Search

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inhibition of copper/quinoprotein amine oxidases (EC 1.4.3.6), AO-I (dimer 2×75 kDa) and AO-II (monomer 80 kDa), from the fungus Aspergillus niger by benzophenanthridine alkaloids sanguinarine, chelerythrine, and fagaronine were studied. For both amine oxidases the alkaloids showed reversible noncompetitive inhibition of n-hexylamine oxidation with Ki 0.6, 0.9 and 2.8 mM for sanguinarine, chelerythrine. and fagaronine, respectively. The values of the inhibition constants corresponded to pKR+ values for the iminium ion/pseudobase equilibrium of the alkaloids. Since thio-compounds protected the enzymes against this inhibition, the inhibition effect was ascribed to the interaction with a sulfhydryl group essential for the enzymatic activity.

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-β-D-glucuronides, B-7-O-β-D-glucuronides, A-20-O-β-D-glucuronides and B-20-O-β-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-β-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Transformation of ten colchicinoids by isolated rat liver microsomes resulted in the mixture of C-2, C-3, and C-10 O-demethylated metabolites. Colchicinoids administered i.p. to rats (2.5 mumol/kg) increased serum and liver activities of alkaline phosphatase and decreased liver microsomal demethylase activity as well as cytochrome P-450 content. The changes of acid phosphatase level were less pronounced. The aspartate and alanine aminotransferase activities were significantly increased only in colchicine treated rats. No relations between enzyme activity changes and colchicinoid hydrophobicities quantified by partition coefficients (log P) were found. However, the enzyme activity changes were related to the type of substitution at C-3, C-7, and C-10 of colchicinoids. Particularly, O-demethylation at C-3 resulted into the fall of alkaline phosphatase response. On the other hand, the microsomal demethylation and cytochrome P-450 content were related to the modification of the nitrogen substituent at C-7.

The Cu(II)-complexes of colchiceine (1) and N-deacetylcolchiceine (2) were prepared. They have been shown to be potent inhibitors of the yeast alcoholdehydrogenase. The results indicate the formation of a ternary enzyme-Cu(II)-alkaloid complex.

The quaternary isoquinoline alkaloid, sanguinarine (SG) plays an important role in both traditional and modern medicine, exhibiting a wide range of biological activities. Under physiological conditions, there is an equilibrium between the quaternary cation (SG+) and a pseudobase (SGOH) forms of SG. In the gastrointestinal tract, SG is converted to dihydrosanguinarine (DHSG). All forms exhibit bright fluorescence. However, their spectra overlap, which limited the use of powerful techniques based on fluorescence spectroscopy/microscopy. Our experiments using a combination of steady-state and time-resolved techniques enabled the separation of individual components. The results revealed that (a) the equilibrium constant between SG+ and SGOH is pKa = 8.06, while fluorescence of DHSG exhibited no changes in the pH range 5–12, (b) the SGOH has excitation/emission spectra with maxima at 327/418 nm and excited-state lifetime 3.2 ns, the spectra of the SG+ have maxima at 475/590 nm and excited-state lifetime 2.4 ns. The DHSG spectra have maxima at 327/446 nm and 2-exponential decay with components 4.2 and 2.0 ns, (c) NADH is able to convert SG to DHSG, while there is no apparent interaction between NADH and DHSG. These techniques are applicable for monitoring the SG to DHSG conversion in hepatocytes.

Periodontitis is a chronic disease associated with inflammation of the tooth-supporting tissues. The inflammation is initiated by a group of gram-negative anaerobic bacteria. These express a number of irritating factors including a lipopolysaccharide (LPS), which plays a key role in periodontal disease development. Plant extracts with anti-inflammatory and anti-microbial properties have been shown to inhibit bacterial plaque formation and thus prevent chronic gingivitis. In this study we tested effects of Prunella vulgaris L. extract (PVE; 5, 10, 25microg/ml) and its component rosmarinic acid (RA; 1microg/ml) on LPS-induced oxidative damage and inflammation in human gingival fibroblasts. PVE and RA reduced reactive oxygen species (ROS) production, intracellular glutathione (GSH) depletion as well as lipid peroxidation in LPS-treated cells. Treatment with PVE and RA also inhibited LPS-induced up-regulation of interleukin 1beta (IL-1beta), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and suppressed expression of inducible nitric oxide synthase (iNOS). The results indicate that PVE and RA are able to suppress LPS-induced biological changes in gingival fibroblasts. The effects of PVE and RA are presumably linked to their anti-inflammatory activities and thus use of PVE and RA may be relevant in modulating the inflammation process, including periodontal disease.

