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Karnozin (β-alanyl-L-histidin) je dipeptid, tvořený aminokyselinou β-alaninem a L-histidinem. Karnozin a obě aminokyseliny se vyskytuji v mozku a kosternim svalstvu vsech obratlovců. Karnozin v buňkach mozkove tkaně chrani intracelularni bilkoviny před jejich poskozenim kyslikovými radikaly, glykaci a tvorbou přicných vazeb mezi jednotlivými řetězci bilkovin. V kosternim svalstvu karnozin snižuje svalovou unavu způsobenou zvýsenou koncentraci kyseliny mlecne, ktera vede k poklesu pH. S rostoucim biologickým věkem klesa koncentrace karnozinu jak v buňkach mozkove tkaně, tak v bilých svalových vlaknech kosterniho svalstva, cimž dochazi k zhorsovani senzorických a kognitivnich funkci a ubytku svaloviny. Tyto degenerativni procesy lze zpomalit konzumaci potravin bohatých na karnozin nebo doplňovanim diety β-alaninem. β-alanin urcuje rychlost tvorby karnozinu a jeho koncentraci v buňkach mozkove a svalove tkaně. Clanek shrnuje soucasne nazory mediciny na roli karnozinu v procesu starnuti lidskeho organismu a významu obohacovani diety β-alaninem.

L-tryptofan je esencialni aminokyselina, kterou clověk ziskava z potravin. V organismu je soucasti řady bilkovin. Kromě teto role je L-tryptofansubstratem pro syntezu serotoninu a dalsich neurotransmiterů v mozku a travicim ustroji. Clanek je přehledem o použiti L-tryptofanu ke zvýsenihladiny serotoninu v mozku a tim ke zlepseni nalady, smyslových schopnosti a socialniho chovani.

U pacientů s roztrousenou sklerozou (RS) se neurologický deficit projevuje sirokým spektrem klinických potiži, vcetně mikcnich dysfunkci. Infekce mocových cest (IMC) zhorsuji kvalitu života pacientů a mohou významně ovlivnit průběh onemocněni. Pilotni, randomizovana a placebem kontrolovana studie ověřila, že sestiměsicni uživani 1,5 g (odpovida 75 g cerstveho plodu) klikvy velkoplode denně mělo vliv na kvalitu života a na možne sniženi rizika onemocněni infekci mocových cest pacientů s RS. Průkaz profylaxe IMC plodem V. macrocarpon vsak vyžaduje rozsahlejsi klinickou studii.

Most research on American cranberry in the prevention of urinary tract infection (UTI) has used juices. The spectrum of components in juice is limited. This study tested whether whole cranberry fruit powder (proanthocyanidin content 0.56%) could prevent recurrent UTI in 182 women with two or more UTI episodes in the last year. Participants were randomized to a cranberry (n = 89) or a placebo group (n = 93) and received daily 500 mg of cranberry for 6 months. The number of UTI diagnoses was counted. The intent-to-treat analyses showed that in the cranberry group, the UTIs were significantly fewer [10.8% vs. 25.8%, p = 0.04, with an age-standardized 12-month UTI history (p = 0.01)]. The Kaplan-Meier survival curves showed that the cranberry group experienced a longer time to first UTI than the placebo group (p = 0.04). Biochemical parameters were normal, and there was no significant difference in urinary phenolics between the groups at baseline or on day180. The results show that cranberry fruit powder (peel, seeds, pulp) may reduce the risk of symptomatic UTI in women with a history of recurrent UTIs.

The aim of this study was to evaluate the fatty acid (FA) profile in the plasma and erythrocyte membranes of apparently healthy men compared to male patients with various cardiovascular diseases (CVD). The plasma FA profiles were analyzed in five groups of subjects aged 37–57 years: groups 1 and 2 were male voluntary blood donors with a body mass index (BMI) 25.1 ± 1.6, (Group 1, N = 12) and a BMI 32.9 ± 4.5, (Group 2, N = 11) respectively, male patients with chronic heart failure due to dilated cardiomyopathy (Group 3, N = 10), chronic atrial fibrillation without heart failure or coronary heart disease (Group 4, N = 10) and patients after myocardial infarction (Group 5, N = 11). The FA profile in plasma and erythrocytes was measured by gas chromatography (GC) with flame ionization detection and basic plasma lipid parameters were then estimated. Deficiencies of eicosapentaenoic acid and docosahexaenoic acid were found in all the groups of CVD patients and in Group 2 (BMI 32.9 ± 4.5) of this Central European population. Unfavorable alterations in FA composition and clinical biochemistry parameters were found in patients with dilated cardiomyopathy (Group 3). Further, 75% of Group 1 (blood donors) had an average plasma PUFA level in the worst quartile for relative risk of cardiovascular events. Tailored n-3 PUFA supplementation should be recommended not only for secondary prevention in patients suffering from CVD but also for primary prevention in overweight and obese persons with a proven deficiency. Practical applications: Plasma and erythrocyte membrane n-3 polyunsaturated fatty acid (PUFA) levels are an important parameter of diet composition as well as a marker of cardiovascular risk. Regular sea fish intake or alternative supplementation with the n-3 PUFA recommendation is in all probability insufficiently realized, particularly in inland countries. In addition to contemporary guidelines and practice, research is providing data on the use of FA analysis for revealing people with an n-3 PUFA deficiency. This approach could benefit people suffering from CVD as well as for overweight and obese populations with demonstrated deficiency. Tailored n-3 PUFA supplementation should be recommended not only for secondary prevention in patients suffering from CVD but also for primary prevention in overweight and obese persons with a proven deficiency.

