St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells.

BACKGROUND: Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. METHODS: Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of ß1-adrenoceptors and changes in cAMP formation were measured by ELISA. RESULTS: Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of ß1-adrenoceptors as well as reduced cAMP formation after stimulation with the ß1-adrenoceptor agonist dobutamine. CONCLUSION: Obviously, the signaling of ß1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.

Metabolic switch induced by Cimicifuga racemosa extract prevents mitochondrial damage and oxidative cell death.

BACKGROUND: Cimicifuga racemosa extract is a well-established therapy for menopausal symptoms. The mechanisms underlying the multiple therapeutic effects of Cimicifuga extract, e.g. reducing hot flushes and profuse sweating are not well defined. Recent studies revealed pronounced effects of Ze 450, a Cimicifuga racemosa extract that was produced by a standardized procedure, on energy metabolism through activation of AMP-activated protein kinase in vitro and beneficial anti-diabetic effects in vivo. PURPOSE: The aim of the study was to investigate the effects of Ze 450 on energy metabolism. Since mitochondria are the key regulators of cellular energy homeostasis, we wanted to elucidate whether Ze 450 affects mitochondrial resilience and can provide protection against oxidative damage in neuronal and liver cells. METHODS/STUDY DESIGN: In this study, we investigated the effects of Ze 450 (1-200 µg/ml) on mitochondrial integrity and function, and cell viability in models of oxidative stress induced by erastin and RSL-3 in neuronal and liver cells. The effects of Ze 450 in control conditions and after induction of oxidative stress were analyzed using FACS for detecting lipid peroxidation (BODIPY), mitochondrial ROS formation (MitoSOX), mitochondrial membrane potential (TMRE) and cell death (AnnexinV/PI staining). Furthermore, we determined metabolic activity (MTT assay), ATP levels and mitochondrial respiration and glycolysis (oxygen consumption rates, extracellular acidification rates; Seahorse). RESULTS: Ze 450 preserved mitochondrial integrity and ATP levels, and prevented mitochondrial ROS formation, loss of mitochondrial membrane potential and cell death. Notably, Cimicifuga racemosa extract alone did not alter mitochondrial ROS levels, and subtle inhibitory effects on cell proliferation were reversed after withdrawal of the extract. In addition, Ze 450 did not exert toxic effects to liver cells, but rather protected these from the oxidative challenge. Further analysis of the mitochondrial oxygen consumption rate and the extracellular acidification rate revealed that Ze 450 mediated a switch from mitochondrial respiration to glycolysis, and this metabolic shift was a prerequisite for the protective effects against oxidative damage. CONCLUSION: In conclusion, the bioenergetic shift induced by Ze 450 exerted protective effects in different cell types, and offers promising therapeutic potential in age related diseases involving oxidative stress and mitochondrial damage.

Effects of Cimicifuga racemosa extract Ze450 on mitochondria in models of oxidative stress in neuronal cells.

This data article describes the influence of Cimicifuga racemosa extract Ze 450 on neuronal cells in models of glutamate-induced excitotoxicity and cell death induced by oxidative stress. Effects of Ze 450 on glutamate-mediated excitotoxicity were assessed in primary cortical rat and mouse neurons and, further, glutamate-mediated oxidative stress was analyzed in HT22 cells lacking ionotropic glutamate receptors. This study especially focusses on mitochondrial parameters like mitochondrial ROS formation, mitochondrial membrane potential, ATP production and mitochondrial integrity. Further the effects of Ze 450 on lipid-peroxidation, metabolic activity, cell proliferation and cell death were assessed under control conditions and oxidative challenge evoked by millimolar concentrations of glutamate in HT22 cells. These data support the findings in HT22, mHypo and HepG2 liver cells (Rabenau et al., 2018) [1].

Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. AIM OF THE STUDY: The aim was to develop an in vitro screening method of their cytotoxicity. MATERIALS AND METHODS: Human and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). RESULTS: Galactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. CONCLUSIONS: If no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Modelling antibiotic and cytotoxic isoquinoline effects in Staphylococcus aureus, Staphylococcus epidermidis and mammalian cells.

Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.

Pharmacokinetic drug interactions involving Ginkgo biloba.

