Sea fennel (Crithmum maritimum L.): phytochemical profile, antioxidative, cholinesterase inhibitory and vasodilatory activity.

Sea fennel, a rediscovered star of the coastal cuisine, has been investigated for its phytochemical profile and biological potential. Sea fennel flowers, stems and leaves were analyzed for essential oils (EOs) isolated by hydrodistillation, as well as non-volatiles obtained by ethanolic extraction. Limonene were found to be a dominant compound in EOs and ethanolic extracts; ranging from 57.5-74.2 % and 0.7-8.1 mg/g dry plant material, respectively. In addition total phenolic content was determined for ethanolic extracts. All samples and their main phytochemicals were tested for various methods. EO and extract obtained from flowers were tested for vasodilatory activity on rat aortic rings. Antioxidant activity of EOs was extremely low in comparison to extracts, on the contrary to cholinesterase inhibition where EOs showed better activity than extracts. Flower extract and chlorogenic acid showed stronger vasodilators in comparison to EO and limonene. The obtained results point out the potential impact of the dominant compounds from EO and extract on the biological properties of the sea fennel.

Seasonal variations of phenolic compounds and biological properties in sage (Salvia officinalis L.).

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed-phase (RP)-HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, Briggs-Rauscher reaction, and ß-carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram-positive (Bacillus cereus and Staphylococcus aureus) and two Gram-negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.

Differences in vasodilatory response to red wine in rat and guinea pig aorta.

We examined and compared mechanisms of the red wine (RW)-induced vasorelaxation in guinea pig (GP) and rat aorta. Acetylcholine-induced relaxation of norepinephrine-precontracted aortic rings was stronger in rat aorta than in GP aorta, whereas RW-induced vasorelaxation was stronger in GP aorta. L-nitro-arginine methyl ester (L-NAME) abolished RW-induced vasorelaxation in rat aorta, whereas in GP aorta, it was only reduced by 50%. To examine mechanisms of the L-NAME-resistant relaxation, GP aortic rings were additionally exposed to indomethacin, clotrimazole, and their combination. Indomethacin insignificantly reduced RW-induced relaxation, but in combination with L-NAME, the relaxation was synergistically decreased (80%). After clotrimazole exposure, the relaxation was reduced by 25%, and addition of indomethacin caused no further reduction. Only the combination of L-NAME, indomethacin, and clotrimazole prevented RW-induced vasorelaxation. RW-induced vasorelaxation in KCl-precontracted GP rings was significantly smaller (Emax 78.31% +/- 6.09%) than the RW-induced relaxation in norepinephrine-precontracted rings (Emax 126.01% +/- 2.11%). L-NAME in KCl-precontracted GP rings prevented RW-induced vasorelaxation. In conclusion, different pathways are involved in the RW-induced vasorelaxation in GP aorta, in contrast to rat aorta, in which NO plays main role. Therefore, the uncritical extrapolation of the results from one species to another could be misleading.

Interactions of resveratrol with other phenolics and activity against food-borne pathogens.

The aim of this study was to investigate the antibacterial activity of individual phenolics and their binary mixtures with resveratrol against selected food-borne pathogens. The antibacterial activity was quantified using the broth microdilution method by the determination of minimal inhibitory concentrations (MICs). Interactions between compounds in the binary phenolic mixtures were determined by calculating the fractional inhibitory concentration index (FICI). The influence of the number of OH groups in the phenols' structure on their antibacterial activity was assessed by principal component analysis (PCA). The most effective compounds were flavone luteolin and flavonol rutin, while the weakest antimicrobial activity was observed for phenolic acid and flavan-3-ols (catechin and epicatechin). The synergistic effect (FICI ≤0.5) of equimolar mixture of resveratrol with kaempferol was confirmed against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, while the mixture of rutin with resveratrol proved synergistic only against S. aureus. The increasing concentrations of resveratrol in the mixtures with kaempferol and rutin resulted in a loss of synergism which indicates that only selected phenolic mixtures, with optimal concentrations of their individual components, result in synergistic antibacterial activity. We did not find an association between total number of OH groups and antibacterial activity of either individual phenolics or their mixtures.

Antioxidant and Antimicrobial Potential of Phenolic Metabolites from Traditionally Used Mediterranean Herbs and Spices.