The aim of this double-blind, placebo controlled clinical trial was to assess the effects of a combination of selenium and silymarin in men with lower urinary tract symptoms, benign prostatic hyperplasia and a prostate specific antigen (PSA) ≤2.5ng/ml. The volunteers were randomized to two groups: the first one (n=26) received 240μg selenium (in the form of yeast l-selenomethionine) plus 570mg silymarin daily for 6 months and the second (n=29) received placebo. Outcome measures were changes in the International Prostate Symptom Score (IPSS), bladder volume (V), urinary flow rate, ultrasound estimated postvoid residual urine volume (RV), serum PSA, testosterone and selenium levels, safety clinical biochemistry, hematology and oxidative stress parameters at baseline and on day 180. The results showed statistically significant differences (p

This study investigated the effect of one-week consumption of 165 g/day fresh blue honeysuckle berries (208 mg/day anthocyanins) in 10 healthy volunteers. At the end of intervention, levels of benzoic (median 1782 vs 4156), protocatechuic (709 vs 2417), vanillic (2779 vs 4753), 3-hydroxycinnamic (143 vs 351), p-coumaric (182 vs 271), isoferulic (805 vs 1570), ferulic (1086 vs 2395), and hippuric (194833 vs 398711 μg/mg creatinine) acids by LC/MS were significantly increased in the urine. Clinical chemistry safety markers were not altered. Oxidative stress markers, erythrocyte glutathione peroxidase (0.73 vs 0.88 U/g Hb) and catalase (2.5 vs 2.8 μkat/g Hb) activities, and erythrocyte/plasma thiobarbituric acid reactive substance (522 vs 612/33 vs 38 μmol/g Hb/protein) levels were significantly increased, without change in plasma antioxidant status. Nonsignificant changes of advanced oxidation protein products and oxidized LDL were observed. The results provide a solid base for further study of metabolite excretion and antioxidant parameters after ingestion of anthocyanins.

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine-DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b(5) on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine-DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b(5) increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N(2)-oxide. CYP3A4 and 1A1 in the presence of cytochrome b(5) are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine-DNA adduct 2. These ellipticine-DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC(50) value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

The fluorescence properties of protopine and allocryptopine in aqueous and organic environments are described for the first time. The fluorescence of alkaloids and their pH-dependent interconversion to cationic forms (transannular interaction) were studied using steady-state and time-resolved fluorescence techniques. For the analysis of tricyclic base and cis/trans tetracyclic cations of the alkaloids, NMR and X-ray crystallography were used.

The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.

Anthocyanins from the fruit Lonicera caerulea L. var. kamtschatica (blueberry honeysuckle, Caprifoliaceae) were studied via (semi)preparative chromatographic fractionation followed by MS and μLC/MS analysis. The extraction procedure was optimized with respect to analytical purposes as well as its potential use for the preparation of nutraceuticals. The highest yield of anthocyanins was obtained using acidified methanol as the extraction medium. A comparable total anthocyanin content was obtained using a mixture of methanol and acetone. However, when Lonicera anthocyanins were in contact with acetone, a condensation reaction occurred to a large extent and related 5-methylpyranoanthocyanins were found. The effect of other extraction media, including ethanol as a "green" solvent, is also discussed. The potential of two fractionation procedures for extract purification differing in their chromatographic selectivity and scale was studied (i.e. using a Sephadex LH-20 gel column and a reversed phase). Fractions obtained by both procedures were used for a detailed analysis. MS and μLC/MS(2) methods were used for monitoring anthocyanin and 5-methylpyranoderivatives content as well as identifying less common and more complex dyes (dimer of cyanidin-3-hexoside, cyanidin-ethyl-catechin-hexosides, etc.). These more complex dyes are most likely formed during fruit treatment.

This paper describes oxidation of the isoquinoline alkaloid, protopine (PR) at a pyrolytic graphite electrode (PGE) using cyclic and square-wave voltammetry. In the alkaline range (pH 7.5–10.5) of a Britton–Robinson (B–R) buffer, a PR oxidation can be observed as a well-developed voltammetric peak around + 0.9 V (vs. Ag j AgCl j 3 M KCl). With increasing pH of the B–R buffer, the PR peak is shifted to less positive potentials. The acquired voltammetric data suggest that PR strongly adsorbs onto the surface of the pyrolytic graphite where it is subjected to irreversible electrochemical oxidation in its uncharged free (tricyclic) base form. The results are discussed in connection with the electrochemical oxidation of other isoquinoline alkaloids and the potential applications of these data.

