P2X7-Receptor Pathway Involvement in the Anti-Inflammatory Activity of Medicinal Plants.

Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye in vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1 µg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.

Varietal Differences of Prokupac, Evita and Cokot Zemun Based on Their Anthocyanins Content in Grape Skin Extract.

In this study, we have analyzed the anthocyanin composition of skin extracts of three red grape varieties Prokupac, Evita and Cokot Zemun in order to distinguish these cultivars based on their anthocyanin profile. Also, mechanical analysis of grape bunches and berries was performed. According to our results, seventeen anthocyanins were identified using LC/MS technique and quantitative differences were recorded using HPLC-DAD method. The highest content of total anthocyanins was obtained for Evita variety and the lowest one was recorded in Prokupac. Also, clear differences were observed in anthocyanins ratios. In comparison to Prokupac and Evita varieties, Cokot Zemun was characterized with a high content of coumaroyl derivatives of anthocyanin compounds, while high levels of acetylated derivatives were recorded in Prokupac. Data reported in this study represent a certain contribution to a database of mechanical properties and chemical composition of grape varieties originating from Balkan.

Immunomodulatory activity and protective effects of chokeberry fruit extract on Listeria monocytogenes infection in mice.

Chokeberry (Aronia melanocarpa) fruit extracts (CE) are rich in polyphenols and usually exhibit immunomodulatory, anti-viral and anti-bacterial effects. We have previously shown that the CE used in this study activated macrophages and stimulated effector T cell differentiation in vitro. When applied orally to healthy mice, CE increased the proportion of CD11c+ dendritic cells in the gut-associated lymphoid tissue. CE-pretreated BALB/c mice readily eradicated orally ingested Listeria monocytogenes as evidenced by a slighter decrease in body weight and number of bacteria recovered from the spleen and reduced spleen size compared to the control infected mice. CE pretreatment in infected mice resulted in higher proportions of CD11b+ macrophages and CD8+ cytotoxic T cells both in the gut and the spleen. Phagocytosis, reactive oxygen species production and the proportions of activated CD86+ macrophages (CD11b+) and dendritic cells (CD11c+) were also enhanced in CE-pretreated infected mice. Furthermore, the expression of inducible nitric oxide synthase and IL-6 was increased in CE-pretreated infected mice and similar results were obtained in peritoneal macrophages in vitro. This effect of CE was associated with increased phosphorylation of IκB and Notch1 production. Finally, CE pretreatment elevated the proportion of perforin-producing cells in the spleen compared to control infected mice. This study demonstrates that prophylactic treatment with CE leads to more rapid eradication of bacterial infection with L. monocytogenes predominantly through increased activity of myeloid cells in the gut and in the spleen.

Effect of pomegranate peel extract on the oxidative stability of pomegranate seed oil.

Pomegranate seed oil is valuable source of bioactive compounds with health-beneficial effects, but it is sensitive to oxidation due to high content of PUFA. In order to improve stability of pomegranate seed oil, the influence of natural antioxidant (0.1% pomegranate peel extract) and a combination of synthetic antioxidant (0.01% butylhydroxytoluene) and pomegranate peel extract (0.05%) was investigated. The oxidation process was followed by monitoring fatty acid composition and measuring the quality and stability parameters of oil (acid value, peroxide value, anisidine value and thiobarbituric acid reactive substances value) during storage for 12 days at 65 °C. Furthermore, total phenolic content and DPPH radical scavenging activity was measured. Pomegranate peel extract as well as a combination of pomegranate peel extract and BHT can have a significant positive impact (P < 0.05) on improvement of the quality and stability parameters of pomegranate seed oil. Furthermore, such activity was better than BHT used alone.

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.

Acute effects of black currant consumption on salivary flow rate and secretion rate of salivary immunoglobulin a in healthy smokers.