The phenolic extracts of fifteen Mediterranean medicinal plants, as well as their antioxidant and antimicrobial activities were investigated to grade their potential as additives in the food industry. Phenolic profiles of plant extracts were determined spectrophotometrically (total phenolics and phenolic subgroups) while individual compounds were identified using chromatographic assays. The biological activity of samples was determined using five antioxidant assays, while the antibacterial potential was determined against six foodborne pathogens (Camplyobacter coli, Escherichia coli, Salmonela Infantis, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus). The results showed significant variations in phenolic profile of plants and consequently their biological activity. Bearberry contained the highest concentration of phenolics, was extremely rich in non-flavonoids and also had the highest amount of catechins that resulted with good reducing and free radical scavenging properties and low chelating activity. All extracts were not effective against tested microorganisms with Gram-positive bacteria being more sensitive (especially S. aureus). The most effective extracts were St. Johns wort against S. aureus with minimal inhibitory concentration (MIC) of 1.00 mg/mL), bay laurel and nettle against B. cereus (MICs of 1.67 mg/mL), and woodland strawberry against L. monocytogenes (MIC of 3.33 mg/mL).

Comparison of chemical composition and free radical scavenging ability of glycosidically bound and free volatiles from Bosnian pine (Pinus heldreichii Christ. var. leucodermis).

The results obtained show that Bosnian pine is rich in glycosidically bound volatile compounds with strong free radical scavenging properties. Since volatiles can be released from nonvolatile glycoside precursors, these compounds can be considered as a hidden potential source of antioxidant substances and may contribute to the total free radical scavenging ability of Bosnian pine.

The antioxidant power and level of lipid peroxidation products in the sera of apparently healthy adult males.

The aim of this study was to determine the antioxidant potential of the serum and the level of lipid oxidation products in the sera of apparently healthy adult males. The "antioxidant power" of the serum, defined as the ability to reduce ferric ions by antioxidants from the serum (FRAP), was taken as the indicator of total antioxidation potential. The formation of lipid oxidation products was evaluated as thiobarbituric reactive species serum test (TBARS). The ferrous oxidation in xylenol orange version 2 (FOX2) assay coupled with triphenylphosphine was used for measurement of total sera hydroperoxides (ROOHs). The following biochemical variables were determined in the sera: aspartat aminotranspherase (AST), alanine aminotranspherase (ALT), gamma-glutamyl transpherase (GGT), bilirubin, glucose, creatinine, cholesterol, triglycerides and hemoglobin. Blood sera from apparently healthy subjects (166 adult males) were analyzed. Median age of study participants was 36 years (range 25-50 years). The X +/- SD sera FRAP level of the 166 apparently healthy adult males was 1047 +/- 131 micromol/L (779-1410 range). The X +/- SD level of sera TBARS was 1.2 +/- 0.3 micromol/L of the sera (0.5-2.2 range). Compared with the level of TBARS in the sera, the level of FOX2-ROOH was significantly higher The X +/- SD level of lipid hydroperoxides in the fresh sera, determined as FOX2, was 3.9 +/- 1.5 micromol/L of the sera (1.9-6.9 range). Observation of correlation between FRAP and determined biochemical variables in the sera have confirmed a statistically significant linear correlation between sera FRAP and bilirubin, hemoglobine, glucose, ALT and triglycerides (p < 0.05). The results of sera FRAP, TBARS and FOX2 levels can help in estimating the antioxidant status of humans. Significant correlation between the antioxidant power of blood serum and particular biochemical parameters indicates the complexity of defence mechanisms and various molecules involved in increasing the reduction power of the serum.

Oxidant/antioxidant properties of Croatian native propolis.

Native propolis was defined as propolis powder collected from the continental part of Croatia and prepared according to a patented process that preserves all the propolis natural nutritional and organoleptic qualities. Nine phenolic compounds (out of thirteen tested) in propolis sample were detected by high performance liquid chromatography (HPLC) analysis. Among them chrysin was the most abundant (2478.5 microg/g propolis). Contrary to moderate antioxidant activity of propolis examined in vitro (ferric reduction antioxidant power; FRAP-assay), propolis as a food supplement modulated antioxidant enzymes (AOE) and significantly decreased lipid peroxidation processes (LPO) in plasma, liver, lungs, and brain of mice. The effect was dose- and tissue-dependent. The lower dose (100 mg/kg bw) protected plasma from oxidation, whereas the higher dose (300 mg/kg bw) was pro-oxidative. Hyperoxia (long-term normobaric 100% oxygen) increased LPO in all three organs tested. The highest vulnerability to oxidative stress was observed in lungs where hyperoxia was not associated with augmentation of AOE. Propolis protected lungs from hyperoxia by increased catalase (CAT) activity. This is of special importance for lungs since lungs of adult animals are highly vulnerable to oxidative stress because of their inability to augment AOE activity. Because of its strong antioxidant and scavenging abilities, native propolis might be used as a strong plant-based antioxidant effective not only in physiological conditions but also in cases that require prolonged high concentration of oxygen.