Lower urinary tract symptoms (LUTS) are a common condition in older men. The objective of the present study was to evaluate the efficacy and tolerability of cranberry (Vaccinium macrocarpon) powder in men at risk of prostate disease with LUTS, elevated prostate-specific antigen (PSA), negative prostate biopsy and clinically confirmed chronic non-bacterial prostatitis. Forty-two participants received either 1500 mg of the dried powdered cranberries per d for 6 months (cranberry group;n21) or no cranberry treatment (control group;n21). Physical examination, International Prostate Symptom Score, quality of life (QoL), five-item version of the International Index of Erectile Function (IIEF-5), basic clinical chemistry parameters, haematology, Se, testosterone, PSA (free and total), C-reactive protein (CRP), antioxidant status, transrectal ultrasound prostate volume, urinary flow rate, ultrasound-estimated post-void residual urine volume at baseline, and at 3 and 6 months, and urineex vivoanti-adherence activity were determined in all subjects. In contrast to the control group, patients in the cranberry group had statistically significant improvement in International Prostate Symptom Score, QoL, urination parameters including voiding parameters (rate of urine flow, average flow, total volume and post-void residual urine volume), and lower total PSA level on day 180 of the study. There was no influence on blood testosterone or serum CRP levels. There was no statistically significant improvement in the control group. The results of the present trial are the first firm evidence that cranberries may ameliorate LUTS, independent of benign prostatic hyperplasia or C-reactive protein level.

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called alpha- and beta-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MS(n) data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H](+) and [M-H](-) ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.

Cranberry (Vaccinium macrocarpon Ait. Ericaceae) fruits and juice are widely used for their antiadherence and antioxidative properties. Little is known however about their effects on clinical chemistry markers after long-term consumption. This study was conducted to evaluate the effect of three commercial cranberry products, NUTRICRAN90S, HI-PAC 4.0, and PACRAN on the antioxidative status of rodents, divided into three experimental groups. The products were given as dietary admixtures (1500 mg of product/kg of stock feed) for 14 weeks to male Wistar rats (Groups 2-4) and a control Group 1 which received only stock feed. There were no significant cranberry treatment-related effects on oxidative stress parameters, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase, superoxide dismutase, total antioxidant capacity, thiobarbituric acid reactive substances, advanced oxidation protein products, total SH-groups, or any other measured clinical chemistry markers. Hematological parameters, body weight, and food consumption were also unaffected by intake of cranberries. Only liver glutathione reductase activity and glutathione levels were significantly lower in Group 4 than in Group 1. Plasma alkaline phosphatase alone was significantly decreased in Group 2. No gross pathology, effects on organ weights, or histopathology were observed. No genotoxicity was found, and total cytochrome P450 level in liver was unaffected in all groups. The levels of hippuric acid and several phenolic acids were significantly increased in plasma and urine in Groups 2-4. The concentration of anthocyanins was under the detection threshold. The dietary addition of cranberry powders for 14 weeks was well tolerated, but it did not improve the antioxidative status in rats.

The plant chemistry and cytotoxic activity of Colchicum baytopiorum CD Brickell (Liliaceae/Colchicaceae), an endemic species growing in Turkey, has been studied for the first time. Nine known alkaloids were isolated and their structures were identified by spectral methods (UV, IR, (1)H-NMR, and ESI/MS), and the presence of three alkaloids, which could not be isolated from the plant, was also detected by LC/MS/MS spectrometry. Phenolic acids were elaborated using LC/MS and 11 phenolic acids were identified. The presence of two flavonoids appeared to be valuable for chemotaxonomic purposes. Guided by the brine shrimp lethality test (BSLT), methanol extracts were tested for cytotoxic activity by colorimetric MTT test on K562 and HL60 cells. Except the seed extract, all methanol extracts showed more cytotoxic activity on HL60 cells (IC(50): 6.5-0.1 microg/mL) than on K562 cells (IC(50):500-44 microg/mL).

Macleaya cordata (Willd.) (Papaveraceae) is used as an active component in the natural feed additive Sangrovit. Sangrovit contains mixture of the intact aerial parts and the fraction of quaternary benzo[c]phenanthridine alkaloids from M. cordata (FQBA). In a 90-day pilot toxicity trial, Sangrovit and the FQBA were tested for safety. Male Wistar rats were fed for 90 days with 100, 7000 or 14000mg of Sangrovit or 600mg of FQBA in 1kg of feed. Body and organ weights, clinical chemistry and hematology markers, oxidative stress parameters, morphological structure of tongue, liver, ileum, kidney and heart samples, and total cytochrome P450 in liver were monitored. The results showed no statistically significant alterations in any parameter between control and treated animals, except for the group treated with 14000ppm Sangrovit that resulted in elevation of reduced glutathione level and superoxide dismutase activity in liver.

The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of Sangrovit, a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and 500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No significant alterations in body or organ weights, macroscopic details of organs, histopathology of liver, ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay, formation of DNA adducts in liver by 32P-postlabeling, modulation of cytochrome P450 1A1/2 or changes in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentration in plasma at the first or second hour following application. DHSG is cleared from the body relatively quickly (its plasma levels drop to zero after 12 or 18 h, respectively).

Yacon and maca are native Andean crops with growing popularity as food supplements often in combination with other components, e.g. silymarin. There are however no published data on their toxicity and safety in humans. The aim of our randomized placebo-controlled 90-day study was to evaluate the effects of yacon and maca in combination with silymarin on plasma and lipoprotein lipids, serum glucose and safety parameters in patients suffering from the metabolic syndrome. No adverse effects were found in volunteers using silymarin (0.8 g/day), silymarin + yacon (0.8 + 2.4 g/day) and silymarin + maca (0.6 + 0.2 g/day). A moderate AST level and diastolic blood pressure increase was found in volunteers using maca (0.6 g/day). In conclusion, the combination silymarin + yacon appears to be promising as a nutraceutical in the prevention of diseases with a proatherogenic lipoprotein profile and liver steatosis. The effect of maca on AST level and blood pressure must be considered when using high doses of maca powder. This effect could be reversed by supplementation with silymarin.