The role of saliva in maintaining oral health and homeostasis is based on its physicochemical properties and biological activities of its components, including salivary immunoglobulin A (IgA). Both salivary rates and immunological status of saliva are found to be compromised in smokers. The aim of this study was to investigate the acute time-dependent effect of smoking and black currant consumption on the salivary flow rate (SFR) and salivary IgA secretion rate (sIgA SR) in healthy smokers. SFR, sIgA levels in saliva, and sIgA SRs were determined in healthy smokers (n=8) at eight times of assessment within three consecutive interventions: at the baseline; 5, 30, and 60 min after smoking; 5, 30, and 60 min after black currant consumption (100 g), followed by smoking; and 5 min after black currant consumption. Smoking induced a significant delayed effect on SFR measured 60 min after smoking (P=.03), while black currant consumption preceding smoking prevented that effect. Salivary IgA concentrations and sIgA flow rates were not acutely influenced by smoking. Black currant consumption preceding smoking induced a significant decrease in sIgA concentrations 5 min after the intervention compared with the baseline (P=.046), with a further increasing trend, statistically significant, 60 min after the intervention (P=.025). Although smoking cessation is the most important strategy in the prevention of chronic diseases, the obtained results suggest that the influence of black currant consumption on negative effects of tobacco smoke on salivary flow and immunological status of saliva could partly reduce the smoking-associated risk on oral health.

Further amphoricarpolides from the surface extracts of Amphoricarpos complex from Montenegro.

Analysis of composition of sesquiterpene lactone fraction of leaf cuticular neutral lipids of Amphoricarpos complex from two different localities in north Montenegro, i.e., canyon of river Tara (A. autariatus ssp. autariatus) and mountain Zeletin (A. autariatus ssp. bertisceus) afforded sesquiterpene lactones with guaianolide skeletons (17 compounds), so called amphoricarpolides, typical for this genus. Nine of them, 9-17, were new compounds, and their structures were elucidated by detailed analyses of IR, NMR, and MS data.

Ethnobotanical study on traditional use of medicinal plants in South-Western Serbia, Zlatibor district.

ETHNOPHARMACOLOGICAL RELEVANCE: This paper provides significant ethnobotanical information on medicinal plant uses in the Zlatibor district, South-Western Serbia. MATERIALS AND METHODS: A survey was performed using questionnaires with 220 informants (mean age 47, 79% female, 21% male). In addition, the use value and the relative importance of species were determined and the informant consensus factor was calculated for the medicinal plants included in the study. Intended plants usage was compared with previous ethnobotanical literature, with reference to the neighboring areas of Zlatibor district. RESULTS: The informants provided data for 69 medicinal plants belonging to 36 families. Rosaceae, Lamiaceae and Asteraceae were the predominant locally used families. The species with the highest use value were Mentha piperita, Matricaria chamomilla, Hypericum perforatum and Achillea millefolium. The most frequently reported medicinal uses were ones for treating gastrointestinal ailments, respiratory problems and skin diseases. Usually, the administration was primarily oral followed by topical applications. All different plant parts were utilized, however leaves were the most exploited parts of the plants. CONCLUSIONS: Folk medicine in South-Western Serbia, Zlatibor district is intended mainly as a mode of primary health care in healing of minor illnesses. The results indicate a slight reduction in the ethnobotanical and medical knowledge in this area, when compared with neighboring regions.

Chemical profile, radical scavenging and cytotoxic activity of yellow gentian leaves (Genitaneae luteaefolium) grown in northern regions of Montenegro.

LC-ESI-MS and HPLC were used for the identification of the constituents from G. lutea leaves collected at different localities, as well as for quantification of the main compounds. Seven secoiridoids, five C-glucoflavones and three xanthones, were identified. Swertiamarin derivatives, namely eustomorusside (2), eustomoside (3) and septemfidoside (5), were detected in G. lutea for the first time. Concentrations of five constituents (swertiamarin, gentiopicrin, isovitexin, mangiferin and isogentisin) were determined. The relationship between concentrations of y-pyrones and altitude was observed with statistically significant correlation (r = 0.94). The extracts were also evaluated for their content of total phenolics, and antiradical and cytotoxic activities. The total phenolics content ranged from 7.7 to 12.7 mg GAE/g, and the IC50 values for DPPH radical scavenging activity varied between 0.45 to 2.02 mg/mL. The leaf extract exhibited moderate cytotoxic effects toward HeLa cells with an IC50 value of 41.1 microg/mL, while gentiopicrin, mangiferin and isogentisin exerted strong activity against HeLa cells, with IC50 values ranging from 5.7 to 8.8 microg/mL. The results confirm the traditional usage of G. lutea leaves and also suggest their possible utilization as hepatoprotective, hypoglycemic and anti-inflammatory agents.

Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts.

Flowers of Helichrysum plicatum were extracted under different experimental conditions, and their antioxidant activity was determined by DPPH radical scavenging assay. Extracts obtained with higher concentration of ethyl acetate (90% or 100%) were found to contain the greatest amount of total phenolics (> 250 mg gallic acid equivalents/g of dried extract), and high correlation between total phenolic content and antiradical activity was observed (r = -0.79). Based on the total phenolic content and antiradical activity, some extracts were selected for investigation of cytotoxic activity toward PC3, HeLa and K562 human cancer cell lines in vitro. All tested extracts exhibited moderate activity against HeLa cells (41.9-42.1 microg/mL), whereas the extract obtained with 100% ethyl acetate was the most active against K562 and PC3 cell lines (25.9 and 39.2 microg/mL, respectively). Statistical analysis revealed significant correlation between total phenolic content and cytotoxic activity against PC3 and K562 cells. HPLC identification of phenolic compounds from the extracts indicated the presence of apigenin, naringenin and kaempferol as free aglycones, and glycosides of apigenin, naringenin, quercetin and kaempferol. Among aglycones, kaempferol displayed moderate cytostatic activity against all cell lines (24.8-64.7 microM).

Radioprotective activity of Gentiana lutea extract and mangiferin.

Radioprotective/sensitizing actions of Gentiana lutea aqueous-ethanol extract and mangiferin on radiation-induced effects on different types of cells were investigated. The study focused on the decreasing survival of normal human immunocompetent cells, the survival of the malignant cells in vitro, and the survival of ex vivo irradiated cells before and after consumption of the extract by healthy volunteers. The in vitro experiments showed that mangiferin could inhibit cytotoxic action of ionizing irradiation (doses of 6 and 8 Gy) only on normal resting human PBMC, not stimulated for proliferation. Orally consumed G. lutea extract showed the potential to reduce the cytotoxic effect of x-ray irradiation on normal human immunocompetent cells PBMC of some healthy people, without changing the susceptibility of malignant cells to be destroyed by irradiation. Since the radioprotective effect was individually dependent, further clinical studies are needed.

Anti-inflammatory and gastroprotective properties of Hypericum richeri oil extracts.

Oil extracts of flowering tops of Hypericum richeri Vill. prepared in three different ways were evaluated for chemical composition, and anti-inflammatory and gastroprotective activities. An HPLC method was developed for determination of two dominant flavonoids, quercetin and I3,II8-biapigenin. The carrageenan-induced rat paw edema test was used for screening the anti-inflammatory activity, while indomethacin-induced rat gastric mucosa damage test was used for evaluation of gastroprotective activity. The oil extract prepared by maceration with 96% ethanol, followed by extraction with sunflower oil by heating on a water bath, exhibited the highest anti-inflammatory (38.4%) and gastroprotective activities (gastric damage score of 0.9). The same oil extract had the highest content of quercetin (49 microg/mL) and I3,II8-biapigenin (60 microg/mL). These results approve the usage of oil extracts of H. richeri as an anti-inflammatory and gastroprotective agent.

Antioxidant, cytotoxic, and antimicrobial activity of Alnus incana (L.) ssp. incana Moench and A. viridis (Chaix) DC ssp. viridis extracts.