A rapid prechromatographic sample preparation and a reversed phase high performance liquid chromatographic (RP-HPLC) ion pair method have been developed for the qualitative and quantitative analysis of the main alkaloids of Eschscholtzia californica, namely allocryptopine, californidine, chelerythrine, eschscholtzine, N-methyllaurotetanine, protopine and sanguinarine. The identity and purity of the component HPLC-peaks were established by photodiode array detection and by comparison with standards. The distribution of alkaloids in the organs of the plant was examined: allocryptopine is the main constituent in roots, whilst californidine is the major alkaloid in all aerial organs except flowers where the main compound is eschscholtzine. HPLC fingerprint chromatograms of extracts of E. californica from different commercial sources were found to be similar.

Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid, exhibits antimicrobial and anti-inflammatory activities and for this reason it is used in dental hygiene products and feed additives. Its metabolism and disposition is the subject of constant scientific discourse. In this paper we summarize current knowledge on sanguinarine metabolism. We show in particular that: (i) Sanguinarine is not transformed to 3,4-benzacridine and that the literature reporting this compound as a metabolite of sanguinarine is based on artifacts and misinterpretations that in course of time have created a dogma; (ii) Sanguinarine is converted to dihydrosanguinarine in vivo, the conversion being tentatively a detoxication pathway; (iii) Aryl hydrocarbon receptor metabolic signaling pathways modulate sanguinarine biological activity.

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name "maca"), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC(50) values of 3.46 +/- 0.16 and 0.71 +/- 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 mug of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.

Silymarin, a standardized mixture of flavonolignans, or its major constituents could be effective for prevention and treatment of hepatic damage or skin cancer. However, their potential side effects, such as modulation of endocrine functions via the disruption of estrogen receptor (ER) and/or aryl hydrocarbon receptor (AhR) activation, are largely unknown. In the present study, we investigated impact of silymarin, its constituents and a series of their synthetic derivatives on ER- and AhR-mediated activities using in vitro reporter gene assays. We found that none of the compounds under study affected the AhR-mediated activity in rat hepatoma cells. Contrary to that, several compounds behaved as either partial or full ER agonists. Silymarin elicited partial ER activation, with silybin B being probably responsible for a majority of the weak ER-mediated activity of silymarin; silybin A and other flavonolignans were found to be inactive and potent ER agonist taxifolin is only a minor constituent of silymarin. To our knowledge, this is probably the first time, when receptor-specific in vitro effects of separated diastereomers have been demonstrated. In contrast to silymarin constituents, the synthetic silybin derivatives, potentially useful as chemoprotective agents, did not modulate the ER-mediated activity, with exception of 23-O-pivaloylsilybin. Interestingly, 7-O-benzylsilybin potentiated ER-mediated activity of 17beta-estradiol despite possessing no estrogenic activity. In conclusion, our data suggest that estrogenicity of some silymarin constituents should be taken in account as their potential side effect when considered as chemopreventive compounds. These results also stress the need to study biological activities of purified or synthesized diastereomers of silybin derivatives.

Capillary zone electrophoresis was applied for the investigation of interactions of some quaternary isoquinoline alkaloids, namely sanguinarine, chelerythrine, berberine, and jatrorrhizine, with DNA constituents and with DNA. None of these alkaloids attach covalently to nucleotides or to the whole DNA under physiological conditions. The interaction with DNA constituents is a noncovalent complexation based on weak intermolecular forces. Electrostatic attraction participates in the interaction but other types of intermolecular forces are involved as well. Cations were identified as the most probable interacting forms of the alkaloids. The interaction with compounds derived from purine was always stronger than those derived from pyrimidine. All alkaloids behaved analogously and similarly to ethidium bromide, the classic DNA intercalator. Stability constants K (in I .mol - 1 ) for sanguinarine and chelerythrine in phosphate buffer of pH 7.4 (/s = 30 mM) ranged from tens to hundreds.