This paper describes a sensitive isocratic HPLC/ECD method developed for the determination of rosmarinic acid (RA) in plant material, animal feed, and pig plasma. The plasma sample preparation only includes protein precipitation and adjustment of the pH. The applicability of the method was tested on plasma samples of pigs that were exposed to a 91-day oral intake of RA via feed enriched by aerial parts of Prunella vulgaris. The plasma was directly analyzed using the method described as well as after enzymatic hydrolysis. When no hydrolysis step was included, RA and caffeic acid (CA) were quantified in the plasma. In hydrolyzed plasma samples, several other metabolites were determined, including dihydrocaffeic, ferulic, and dihydroferulic acid. The dual-channel coulometric detection employed, as an alternative to mass spectrometry, offers good selectivity and sensitivity owing to the electrochemical properties of the phenolic constituents.

Silybin (a flavonolignan, the main component of silymarin, an extract from the seeds of Silybum marianum) has been used to date mostly as a hepatoprotectant. However, it also has other interesting activities, e.g., anticancer and hypocholesterolemic effects. It is also known that silybin can inhibit the activities of the cytochrome P450 (P450) enzymes. In this study, a weak interaction of silybin with human microsomal CYP2E1, 2A6, 2B6, 2C19, and 2D6 (IC(50) > or = 250 microM) was found; a moderate inhibition was observed for CYP1A2 and 2C8. The most prominent inhibition effect was found with CYP3A4 and CYP2C9 (IC(50) < or = 50 microM). Using mass spectometry detection, production of O-demethylated (the main metabolite) as well as hydroxylated derivatives of silybin formed by P450 enzymes was detected. The effect of different P450 inhibitors on the formation of O-demethylated product was also studied. In particular, a relatively specific inhibitor of CYP2C8 (quercetin) markedly inhibited the formation of this metabolite. With the help of recombinant enzymes (bactosomes), it was confirmed that the CYP2C8 enzyme is responsible for the reaction leading to O-demethylated silybin.

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 °C and 10 MPa (15 MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO2 modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75 mm × 4.6 mm, 3.5 μm particle size) using methanol (organic phase) and 50 mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0–1 min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0 min, 60–71% organic phase (v/v); from 7.0 to 18.0 min, 71–60% organic phase (v/v) was applied. The limit of detection was 1.22 ng (3.50 pmol) for CHE and 0.95 ng (2.72 pmol) for DHCHE per 1 ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0–1.5%, accuracy was in the range 99.7–100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.

This study assessed the effect of an 8 week consumption of dried cranberry juice (DCJ) on 65 healthy young women. Basic biochemical and hematological parameters, antioxidant status, presence of metabolites in urine, and urine ex vivo antiadherence activity were determined throughout the trial. A 400 mg amount of DCJ/day had no influence on any parameter tested. A 1200 mg amount of DCJ/day resulted in a statistically significant decrease in serum levels of advanced oxidation protein products. This specific protective effect against oxidative damage of proteins is described here for the first time. Urine samples had an inhibitory effect on the adhesion of uropathogenic Escherichia coli strains, but no increase in urine acidity was noted. Hippuric acid, isomers of salicyluric and dihydroxybenzoic acids, and quercetin glucuronide were identified as the main metabolites. In conclusion, cranberry fruits are effective not only in the prevention of urinary tract infection but also for the prevention of oxidative stress.

The effect of a bilberry extract (BE, 25% anthocyanins) against oxidative damage in primary cultures of rat hepatocytes, induced by tert -butyl hydroperoxide and allyl alcohol, was investigated. BE displayed cytoprotective effects at 100 and 500 μg/ml in the MTT viability test. It protected the cells against lactate dehydrogenase leakage and lipoperoxidation products formation. Maximum protection (58%) was noted using 500 μg/ml of BE and intoxication by allyl alcohol. The observed cytoprotective effect is probably due to the antioxidant properties of its constituents, mainly anthocyanins. BE scavenged DPPH (IC 50 3.99 ± 0.14 μg/ml) and enzymatically generated superoxide radical with an activity equivalent to 108 ± 7.2 U of superoxide dismutase per mg of extract. Our results support the use of bilberry and bilberry extracts in functional foods and food supplements designed for the prevention of chronic diseases associated with oxidative stress.

