence and content of 7 phenolics in ethanolic extracts of leaves and berries were studied using HPLC-DAD, where the most dominant compounds were hyperoside, isoquercitrin and chlorogenic acid. The leaf extract was more effective as a DPPH radical scavenger (IC50 = 29.7 µg/g) than the berry extract, as well as in the relative reducing power on Fe³âº. Anti-inflammatory potential was studied by means of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibitory activity; both extracts evinced activity. Furthermore, C. orientalis leaf extract showed a concentration dependent inhibition of COX-1 pathway products 12-HHT and TXB2, reaching IC50 values below the lowest applied concentration (68.9% and 55.2% of 12-HHT and TXB2 production inhibition, respectively, at concentration of 0.4 mg/mL). Although inhibitors such as acetylsalicylic acid and quercetin showed higher activity, this study demonstrates that the investigated extracts are potential anti-inflammatory agents.

Comparative study of biological activities and phytochemical composition of two rose hips and their preserves: Rosa canina L. and Rosa arvensis Huds.

The aim of this study was to compare phenolic profile, vitamin C content, antioxidant, anti-inflammatory and cytotoxic activity of rose hips and the preserves (purée and jam) of two Rosa species: renowned Rosa canina L. and unexplored Rosa arvensis Huds. The liquid chromatography-tandem mass spectrometry analysis of 45 phenolics resulted in quantification of 14 compounds, with quercitrin, gallic and protocatechuic acids as the most dominant. High antioxidant potential of R. canina and a moderate activity of R. arvensis extracts were determined through several assays. Purée of both species and methanol extract of air-dried R. canina hips showed some anti-inflammatory (cyclooxygenase-1 and 12-lipooxygense inhibition potency) activity. Purée of R. canina exerted cytotoxic activity only against the HeLa cell line among several others (HeLa, MCF7, HT-29 and MRC-5). The presented results support traditional use of rose hips and their fruit preserves as food with health and nutritional benefits.

Binary and Tertiary Mixtures of Satureja hortensis and Origanum vulgare Essential Oils as Potent Antimicrobial Agents Against Helicobacter pylori.

Essential oils possess strong antimicrobial activity, even against multiresistant Helicobacter pylori. Available therapies against H. pylori infection have multiple disadvantages, indicating a great need for a development of new therapeutics. The purpose of this study was to develop a potent natural product based anti-H. pylori formulation. First, anti-H. pylori activity of nine essential oils was determined, after which the most active oils were mixed in various ratios for further testing. Satureja hortensis, Origanum vulgare subsp. vulgare and O. vulgare subsp. hirtum essential oils expressed the highest activity (MIC = 2 µL mL(-1)). Their binary and ternary mixtures exhibited notably higher antimicrobial activity (MIC ≤ 2 µL mL(-1)). The most active was the mixture of S. hortensis and O. vulgare subsp. hirtum oils in volume ratio 2:1, which expressed 4 times higher activity than individual oils (MIC = 0.5 µL mL(-1)). According to GC-MS, both oils in the mixture were characterized by high content of phenols (48-73%), with carvacrol as the main carrier of antimicrobial activity. Presented in vitro study pointed out binary mixture of S. hortensis and O. vulgare subsp. hirtum essential oils in volume ratio 2:1 as promising candidate for further in vivo studies targeting H. pylori infection.

Phenolic profile, antioxidant, anti-inflammatory and cytotoxic activities of black (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles.

The aim of this study was a comprehensive investigation on phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of the still insufficiently explored black summer truffles (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles. Methanol and water extracts, obtained by maceration and Soxhlet extraction, were examined. Forty-five phenolics was studied using LC-MS/MS: presence of 14 compounds was confirmed, with the most dominant being p-hydroxybenzoic acid, baicalein and kaempferol (T. aestivum), epicatechin and catechin (T. magnatum). Moderate antioxidant activity of both species was determined through several assays. Only T. magnatum showed anti-inflammatory potential by inhibiting COX-1 and 12-LOX pathway products synthesis. Methanol extracts exerted cytotoxicity against some tumour cell lines (HeLa, MCF7, HT-29), besides the prominent activity of water extracts towards breast adenocarcinoma (MCF7). To conclude, these results support further investigations of phytochemicals and biological activity towards verification of nutraceutical use of both species.

Quantitative determination of plant phenolics in Urtica dioica extracts by high-performance liquid chromatography coupled with tandem mass spectrometric detection.

A method for quantification of 45 plant phenolics (including benzoic acids, cinnamic acids, flavonoid aglycones, C- and O-glycosides, coumarins, and lignans) in plant extracts was developed, based on reversed phase HPLC separation of extract components, followed by tandem mass spectrometric detection. The phenolic profile of 80% MeOH extracts of the stinging nettle (Urtica dioica L.) herb, root, stem, leaf and inflorescence was obtained by using this method. Twenty-one of the investigated compounds were present at levels above the reliable quantification limit, with 5-O-caffeoylquinic acid, rutin and isoquercitrin as the most abundant. The inflorescence extracts were by far the richest in phenolics, with the investigated compounds amounting 2.5-5.1% by weight. As opposed to this, the root extracts were poor in phenolics, with only several acids and derivatives being present in significant amounts. The results obtained by the developed method represent the most detailed U. dioica chemical profile so far.

Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of plantain (Plantago L.) species.

To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12-LOX enzymes and highly sensitive and specific LC-MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well-known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC(50)) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC(50)=0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents.

Plantain (Plantago L.) species as novel sources of flavonoid antioxidants.

To examine the antioxidant properties of methanol extracts of selected Plantago species (P. argentea Chaix., P. holosteum Scop., P. major L., P. maritima L., and P. media L.), various assays that measure free radical scavenging ability were carried out: DPPH, hydroxyl radical, superoxide anion, and nitric oxide scavenger capacity tests, reducing power (FRAP) assay, and Fe(2+)/ascorbate induced lipid peroxidation. In all of the tests extracts showed a potent antioxidant effect compared with BHT, a well-known synthetic antioxidant, and the extract of P. major, accepted as an official remedy. Besides, in examined extracts the total phenolic amount (ranging from 38.43 to 70.97 mg of GAE/g of dw) and the total flavonoid content (5.31-13.10 mg of QE/g of dw) were determined. Furthermore, the presence and content of selected flavonoids (luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin, rutin, and quercetin) were studied using LC-MS/MS technique. LC-MS/MS analysis showed noticeable qualitative and quantitative differences between the species according to which the examined Plantago species could be regarded as a possible new source of natural antioxidants. In this study three of the species examined, P. maritima, P. argentea, and P. holosteum, have been analyzed for the first time.