Flavonoids are found universally in plants and act as free radical scavenging and chelating agents with antiinflammatory, antiischemic, vasodilating and chemoprotective properties. In this study, the antilipoperoxidative and cytoprotective effects of apigenin, baicalein, kaempferol, luteolin and quercetin against doxorubicin-induced oxidative stress were investigated in isolated rat heart cardiac myocytes, mitochondria and microsomes. After preincubation of cardiomyocytes with the test compounds for 1 h the cardiomyocytes were treated with the toxic agent, doxorubicin (100 micro M for 8 h). Cardiomyocyte protection was assessed by extracellular LDH and cellular ADP and ATP production. Cytoprotection was concentration dependent for baicalein > luteolin congruent with apigenin > quercetin > kaempferol. All test compounds had signi fi cantly better protective effects than dexrazoxan, an agent currently used for adjuvant therapy during anthracycline antibiotic therapy. In microsomes/mitochondria the IC(50) values of lipid peroxidation inhibition for quercetin, baicalein, kaempferol, luteolin, and apigenin were 3.1 +/- 0.2/8.2 +/- 0.6, 3.3 +/- 0.3/9.6 +/- 0.5, 3.9 +/- 0.3/10.1 +/- 0.8, 22.9 +/- 1.7/18.2 +/- 0.7, and 338.8 +/- 23.1/73.1 +/- 6.4 mM, respectively. The antilipoperoxidative activity of apigenin differed from its cytoprotective effects, but correlated with the free radical scavenging of 2,2-diphenyl-1-picrylhydrazyl radical and half peak oxidation potential (E(p/2)). Apigenin was the least effective of the flavonoids studied in all models except the cardiomyocyte model where its cardiomyocyte cytoprotective effect was comparable to other compounds.

The chemoprotective effects of caffeic (CA), chlorogenic (CHA) and rosmarinic (RA) acids were tested against the toxicity of doxorubicin (DOX) in neonatal rat cardiomyocytes and the iron-dependent DOX induced lipid peroxidation of heart membranes, mitochondria and microsomes. The protectivity of these acids was compared with dexrazoxan, used as an adjuvant during DOX chemotherapy. The cytoprotective effects were assessed by enzyme (LDH and ASAT) and troponin I leakage, secondly by intracellular ATP content. All hydroxycinnamic acids proved non-cytotoxic, and they stabilized both membranes and the energetic status of cardiomyocytes. After preincubation of cardiomyocytes with the test compounds (100, 200 µm; 1 h) the cardiomyocytes were treated with the toxic agent, DOX (100 µm; 8 h). The test compounds protected cardiomyocytes against DOX induced oxidative stress (RA > CHA ≥ CA) on all monitored parameters. Substantial preservation of monolayer integrity of the cardiomyocytes by test compounds was also found microscopically. All the acids were more effective in the assays used than dexrazoxan. RA showed the most effective cytoprotectivity. All the acids significantly reduced the iron-dependent DOX induced lipid peroxidation of heart membranes, although of the test compounds, CHA was found to be the most effective (IC50 = 8.04 ± 0.74/6.87 ± 0.52 µm for microsomes/mitochondria). Copyright © 2004 John Wiley & Sons, Ltd.

The cardioprotective effect of Prunella vulgaris ethylacetate fraction (PVEF) and its constituent rosmarinic acid (RA) was evaluated on isolated rat cardiomyocytes subjected to doxorubicin-induced oxidative stress. The cytoprotectivities of PVEF and RA were concentration-dependent in the range of 0.005 to 0.05 mg/ml and the effect of PVEF correlated with the RA content. Dexrazoxan (DE), used as positive control, was less effective than PVEF or RA.

Using mercaptoethanol and ( l )-cysteine as representatives of mercapto compounds and capillary zone electrophoresis as experimental technique, it was evidenced that sanguinarine and chelerythrine do not react with the mercapto group of organic compounds at pH 7.4. Their interaction is fast and reversible complexation based on non-bonding intermolecular interaction in which enter uncharged forms of sanguinarine or chelerythrine. A negatively charged group, either bound to the mercapto ligand or supplied from solution, participates in the complexation. Simple 1:1 interaction scheme reported in literature holds therefore only for mercapto compounds bearing anionic group. Stoichiometric binding constants corrected for the abundance of the uncharged alkaloid form at pH 7.4 are of the order of magnitude of 10 4 l/mol and depend on both cations and anions of the background electrolyte. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins does not qualitatively differ from their interaction with simple mercapto compounds. Stoichiometric binding constants for the binding of sanguinarine with human and bovine serum albumins in sodium phosphate buffer pH 7.4, corrected for the abundance of the interacting uncharged form, are 332 000±38 400 and 141 000±14 400 l/mol, respectively. The former agrees well with the value K =385 000 (or log K =5.59) from static photometric experiments. Constants for the complexation of uncharged chelerythrine with human and bovine serum albumins are 2 970 000±360 000 and 1 380 000±22 600 l/mol, respectively.