Putative interactions between quaternary benzo[c]phenanthridine alkaloid sanguinarine (SA) and aryl hydrocarbon receptor/cytochrome P450 CYP1A (AhR/CYP1A) regulatory pathway are the subject of perpetual disputations. The role of CYP1A enzymes and AhR receptor in SA cytotoxicity was anticipated. In this paper, we tested, whether selected inducers of CYP enzymes modulate cytotoxicity of SA in primary cultures of rat hepatocytes. Cells were challenged 48h with dioxin (TCDD; 5nM), phenobarbital (PB; 500microM) or DMSO prior to the treatment with SA. SA itself displayed time- and dose-dependent cytotoxicity as revealed by lactate dehydrogenase leakage into the medium and MTT test. Pre-treatment of hepatocytes with TCDD and/or PB significantly attenuated SA cytotoxicity, the effects being more pronounced at lower concentrations of SA and shorter periods of incubation. We assumed involvement of CYP1A enzymes in diminution of SA cytotoxicity. Surprisingly, co-treatment with SA and furafylline, an inhibitor of CYP1A enzymes, further attenuated SA cytotoxicity instead of expected reversal of this effect. We conclude that TCDD- and PB-inducible genes attenuate cytotoxicity of SA in rat hepatocytes. CYP1A enzymes are not involved in this attenuation, but they rather augment SA cytotoxicity. Future research should focus on analyses of the involvement of other CYPs in SA cytotoxicity and on identification of TCDD-/PB-controlled genes responsible for observed phenomenon.

Adult rats were orally administered with a single dose of sanguinarine (10 mg SA per 1 kg body weight) in 1.0 ml water. In the plasma and the liver, dihydrosanguinarine (DHSA) was identified as a SA metabolite by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS). Significantly higher levels of DHSA were found in both the plasma and the liver in comparison with those of SA. SA and DHSA were not detected in the urine. The formation of DHSA might be the first step of SA detoxification in the organism and its subsequent elimination in phase II reactions. Benz[c]acridine (BCA), in the literature cited SA metabolite, was found neither in urine nor in plasma and liver.

UNLABELLED The glycaemic index (GI) is a measure of the food power to raise blood glucose (B-glucose) concentration after a meal. For healthy eating, foods with low GI are recommended. However, for many foods in the European Union the GI has not been defined yet. The aims of this prospective open-label study were: (1) to determine the GI of white bread and juicy cereal bars FIT (Usovsko, Czech Republic) by means of the glucometer Optium (Abbott/Medisense); (2) to compare the GI of tested foods determined in the morning and in the evening hours; (3) to compare the GI of tested foods in men and women and (4) to assess the variability of the GI. METHODS To determine the GI, measured portions of food containing 50 g of carbohydrates were eaten by 11 healthy volunteers. B-glucose curves were constructed from B-glucose values at time 0, 15, 30, 45, 60, 60, 120 min after the meal. The GI was calculated by dividing the incremental area under the curve (IAUC) for the tested food by that for the standard food (IAUCS). In each volunteer each food was tested 5 times so that 5 GI's was obtained and the average was calculated. The GI for each tested food was calculated as the mean from the respective average GI's of the 11 volunteers. MS Excel and the statistical program SPSS v. 10.1 were used to analyze the data. RESULTS (1) The mean values of the GI for white bread was 70.3 % and for juicy cereal bars was 101.0 %, as determined in a total of 139 tests in the whole group of 11 volunteers. There was a difference when comparing white bread vs. glucose (p = 0.012) and white bread vs. cereal bars (p = 0.026) but no difference between glucose and cereal bars. (2) There was no significant difference between the GI determined in the morning and in the evening hours either for the total of 139 tests or for the individual tested foods. (3) No significant difference could be seen between the GI in men and women when comparing glucose, cereal bars and white bread. (4) There was a wide variability of GI in all tested foods: the standard deviation of GI for white bread was 30.7 %, for juicy cereal bars 38.0 %. CONCLUSIONS The GI's for white bread and juicy cereal bars were determined. There was no difference either between the GI values determined in the morning vs. the evening hours or between the values in men vs. women. The results show wide variability. An accurate standard method for the determination of GI needs to be defined, carefully used and re-evaluated to enable a comparison of the results with various methods of other working groups.

Silymarin, an extract of flavonolignans from the dried fruits of milk thistle (Silybum marianum L. Gaertneri) and its constituents silibinin, dehydrosilibinin, silychristin and silydianin were tested for protective effects on rat cardiomyocytes exposed to doxorubicin. Silymarin and individual flavonolignans did not exert cytotoxicity in the range 25–100 µm (incubation 9 h). Dehydrosilibinin was tested only at 25 µm concentration due to its low solubility. All substances increased the cell ATP level. Silymarin and flavonolignans displayed a dose-dependent cytoprotection against doxorubicin (100 µm, incubation 8 h). The protective effects of silymarin, silibinin, dehydrosilibinin and silychristin were comparable to that of dexrasoxane, while silydianin exerted the best protective effect. The ability of silymarin complex and its components to protect cardiomyocytes against doxorubicin-induced oxidative stress is due mainly to their cell membrane stabilization effect, radical scavenging and iron chelating potency. Copyright © 2004 John Wiley & Sons, Ltd.

