Pavel Kosina

Primary cultures of human hepatocytes as a tool in cytotoxicity studies: cell protection against model toxins by flavonolignans obtained from Silybum marianum

The aim of this study was to evaluate the cytoprotective effects upon primary human hepatocytes of silymarin extract and its main flavonolignans following exposure to the cytotoxic actions of model toxins. The conditions for the hepatocyte intoxication were optimised for allyl alcohol, carbon tetrachloride, D-galactosamine and paracetamol. Silymarin extract, silychristin and silydianin did not exert cytotoxicity (10-100 microM), whereas silybin and isosilybin at higher concentrations and after longer incubation periods were cytotoxic. All main flavonolignans of silymarin tested displayed concentration-dependent cytoprotection against the toxic effects of both allyl alcohol and carbon tetrachloride but neither paracetamol nor galactosamine. The best protection was achieved by silydianin and silychristin and to a lesser degree by silymarin, while silybin and isosilybin were less effective. It is concluded that these differing outcomes result from the varying abilities of the Silybum marianum substances tested to stabilize the cell membrane, exert antioxidant properties and exhibit intrinsic toxicity.

Phytochemical and antimicrobial characterization of Macleaya cordata herb

Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline alkaloids which are used in phytopreparations with anti-inflammatory and antimicrobial activities. In this study, the alkaloids sanguinarine, chelerythrine, their dihydro derivatives, protopine and allocryptopine and phenolics, gallic, protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in extracts prepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV detection and/or LC/MS with electrospray ionization. The highest content of sanguinarine and chelerythrine was found in capsules. Protopine and allocryptopine were major alkaloids in leaves including footstalks. The seed oil contained dihydrosanguinarine, dihydrochelerythrine and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In addition, sanguinarine reductase, a key enzyme in sanguinarine/dihydrosanguinarine equilibrium in plants, was found for the first time, in the soluble proteins of leaves. Finally, extracts were tested for antimicrobial activity using the microdilution method on standard reference bacterial strains.

Natural feed additive of Macleaya cordata: safety assessment in rats a 90-day feeding experiment.

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.

Metabolic Profiling of Phenolic Acids and Oxidative Stress Markers after Consumption of Lonicera caerulea L. Fruit

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.

Disposition of sanguinarine in the rat.

Sanguinarine is an alkaloid with known antibiotic and anti-inflammatory activity and its pharmacokinetics have been studied in the rat after a single oral dose (10 mg kg−1 body weight). Alkaloid determination in the plasma and liver was carried out by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC/ESI-MS). The pharmacokinetic parameters (tmax, cmax, AUC0→t and AUC0→∞) were determined for sanguinarine and dihydrosanguinarine, the major components detected in plasma. The first step in sanguinarine metabolism in the rat was the reduction of the iminium bond resulting in formation of the less toxic dihydrosanguinarine. Both compounds were completely eliminated from the plasma and liver after 24 h and not detected in urine. After a single oral dose of 3H-sanguinarine, more than 42% of the ingested radioactivity was present in gastrointestinal tract. Benz[c]acridine, up to date the only sanguinarine metabolite referred to in the literature, was not detected in the plasma, liver or urine.

Long-term effects of three commercial cranberry products on the antioxidative status in rats: a pilot study.

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 toxicity and pharmacokinetics of dihydrosanguinarine in rat: A pilot study

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).

Biotransformation of flavonols and taxifolin in hepatocyte in vitro systems as determined by liquid chromatography with various stationary phases and electrospray ionization-quadrupole time-of-flight mass spectrometry

Liquid chromatography (LC) on various stationary phases was used for the metabolite profile analysis of quercetin, rutin, isoquercitrin and taxifolin. The metabolites were obtained using an in vitro model system of human and rat hepatocytes in the form of cell suspensions and the primary cultures. For separations of the parent compounds and their metabolites, stationary phases based on C₁₈, C₈, cyanopropyl (CNP) or phenyl (PHE) modifications of silica were tested. CNP and PHE stationary phases operating in reversed-phase mode have been shown to be efficient for separation of parent flavonoids and their polar metabolites. Individual metabolites were identified on the basis of an elemental composition determination using electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) on-line connected with an LC system. Detailed analytical parameters such as retention times, selectivity, resolution of chromatographic peaks, MS fragmentation and UV-vis absorption maxima were determined for individual metabolites, namely for phase II biotransformation products. The predominant metabolites were methylated flavonols and flavonol glucuronides. The highest biotransformation rate was found with taxifolin, which was mainly converted to sulfates. The HPLC/ESI-QqTOF MS analyses revealed that quercetin and taxifolin were metabolized more extensively than the studied glycosides, rutin and isoquercitrin.

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO2 extracts from real and spiked plasma samples

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 degrees C and 10MPa (15MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO(2) 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 (75mmx4.6mm, 3.5mum particle size) using methanol (organic phase) and 50mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0min, 60-71% organic phase (v/v); from 7.0 to 18.0min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22ng (3.50pmol) for CHE and 0.95ng (2.72pmol) for DHCHE per 1ml 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.

Mass spectrometric investigation of chelerythrine and dihydrochelerythrine biotransformation patterns in human hepatocytes.

The quaternary benzo[c]phenanthridine alkaloids (QBAs) are an important subgroup of plant secondary metabolites. Their main representatives, sanguinarine (SG) and chelerythrine (CHE), have pleiotropic biological effects and a wide spectrum of medicinal applications. The biotransformation of SG and CHE has only been partially studied while subsequent oxidative transformation of their dihydro derivates, the main metabolites, is practically unknown. The aim of this study was to characterize the biotransformation of CHE and dihydrochelerythrine (DHCHE) in detail, with respect to their more extensive biotransformation than SG. Phase I as well as phase II biotransformation of both compounds was examined in human hepatocyte suspensions. Liquid chromatography with electrospray-quadrupole time-of-flight mass spectrometry (LC-ESI-QqTOF MS) was used for analysis of the metabolites. Using the LC-ESI-QqTOF MS method, we analyzed and then suggested the putative structures of 11 phase I and 5 phase II metabolites of CHE, and 11 phase I and 6 phase II metabolites of DHCHE. For the most abundant metabolites of CHE, DHCHE and O-demethylated DHCHE, their cytotoxicity on primary cultures of human hepatocytes was analyzed. Both metabolites were nontoxic up to 50μM concentration and this indicates decreasing toxic effects for CHE biotransformation products, i.e. DHCHE and O-demethylated DHCHE.