Insight into the Presence of Stilbenes in Medicinal Plants Traditionally Used in Croatian Folk Medicine.

Over the last years, great interest has arisen concerning plant stilbenes, especially resveratrol, which has a whole spectrum of positive biological activities. In this study, we investigated the presence of resveratrol monomers (trans- and cis- form) and naturally occurring derivatives of trans-resveratrol (piceid, astringin and isorhapontin) in phenolic extracts of twenty medicinal plants traditionally used in Croatian folk medicine. The investigated compounds were present in the samples, in free form or as glucosides, and the highest share was found in immortelle, common yarrow and Lamiaceae plants. The obtained results indicate that biological activity of selected medicinal plants can be related to the presence of this valuable group of phytochemicals.

[Some epidemiologic characteristics of foodborne intoxications in Croatia during the 1992-2001 period]. / Neke epidemioloske osobitosti alimentarnih toksoinfekcija u Hrvatskoj u razdoblju 1992.-2001. godine.

Epidemiologic characteristics of foodborne intoxications registered in Croatia during the 1992-2001 period are described. Salmonellosis, as well as staphylococcal food intoxication, foodborne botulism, Clostridium perfringens food intoxication, histamine poisoning and food intoxication caused by other pathogens, especially conditionally pathogenic bacteria, were analyzed. During the study period, 87,782 cases were reported, 45,721 (52.1%) of them related to salmonellosis and the rest of 42,061 (47.9%) cases to foodborne intoxications of various etiology. The diseases occurred throughout the year, with a higher proportion in summer than at any other time of year. During the study period, 480 outbreaks of foodborne intoxications with 10,567 cases were reported, 405 (84.4%) of them with 8476 cases related to salmonellosis. The most frequently isolated pathogen in salmonellosis outbreaks was Salmonella enteritidis with 345 (85.2%) outbreaks. The outbreaks of salmonellosis were primarily associated with the intake of pastries (40.1%). Ice-cream was associated with a high risk of staphylococcal food intoxication, and bean salad of Clostridium perfringens food intoxication. The food vehicle of transmission most often associated with foodborne botulism outbreaks was smoked ham. Histamine poisoning, recorded in four outbreaks, occurred after consumption of fish and fish products. Salmonellosis outbreaks most frequently occurred at home (n=149; 36.8%), followed by pastry shops with 47 (11.6%) outbreaks, the latter also ranking first in the outbreaks of staphylococcal food intoxication (40%).

[Antioxidative activity of propolis from Dalmatia (Croatia)]. / Antioksidacijski kapacitet dalmatinskog propolisa.