Sanguinaria canadesis, Chelidonium majus and Macleya cordata have been used for centuries as alternative medicines. Currently the extracts from these medicinal plants are components of veterinary and human phytopreparations, and of oral-hygiene agents. Sanguinarine and chelerythrine (SA/CHE) are biologically active components of these extracts. They display distinct antibacterial and anti-inflammatory properties, but, on the other hand, they have been reported as having adverse effects - genotoxicity and hepatotoxicity. This paper is aimed at evaluation of the effects of daily administration of the extract from Macleya cordata (2 mg and 100 mg in 1 kg feed, sanguinarine:chelerythrine 3:1) in the diet on the health status of swine. After 90-day administration, alkaloids were retained to a different extent in tissues. The highest SA/CHE retention was detected in the gingiva (0.55 microg/g) and liver (0.15 microg/g), no SA/CHE were detected in muscles. Plasma SA levels attained 0.11 microg/ml. Treated animals did not display any results of hematological, biochemical or histological assay different from controls. A (32)P-postlabeling assay proved that no DNA-adducts with SA/CHE were detected in pig livers. We did not observe any symptom linked to epidemic dropsy syndrome often attributed to sanguinarine. In conclusion, an average daily oral dose of alkaloids up to 5 mg per 1 kg animal body weight proved to be safe.

Capillary zone electrophoresis was applied to the investigation of the interaction of sanguinarine and chelerythrine with mercapto compounds including albumins at pH 7.4. Mercaptoethanol and L-cysteine were chosen for the identification of the type of interaction and for the identification of the interacting chemical form of these alkaloids. It was evidenced that sanguinarine and chelerythrine do not react chemically with these mercapto compounds at pH 7.4 and that non-covalent products form in this interaction. Their interaction is a fast and reversible complexation and is based on non-bonding intermolecular interactions. Conditional binding constants measured at pH 7.4 and 5.0 indicate that only uncharged forms of sanguinarine and chelerythrine (pseudobases) participate in complexation. A negatively charged group, either bound to the mercapto ligand or supplied by the solution, enters in the complexation. The simple 1 : 1 interaction scheme holds, therefore, only for mercapto compounds bearing an anionic group. Constants corrected for the abundance of the uncharged alkaloid form are of the order of magnitude of 104 l mol−1 and depend on the chemical composition of buffer. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins is qualitatively identical with interaction of these alkaloids with simple mercapto compounds. Constants for the binding of uncharged form of sanguinarine with human and bovine serum albumins in sodium phosphate buffer at pH 7.4, corrected for abundance of the interacting uncharged form, are 332 000±38 400 and 141 000±14 400 l mol−1, respectively. The former agrees well with the value K=385 000 (or log K=5.59) reported from static experiments. For the uncharged form of chelerythrine, the constants are 2 970 000±360 000 and 1 380 000±22 600 l mol−1 for human and bovine serum albumins, respectively. Copyright © 2003 John Wiley & Sons, Ltd.

To assess the role of plasma lipoproteins in the transport of silibinin, an antioxidant flavonolignan, (125)I-labelled silibinin ((125)I-SB) administered perorally to the rat was used. The plasma (125)I-SB derived radioactivity was distributed among plasma lipoproteins according to their lipophilicity (TAG-rich lipoproteins 30-40% > LDL 15% > HDL 5%), and in the fraction of d > 1.215 containing albumin and other proteins a minority amount of radioactivity was found. Administration of (125)I-SB in a complex with phosphatidylcholine resulted in proportionally higher radioactivities in all fractions as well as in tissues. Dietary olive oil had a slightly decreasing effect on plasma concentrations of silibinin measured by HPLC as well as on (125)I-SB derived radioactivity in plasma and liver. In the TAG-rich lipoprotein fraction and HDL no effects of olive oil on the levels of (125)I-SB derived radioactivities were observed, however, at a 30 min interval the levels of (125)I-SB derived radioactivity in LDL and the heart were significantly decreased in the olive oil group. These results suggest that (i) silibinin is not resorbed by the chylomicron pathway, and (ii) the endogenous lipoprotein pathway VLDL --> LDL may play a role in the transport of silibinin from the liver to the extrahepatic tissues concurrently facilitating the lipoprotein antioxidant influence of silibinin.

Background & Aims: Yacon (Smallanthus sonchifolius, Asteraceae) is a native Andean plant, cultivated for its tubers throughout South America. The leaves are used in folk medicine as a medicinal tea for hypoglycemia. This paper describes the antioxidant activity of various extracts from S. sonchifolius leaves for their content of phenolic components. Methods: The dried leaves were extracted in several ways. Two fractions were selected for their high content of phenolic compounds and analyzed by RP-HPLC. The antioxidant activity of these fractions was tested in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and xanthine/XOD superoxide radical scavenging assays, as inhibition of lipoperoxidation of subcellular membranes and as protective activity against oxidative injury of rat hepatocytes in primary cultures. Results and conclusions: The presence of protocatechuic (2.5 and 0.12 mg/g), chlorogenic (9.9 and 1.7 mg/g), caffeic (14.7 and 0.09 mg/g) and ferulic (traces) acids were determined in the two fractions. Both fractions showed potent antioxidant activity in DPPH (IC50 = 16.1 ± 3.4 and 24.3 ± 2.7 mg/ml) and xanthine/XOD superoxide radical scavenging (42.0 ± 20.3 and 34.3 ± 11.4 SOD equivalents (U/mg)) tests, they inhibited the lipoperoxidation of rat liver subcellular membranes and they protected rat hepatocytes against oxidative injury. Our results may predetermine the use of S. sonchifolius leaves in human diet as a potential remedy in the prevention of chronic diseases caused by radicals, e. g., arteriosclerosis.