Silymarin, an extract of Silybum marianum seeds, and the constituent flavonolignans silybin, silydianin and silychristin, as well as the flavonol quercetin, protected rat heart microsomes and mitochondria against iron-dependent doxorubicin induced lipid peroxidation. Quercetin was found to be more potent than either silymarin or its three constituents, whose cytoprotectivity was comparable. The radical scavenging activity of the compounds was investigated using a DPPH colour reduction assay and cyclic voltametry to assess their antioxidant activities. In contrast to quercetin, silybin, silydianin and silychristin did not chelate iron in aqueous solution. The results suggest that silymarin may prevent doxorubicin-mediated damage to rat heart membrane primarily through a free radical scavenging mechanism.

Sweeping preconcentration and electrokinetic injection was used for the capillary electrophoretic analysis of trace amounts of biologically active anthracyclines with UV absorption detection. Phosphate buffer (100 mM), pH 2.5, with addition of 40% v/v methanol was used as background electrolyte (BGE). Sodium dodecyl sulfate (150 mM) was added to BGE in the inlet vial as the sweeping agent. The system enables effective separation of anthracyclines as well as cleanup from matrix impurities. Sweeping preconcentration of sample provides an excellent detection limit (1 x 10(-9) mol L(-1)). The method was applied for the determination of therapeutic levels of doxorubicin in real plasma samples.

Silymarin, a standardized extract from Silybum marianum, inhibited in vitro the copper-induced oxidation of human LDL in a concentration-dependent manner. Silybin, a main flavonolignan of silymarin, appeared to be responsible for this LDL antioxidant effect. Silychristin and silydianin, other flavonolignans of silymarin, acted rather as pro-oxidants, but with regard to their content in silymarin, it did not contribute significantly to the reduction of the total LDL antioxidant capacity of silymarin.

The alkaloid content (3-demethylcolchicine, colchifoline, N -deacetyl- N -formylcolchicine, colchicine, cornigerine, 2-demethyldemecolcine, 3-demethyldemecolcine, demecolcine) of corms, leaves and seeds of C. turcicum was determined by HPLC. In addition to these compounds, corms contained β-lumicolchicine, luteolin, and vanillic acid. Complete spectral data of colchifoline are presented.

The interaction between horse liver alcohol dehydrogenase (ADH) and aporphine alkaloids has been studied by kinetic and fluorescence methods. The aporphine alkaloids inhibit ADH. The inhibitory effect depends on the position and type of the substituents in the aporphine nucleus. Aporheine shows the strongest binding to the enzyme, and that irrespective of the configuration of the molecule. The dissociation constant of the complex enzyme-aporpheine is 20-25μmol l-1. The results indicate that the aporphine alkaloids bind to the active center of alcohol dehydrogenase with stechiometry 1 : 1.

2-Methylpapaverinium iodide (I), 2'-hydroxymethyl-2-methylpapaverinium iodide (IX), and 2-methyl-3,4-dihydropapaverinium iodide (X. CH3I) form pseudobase by addition of hydroxide ions to the C(1)=N(+) bond. 2'-Hydroxymethyl-2-methyl-3,4-dihydropapaverinium iodide (XV) and 2'-hydroxymethyl-2-methyl-9-oxo-3,4-dihydropapaverinium iodide (XVI) react with hydroxide ions in aqueous medium under formation of cyclic pseudobases XVII and XVIII. The equilibrium constants (KR+) of pseudobase formation have been measured in aqueous ethanol (1 : 1 w/w, 25°C, ionic strength 0.1). The quaternary papaverinium derivatives are metabolized to isoquinilones and carbonyl compounds by means of rat liver enzymes. The role of pseudobases in these biotransformations has been discussed and biogenetic conclusions have been drawn.