Antioxidant, cytotoxic, and antimicrobial activities of leaves, bark, and cone extracts of Alnus incana (L.) Moench ssp. incana and endemic species A. viridis (Chaix) DC ssp. viridis were evaluated. All extracts were found to be strong 1,1-diphenyl-2-picrylhydrazyl free radical scavengers, exhibiting 50% inhibitory concentration (IC(50)) values of 3.3-18.9 microg/mL, and also showed activity in inhibition of lipid peroxidation with IC(50) values ranging from 38.5 to 157.4 microg/mL. A. incana and A. viridis extracts exhibited significant cytotoxic effects toward HeLa cells, with IC(50) values ranging from 26.02 to 68.5 microg/mL. The most active extract of A. incana bark also contained great amounts of total phenolics (316.2 mg of gallic acid equivalents/g). In our experiment all extracts were virtually nontoxic on brine shrimps. Extracts were screened for activity against 15 microorganisms, and all extracts investigated showed antimicrobial activity. The most active were dry extracts of cones of A. incana and A. viridis with minimum inhibitory concentration values ranging from 0.117 to 0.129 mg/mL.

Cytotoxic and cell cycle effects induced by two herbal extracts on human cervix carcinoma and human breast cancer cell lines.

In recent times interest has increased in the complementary medicine of cancer patients. Two herbal mixtures were prepared from 17 and 12 plants, respectively. The goal of this study was to examine the in vitro cytotoxic and cell cycle effects of the aqueous-ethanol extracts (Extract 1 and Extract 2) obtained by maceration of the mixtures. The two extracts investigated exhibited significant antiproliferative activity toward two human breast cancer cell lines (MDA-MB-361 and MDA-MB-453) and a human cervix carcinoma cell line (HeLa) with 50% inhibitory concentration (IC(50)) values ranging from 9.92 to 17.38 microL/mL. The extracts did not exert any significant cytotoxicity toward healthy human peripheral blood mononuclear cells. In vitro antitumor activities were accompanied by an important apoptotic fraction of all cell lines after treatment with the extracts. The amount of total phenols was similar in both extracts, whereas the concentration of total tannins was significantly higher in Extract 1. Extract 1 was also found to be a stronger free radical scavenger, with an IC(50) value of 13.4 microg/mL. Both extracts contained rosmarinic acid, while ursolic acid was identified in Extract 2.

In vitro cytotoxic and antioxidative activity of Cornus mas and Cotinus coggygria.

Cytotoxicity and antioxidant properties of the methanol extracts of leaves and flowers of Cornus mas and Cotinus coggygria were studied, together with their chemical composition. Extracts of C. coggygria flowers and leaves showed better antioxidant activity in reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as in inhibition of lipid peroxidation (LP) than C. mas extracts. Preliminary results showed that all extracts possessed potential cytotoxic activity towards HeLa and LS174 human cancer cell lines in vitro, with stronger inhibition against growth of HeLa cell growth than against LS174 cell growth. Cytotoxic activity of C. coggygria extracts showed good correlation with their antioxidative activity in vitro. In the brine shrimp lethality test, there were no significant changes in toxicity.

Antimicrobial activity of Gentiana lutea L. extracts.

Methanolic extracts of flowers and leaves of Gentiana lutea L., together with the isolated compounds mangiferin, isogentisin and gentiopicrin, were used to investigate the antimicrobial activity of the plant. A variety of Gram-positive and Gram-negative bacteria as well as the yeast Candida albicans has been included in this study. Both extracts and isolated compounds showed antimicrobial activity with MIC values ranging from 0.12-0.31 mg/ml. Our study indicated that the synergistic activity of the pure compounds may be responsible for the good antimicrobial effect of the extracts. Quantification of the secondary metabolites was performed using HPLC.

Phenolic content and radical scavenging capacity of berries and related jams from certificated area in Serbia.

Chemical composition and the effects of domestic processing and storage were studied in organically cultivated or wild growing berries, harvested in certificated area in Serbia. The total phenolics content in fresh berries ranged from 380 to 1660 mg GAE/100 g. The phenolic content decreased during the processing to jams, and storage in almost all samples, except in black currant. Among fresh berries, the highest amount of total anthocyanins was found in bilberry (0.61 +/- 0.03 %). Processing and storage caused decrease in anthocyanin content. The most ubiquitous anthocyanin aglycone was found to be cyanidin. Ellagic acid was detected in fresh and frozen black raspberries and related jams, and its content increases during thermal processing to jam. Among fresh berries, bilberry showed the strongest DPPH radical scavenging activity with an IC(50) value of 0.82 mg/ml. However, frozen as well as processed berries in our study still showed comparable high values of radical scavenging activity. After 9 months of storage all investigated jams were microbiologicaly correct. Regardless of the losses in the amount of total phenolics, total anthocyanins and radical scavenging activity, frozen berries and related jams are still excellent sources of nutritional substances with antioxidant potential.