Silybin and related flavonolignans form a major part of the Silybum marianum extract, silymarin, which has been used to treat liver diseases for hundreds of years. Although regarded as safe, many of the extract constituents remain thus far untested for their possible effects on liver biotransformation enzymes. Cytochromes P450 (CYP) are very important in this regard. We tested the effect of four flavonolignans: silybin, its hemisynthetic derivative dehydrosilybin, silydianin, and silycristin on three specific CYP activities: bufuralol 1'-hydroxylation (CYP2D6), p-nitrophenol hydroxylation (CYP2E1), and nifedipine oxidation (CYP3A4). All flavonolignans displayed dose-dependent inhibition of these activities with IC(50) values in the micromolar range. The inhibition was competitive or mixed as revealed by double reciprocal plots of kinetic experiments. However, the inhibition is not considered to be relevant for therapy because physiological concentrations of the individual flavonolignans do not exceed 0.5 microM. The data support the use of the extract as a dietary supplement.

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.

Chelerythrine, sanguinarine and an alkaloid extract from Macleaya cordata--sanguiritrin--were found to be inhibitors of aminopeptidase A and dipeptidyl peptidase IV, while fagaronine inhibited dipeptidyl peptidase IV only. At 50 microM, chelerythrine, sanguinarine and sanguiritrin inhibited aminopeptidase N by 82%, 82%, 88%, DPP IV by 38%, 62%, 57%, and fagaronine by 34%, respectively. When bovine serum albumin (500 microg/mL) was added, the inhibition of both proteases by quaternary benzo[c]phenanthridine alkaloids (QBA) (50 microM) was significantly diminished. Strong interaction of chelerythrine and sanguinarine with bovine and human serum albumin was proved by electrophoretic determination of their respective conditional binding constants.

Cranberry (Vaccinium macrocarpon) is being studied for use in the prevention of recurrent urinary tract infection (rUTI) where it affects the adhesion Escherichia coli to the uroepithelium. This 6 month, randomized, double-blind, placebo-controlled trial aimed to evaluate whether a daily dose of 500 mg Pacran® could prevent UTI occurrences in sexually active women with a medical history of at least 2 symptomatic UTI in the previous 12 months. Study participants were randomized to the Pacran® or the placebo group and were followed up for 6 months. The intent-to-treat analysis included 176 participants (n=93 in placebo and n=83 in Pacran® group). In the Pacran® group, the proportion of women experiencing at least one UTI was significantly lower than in the placebo group [10.8% versus 25.8%, respectively, p=0.04, with age adjusted 12-month UTI history (p=0.01), age (p=0.73), and age-squared (p=0.05) also included in the model]. There was a similar finding when risk of an E. coli UTI was assessed (p=0.03). Wh...

This work was aimed to investigate the currant oil-induced modulation of antihypercholesterolemic and LDL antioxidant effects of silymarin (SM), the extract from Silybum marianum L. containing silibinin as the main flavonolignan, and of better bioavailable silibinin-phosphatidylcholine complex (SPC) in rats fed on high-cholesterol (HC) high-fat diet. Feeding of rats on HC-diet supplemented with 10 % of currant oil (from Ribes nigrum L.) containing polyunsaturated fatty acids (PUFA, 61.1% of n–6 and 15.4 % of n–3) and lower amounts of saturated (SFA, 7.7%) and monounsaturated (MUFA, 14.3%) fatty acids caused a significant decrease in plasma cholesterol associated with a mild decrease in VLDL-C and an increase in HDL-C, when compared to rats fed on HC-diet with 10 % of lard fat containing low amounts of PUFA (7.7% of n–6 and 0.7 % of n–3) and higher amounts of SFA (42.7%) and MUFA (47.5%). However, currant oil feeding led to the increased oxidizability of LDL. Silymarin, but not SPC, was effective in prevention of development of dietary induced hypercholesterolemia in the both dietary fats with slightly better result in diet containing the currant oil. On the other hand, SPC supressed more effectively than silymarin LDL oxidizability. The results suggest that antihypercholesterolemic effect of SM in rats fed on HCdiet is improved by dietary currant oil, but the currant oil induces an increased oxidizability of LDL. This can be supressed by improvement of bioavailability of silibinin, as shown here for silibinin-phosphatidylcholine complex.

Capillary electrophoresis was employed to determine the principal quaternary benzo[ c ]phenanthridine alkaloids, sanguinarine and chelerythrine, in two plant extracts and one oral hygiene product. Phosphate–Tris buffer of pH 2.5 was used as a background electrolyte, limits of detection were 3 μmol l −1 (sanguinarine) and 2.4 μmol l −1 (chelerythrine) using UV detection at 270 nm. The method, which correlated well with HPLC, is suitable for serial determination of sanguinarine and chelerythrine in plant products and pharmaceuticals.

The isoquinoline alkaloids adlumiceine, adlumidiceine, coptisine, corytuberine, cryptopine, fumaricine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, palmatine, parfumine, protopine, sinactine, stylopine, and N-methylstylopine were determined by reversed phase high performance liquid chromatography in Fumaria agraria, F. capreolata, F. densiflora, F. muralis, F. officinalis, F. parviflora, and F. vaillantii. Organic acids, namely citric, coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic, protocatechuic acid and caffeic acid (and its methylester) were identified by gas chromatography—mass spectrometry. The total content of phenolic constituents was quantified by a colorimetric method using a phosphomolybdic–phosphotungstic reagent. The chemotaxonomic significance of the results is discussed. Copyright © 1999 John Wiley & Sons, Ltd.