AIM: The aim of this study was to determine the antioxidative activity of propolis from ecologically clean parts of Dalmatia. METHODS: Phenol concentration in ethanolic propolis extracts was determined by Folin-Ciocalteu reagent using gallic acid as the standard. Flavonoid phenolic compounds were analyzed after precipitation with formaldehyde. The residual non-flavonoid phenolics were also determined by Folin-Ciocalteu method. By determining the change of peroxide number (PN), of tiobarbiture acid reactive species (TBARS), and of DPPH-radical activity, antioxidative efficiency of propolis was tested and compared with well known and widely used synthetic antioxidants. Values of PN and TBARS were determined at 60 degrees C in samples of trigyceride substrate (lard) without and with the addition of antioxidants. Compared was the efficiency of three antioxidants: propolis (alcoholic extract), vitamin E, and (+)-catechin in a concentration of 1%. PN was monitored during 50 days. By the method of Sedlacek, TBARS were measured during 30 days. Antioxidative activity of propolis extract was also measured in terms of hydrogen donating ability using stable radical alpha,alpha-diphenyl-beta-picril hidrazyl (DPPH*) and compared with commercial synthetic antioxidants of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and (+)-cathecin. Inhibition degree of DPPH* was calculated by the formula of Yen and Duh. RESULTS: Total phenol content, expressed as gallic acid, in propolis extracts varied from 75.2 to 90.2 g/kg propolis. The proportion of flavonoids in total phenols ranged from 62% to 65%. Values of TBARS were not increased only in samples with added propolis. The inhibition of DPPH-radical by propolis extracts ranged from 93% to 96%, by catechin 95%, by BHT 49%, and by BHA 64%. Compared to BHT and BHA, propolis extracts showed greater reducing activity against DPPH-radical. DISCUSSION: The chemical composition of propolis, and thus its biological activity depend on the plant from which it has been collected, and on the macro- and microclimatic conditions. Many compounds in propolis exert antioxidative activity. A belief was expressed that the biological activity of propolis is very probably based mostly on its antioxidative efficiency. Dalmatian propolis showed high efficiency in the prevention of oxidative processes. This could be explained by the high proportion of polyphenol constituents, especially flavonoids. A very low and equal degree of increase of PN, as a measure of oxidative processes, was noticed in the samples of triglyceride substrate with the addition of propolis and (+)-catechin. The greatest rise of TBARS was measured in the samples of pure lard. There was no increase of TBARS only in the samples with added propolis. Propolis and (+)-catechin showed great efficiency in the inhibition of DPPH-radical, greater than BHT and BHA, which are widely used in food industry. CONCLUSION: The results indicate that Dalmatian propolis could be an efficient protective agent against oxidative processes in food. The high antioxidative activity of propolis, its natural origin, and present knowledge about its biological properties, make it a very promising nutritional additive for human diet.

[Nutritive density of zinc, copper, iron and manganese in daily diet of adults]. / Nutritivna gustoca cinka, bakra, zeljeza i mangana u dnevnoj prehrani odraslih osoba.

The aim of the present study was to determine the actual daily dietary intake of zinc, copper, manganese and iron in daily meals. The mass amounts of this micronutrients were determined by flame atomic absorption techniques. The daily meals from public organised nutrition were collected using random sampling during 1998-1999. The energetic and nutritive quality of total diet and nutritive density of zinc, copper, iron and manganese were evaluated and compared with WHO/FAO recommendations.

Phenolic acids profile, antioxidant and antibacterial activity of chamomile, common yarrow and immortelle (Asteraceae).

Chamomile, common yarrow and immortelle ethanolic extracts were chemically analysed with respect to phenolics. Twelve phenolic acids were separated and identified by HPLC-DAD and the presence of rosmarinic acid was additionally confirmed by LC-MS. Five methods were applied for the evaluation of extracts' antioxidant properties (FRAP, DPPH, ABTS, chelating activity, Briggs-Rauscher reaction), while the antibacterial activity was tested against some of the major food-borne pathogens (Campylobacter coli, Escherichia coli, Salmonella Infantis, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus). Immortelle extract was the richest in phenolics with a dominant flavonoid fraction, while the other two extracts contained higher amount of non-flavonoids. The phenolic acid profile also varied; high concentration of rosmarinic acid was found in chamomile and common yarrow, while caffeic acid was dominant in immortelle. The best antioxidant properties were obtained for chamomile extract, while good antimicrobial activity, especially against Gram-positive bacterial species, was detected for immortelle. The obtained results could be used as a tool for chemotaxonomic classification of the investigated plants or for their potential application as natural antioxidants/antimicrobials.

Antioxidative/acetylcholinesterase inhibitory activity of some Asteraceae plants.

The extracts obtained by 80% EtOH from some Asteraceae plants (Calendula officinalis, Inula helenium, Arctium lappa, Artemisia absinthium and Achillea millefolium) were studied. Rosmarinic acid, one of the main compounds identified in all extracts, was determined quantitatively by using HPLC. In addition, spectrophotometric methods were evaluated as an alternative for rosmarinic acid content determination. Total phenolic content was also established for all extracts. A. millefolium extract was found to have the highest content of rosmarinic acid as well as total phenols. All extracts were tested for antioxidant and acetylcholinesterase inhibitory activity. A. millefolium was shown to possess the best antioxidant activity (for all tested methods) as well as acetylcholinesterase inhibitory activity. Highly positive linear relationships were obtained between antioxidant/acetylcholinesterase inhibitory activity and the determined rosmarinic acid content indicating its significance for the observed activities.

Antioxidant and vasodilatory effects of blackberry and grape wines.