Anti-inflammatory, gastroprotective, free-radical-scavenging, and antimicrobial activities of hawthorn berries ethanol extract.

Hawthorn [Crataegus monogyna Jacq. and Crataegus oxyacantha L.; sin. Crataegus laevigata (Poiret) DC., Rosaceae] leaves, flowers, and berries are used in traditional medicine in the treatment of chronic heart failure, high blood pressure, arrhythmia, and various digestive ailments, as well as geriatric and antiarteriosclerosis remedies. According to European Pharmacopoeia 6.0, hawthorn berries consist of the dried false fruits of these two species or their mixture. The present study was carried out to test free-radical-scavenging, anti-inflammatory, gastroprotective, and antimicrobial activities of hawthorn berries ethanol extract. Phenolic compounds represented 3.54%, expressed as gallic acid equivalents. Determination of total flavonoid aglycones content yielded 0.18%. The percentage of hyperoside, as the main flavonol component, was 0.14%. With respect to procyanidins content, the obtained value was 0.44%. DPPH radical-scavenging capacity of the extract was concentration-dependent, with EC50 value of 52.04 microg/mL (calculation based on the total phenolic compounds content in the extract). Oral administration of investigated extract caused dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. The obtained anti-inflammatory effect was 20.8, 23.0, and 36.3% for the extract doses of 50, 100, and 200 mg/kg, respectively. In comparison to indomethacin, given in a dose producing 50% reduction of rat paw edema, the extract given in the highest tested dose (200 mg/kg) showed 72.4% of its activity. Gastroprotective activity of the extract was investigated using an ethanol-induced acute stress ulcer in rats with ranitidine as a reference drug. Hawthorn extract produced dose-dependent gastroprotective activity (3.8 +/- 2.1, 1.9 +/- 1.7, and 0.7 +/- 0.5 for doses of 50, 100, and 200 mg/kg, respectively), with the efficacy comparable to that of the reference drug. Antimicrobial testing of the extract revealed its moderate bactericidal activity, especially against gram-positive bacteria Micrococcus flavus, Bacillus subtilis, and Lysteria monocytogenes, with no effect on Candida albicans. All active components identified in the extract might be responsible for activities observed.

Radioprotective effects of Gentianella austriaca fractions and polyphenolic constituents in human lymphocytes.

The aim of this study was to identify active principles of Gentianella austriaca responsible for the reduction of the incidence of micronuclei in irradiated lymphocytes in vitro. The radioprotective effects of ether (EF) and methanolic (MeF) fractions, water-soluble xanthones demethylbellidifolin (1), demethylbellidifolin 8-O-glucoside (2), bellidifolin 8-O-glucoside (3), and flavonoid swertisin (4) against chromosomal damage induced by gamma-rays were determined using the micronucleus test. EF and MeF showed better protection in treatment of human lymphocytes after gamma-irradiation than did isolated compounds. Among the isolated compounds, the effectiveness in reduction of the frequency of micronuclei followed the order 4>3>2>1. The anti-lipoperoxidant activity was in the order 2>4>1, while 3 slightly increased the level of malondialdehyde. In addition, the effectiveness in induction of apoptosis followed the order, 3>2>4, while 1 had no proapoptotic effect. These results suggest that the antioxidative properties of the polyphenols tested may contribute to the radioprotective effects of G. austriaca.

Antioxidant activity of nine Fabaceae species growing in Serbia and Montenegro.

The aim of this study was to investigate antioxidant capacity of nine Fabaceae species collected on the mountains of Serbia and Montenegro. Antioxidant assays with various reaction mechanisms were used, including total phenolic content by Folin-Ciocalteu, DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) values by ABTS radical cation and inhibition of liposome peroxidation. The investigated plants exhibited strong antioxidant capacity in all the tested methods, and among them, Lathyrus binatus, Trifolium pannonicum, and Anthyllis aurea were found to be the most active.