To study the ability of silymarin, a standardized mixture of antioxidant flavonolignans from the medicinal plant Silybum marianum, and of silybin, the main flavonolignan of silymarin, to inhibit the development of diet-induced hypercholesterolemia the rats were fed high cholesterol diet (HCD). Silymarin or silybin were given as dietary supplements, and their influences on serum cholesterol levels were compared to those of probucol, an antioxidant hypocholesterolemic drug. Anticholesterolemic effect of silymarin was parallel to that of probucol, and dose-dependent at dietary drug concentrations of 0.1-0.5-1.0% (w/w). However, in contradistinction to probucol, silymarin caused an increase in high density lipoprotein (HDL)-cholesterol and a decrease in liver cholesterol content, changes considered to be of benefit. In addition to its anticholesterolemic effect silymarin partially prevented the HCD-induced decrease in liver reduced glutathione, an endogenous antioxidant. Silybin was not so effective as silymarin suggesting that either other constituent(s) of silymarin may be responsible for its anticholesterolemic effect or the bioavailability of silybin alone might be lower than that of silybin as a compound of silymarin.

Silybin glycosides—23-O-β-glucoside 2b, β-galactoside 3b, β-lactoside 4b and β-maltoside 5b—have been synthesized by different methods (Helferich glycosylation, Lewis acid catalysis). Separation of two silybin diastereoisomers in the form of acetylated monoglycosides has been achieved for the first time. These new silybin glycosides are 4–30 times more water-soluble, and their hepatoprotectivity is increased compared with that of the parent compound silybin 1.

The study objective was to investigate the potential of a beverage containing silymarin and L-arginine to alter basic physiological and urodynamic parameters in 22 normal healthy men aged 38-59 years. The volunteers drank 500 ml/day beverage without silymarin and L-arginine for 10 days followed, after a 7-day washout period, by the beverage with 400mg silymarin and 295 mg L-arginine for 10 days. Blood and urine samples were collected on days 0, 10 and 27. The beverages were well-tolerated with no adverse effects. Most of the biochemical, hematological and urodynamic parameters remained unchanged. Total antioxidant capacity, total level of antioxidants, lipoperoxidation products (malondialdehyde), advanced oxidation products of proteins in plasma and glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase levels in erythrocytes were not influenced. Serum γ-glutamyl transferase, malondialdehyde level and activity of glutathione S-transferase in erythrocytes were lowered at day 27 and the concentration of total plasma SH-groups was higher on day 10. Using an ex vivo system, we found that silymarin/silybin at 10-100 μM is able to adsorb onto human erythrocytes and the complexes displayed antioxidant properties as studied using ex situ square-wave voltammetry. The trial showed that silymarin in vivo may protect erythrocytes against oxidative damage.

Sanguinarine, chelerythrine and fagaronine were examined for hepatotoxic effect. Administration of sanguinarine and chelerythrine (10 mg/kg/day; i.p.) produced marked parenchymal injury not found in the livers of fagaronine treated rats. In chronic administration of sanguinarine (0.2 mg/kg/56 days; i.p.) degeneration and necrosis were not observed in the liver. The effect of the alkaloids was tested by enzyme leakage (LDH and ASAT) and changes in glutathione levels in isolated rat hepatocytes. Sanguinarine produced the greatest toxicity followed by lesser toxicity in the case of chelerythrine while no toxicity was displayed with fagaronine. These effects correlate with pK R values for pseudobase formation. Finally, no increased lipid peroxidation or mitochondrial membrane damage were found.

Twenty isoquinoline alkaloids were identified in the aerial part of Fumaria densiflora DC. In addition to bicuculline, densiflorine, fumaritine, fumariline, scoulerine, coptisine, cryptopine, parfumine, protopine, and sinactine which have been found previously, the following alkaloids were isolated for the first time from this species: adlumine, fumaricine, fumaritridine, fumarophycine, fumarofine, corytuberine, cis-N-methylstylopinium iodide, stylopine, and two new 1-benzylisoquinoline alkaloids, viz. fumaflorine and fumaflorine methyl ester. The cytoprotectivity of extracts of F. densiflora and F. officinalis, and that of protopine, cryptopine, and parfumine was tested on primary cultures of rat hepatocytes intoxicated with carbon tetrachloride. The cytoprotective effect of the extracts is associated with the alkaloids.

A high-performance liquid chromatographic method for the determination of colchicinoid alkaloids in plant material is described. The determination is performed separately in neutral and basic alkaloid fractions using a C18-bonded silica column. Nine alkaloids, i.e., 3-demethylcolchicine, 2-demethylcolchicine, colchifoline, N-deacetyl-N-formylcolchicine, colchicine, cornigerine, 2-demethyldemecolcine, 3-demethyldemecolcine and demecolcine, in seven Colchicum plants were assayed. For identification of phenolic compounds, a method using gas chromatography-mass spectrometry was elaborated and twenty phenolic compounds were identified in extracts from five Colchicum species. As the presence of luteolin appeared to be of value for chemotaxonomic purposes, its simple densitometric determination was developed.