In contrast to the well-described various biological effects of grape wines, the potential effects of commonly consumed blackberry wine have not been studied. We examined in vitro antioxidant and vasodilatory effects of four blackberry wines and compared them with the effects of two red and two white grape wines. Although some blackberry wines had lower total phenolic content relative to the red grape wines, their antioxidant capacity was stronger, which may be related to a higher content of non-flavonoid compounds (most notably gallic acid) in blackberry wines. Although maximal vasodilation induced by blackberry wines was generally similar to that of red wines, blackberry wines were less potent vasodilators. Vasodilatory activity of all wines, in addition to their flavonoid and total phenolic content, was most significantly associated with their content of anthocyanins. No association of vasodilation with any individual polyphenolic compound was found. Our results indicate the biological potential of blackberry wines, which deserves deeper scientific attention.

Influence of the phenophase on the phenolic profile and antioxidant properties of Dalmatian sage.

This study aimed to research the influence of phenophase on the phenolic profile (phenolic acids, flavonoids and stilbenes) and related antioxidant properties of sage, one of the most characteristic plants of Dalmatian karst. The total phenol and flavonoid contents in sage leaves were determined spectrophotometrically, while the principal phenolics were determined using HPLC-RP-DAD. Antioxidant properties of this well known medicinal plant were determined as free radical scavenging activity (DPPH), ferric reducing/antioxidant power (FRAP) and by Briggs-Rauscher oscillating reaction (BR). The results strongly indicate that Dalmatian sage leaves are rich source of valuable phenolics, mainly phenolic acids, with extremely good antioxidant properties. The presence of resveratrol or its derivatives was confirmed in all extracts. The best results for total phenols and flavonoids, as well as the best antioxidant properties were obtained for May sage, while the highest amounts of catechin monomers and compounds from group of stilbenes were found in February extract.

The increase in human plasma antioxidant capacity after red wine consumption is due to both plasma urate and wine polyphenols.

By using red wine, dealcoholized red wine, polyphenols-stripped red wine, ethanol-water solution and water, the role of wine polyphenols and induction of plasma urate elevation on plasma antioxidant capacity was examined in humans (n=9 per beverage). Healthy males randomly consumed each beverage in a cross-over design. Plasma antioxidant capacity (measured by ferric reducing antioxidant power, FRAP), ethanol, catechin and urate concentrations were determined before and 30, 60, 90, 120 and 180 min after beverage intake. Dealcoholized red wine and polyphenols-stripped red wine induced similar increase in FRAP values which represented nearly half the effect of the original red wine. This indicates that consumption of red wine involves two separate mechanisms in elevation of plasma FRAP values and both wine phenols and plasma urate contribute to that effect.

Comparison of protective effects of catechin applied in vitro and in vivo on ischemia-reperfusion injury in the isolated rat hearts.

AIM: To determine overall cardiac effects of flavonoid catechin on ischemia-reperfusion injury in isolated rat hearts in vitro and in vivo. METHODS: After perfusing hearts at the pressure of 70 mm Hg, coronary perfusion was interrupted for 30 minutes and then re-established. There were four experimental groups, with 10 rats each. The first group received 100 micromol/L of catechin added to the perfusate 10 minutes before, during, and 10 minutes after ischemia; the second group underwent in vivo pretreatment with catechin (250 mg/kg body weight) applied intragastrically for 10 days; the third group received it as a single dose 1 h before sacrifice; and the fourth group received saline. An additional group of 10 hearts served as untreated, nonischemic time control. The variables included heart rate, atrioventricular conduction time, cardiac rhythm, isovolumetric left ventricular pressure, coronary flow and responsiveness, oxygen consumption, relative cardiac efficiency, lactate dehydrogenase release, and myocardial lipid peroxidation. RESULTS: Catechin added to the perfusate increased the coronary flow and ratio of oxygen delivery to myocardial oxygen consumption before ischemia and during reperfusion. Following 30 minutes of reperfusion, isovolumetric left ventricular developed pressure recovered to 42+/-3%, 63+/-3%, 71+/-2%, and 55+/-3% of the initial control values in the control, catechin 1 h, catechin for 10 days, and catechin in vitro group, respectively. Cardiac efficiency and coronary responsiveness were also best preserved in the group receiving catechin for 10 days. CONCLUSION: Application of catechin in vitro and in vivo, irrespective of duration of application, resulted in cardioprotection against ischemia-reperfusion injury, but long-term pretreatment provided more favorable effects. Directly applied, catechin acted as a vasodilator.