Ginkgo biloba leaf extracts (GLEs) are popular herbal remedies for the treatment of Alzheimer's dementia, tinnitus, vertigo and peripheral arterial disease. As GLEs are taken regularly by older people who are likely to also use multiple other drugs for the treatment of, e.g. hypertension, diabetes, rheumatism or heart failure, potential herb-drug interactions are of interest. Preclinical studies of high doses/concentrations of GLEs of varying quality and standardization hinted at both an inhibition and induction of metabolic enzymes and transporters. However, in humans, positive in vitro-findings could not be replicated in vivo. At maximum recommended doses of 240 mg/day, a clinically relevant interaction potential of the standardized GLE EGb 761 could not be shown. GLE doses higher than the recommended ones led to a weak induction of the CYP2C19-mediated omeprazole 5-hydroxylation, and a weak inhibition of the CYP3A4-mediated midazolam 1'-hydroxylation, respectively. Also, the regular intake of a poorly characterized GLE at a dose of 360 mg/day slightly increased the bioavailability of talinolol, a substrate of P-glycoprotein and various organic anion-transporting polypeptides. Thus, regarding pharmacokinetic herb-drug interactions, the intake of the standardized GLE, EGb 761, together with synthetic drugs appears to be safe as long as daily doses up to 240 mg are consumed. If this applies to other extracts prepared according to the European Pharmacopoeia remains uncertain. Also, a relevant potential for drug interactions cannot be excluded for poorly standardized GLEs used in many food supplements.

Modeling antibiotic and cytotoxic effects of the dimeric isoquinoline IQ-143 on metabolism and its regulation in Staphylococcus aureus, Staphylococcus epidermidis and human cells.

BACKGROUND: Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. RESULTS: Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. CONCLUSIONS: The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics.

Daily honey consumption does not change CYP3A activity in humans.

Several studies investigating the interaction of honey and drug-metabolizing enzymes showed controversial results, with some suggesting that honey induces CYP3A-mediated metabolism in mammals and humans. This clinical trial was conducted to determine the effect of repeated honey administration on human CYP3A enzyme activity using midazolam as a marker substance. In a randomized, single-blind, parallel-group study, 20 healthy volunteers were randomly assigned to receive either honey (2 × 20 g/d) or artificial honey (2 × 20 g/d) over a period of 10 days. To determine intestinal and hepatic CYP3A activity, oral (4 mg) and intravenous (2 mg) midazolam was administered in a semi-simultaneous way before honey administration, after the last honey administration, and 1 and 6 days thereafter. At baseline after oral midazolam, the partial metabolic clearance was similar in both groups (honey: 917.8 ± 234.6 mL/min vs artificial honey: 973.5 ± 373.8 mL/min). Ten days of honey administration did not change partial metabolic clearance (honey: 1016 ± 268 mL/min vs artificial honey: 1043 ± 450 mL/min), which was also true 1 and 6 days later. Neither honey nor artificial honey in amounts usually consumed affected the intestinal and hepatic CYP3A activity in healthy volunteers.

[Pharmacokinetic drug interactions by herbal drugs: Critical evaluation and clinical relevance]. / Pharmakokinetische Arzneimittelinteraktionen durch pflanzliche Arzneimittel : Kritische Bewertung und klinische Relevanz.

Pharmacokinetic herb-drug interactions are caused by an induction or inhibition of cytochrome P450 (CYP) enzymes or transporters e.g. P-glycoprotein. St. John's wort extracts containing hyperforin increase the expression of CYP-enzymes and P-glycoprotein mainly in the gut and liver which leads to a clinically relevant decrease of the bioavailability of CYP and P-glycoprotein substrates. Contrarily, the bioactivation of the prodrug losartan is reduced by milk thistle extracts which is due to an inhibition of CYP2C9. However, the 15 % reduction of the bioavailability of the active metabolite E-3174 is clinically not relevant. Also, minor changes in drug bioavailability observed in clinical studies for valerian, echinacea, ginkgo and hawthorne are clinically not relevant, although in vitro studies point to drug interactions in vivo. Since for herbal extracts a positive in vitro - in vivo correlation regarding the impact on drug bioavailability is rare, results from in vitro studies should be carefully interpreted.

Bioconversion of the antihistaminc drug loratadine by tobacco cell suspension cultures expressing human cytochrome P450 3A4.

In this study we have expanded the metabolic potential of plant cell suspension cultures by introducing active human cytochrome P450 monooxygenase 3A4 into tobacco cells. Exogenously supplied loratadine was metabolized in a 3A4-specific manner, showing the capacity of this system for the generation of metabolites.

O-glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC-ESI/multi-stage tandem mass spectrometry.

INTRODUCTION: The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. OBJECTIVE: To apply high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI/MS(n)) to identify the O-glycoside sequence of saponins from the roots of Phytolacca bogotensis. METHODOLOGY: Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision-induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid-phase extraction (SPE) and HPLC/ESI-MS(n) for the characterisation of saponins and identification of novel molecules is described. RESULTS: Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC-ESI/MS(n). CONCLUSION: Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts.

Capillary electrophoresis of natural products: current applications and recent advances.

Capillary electrophoresis (CE) was introduced as a new analytical technique in the 1970s and rapidly proved to be a powerful tool for the separation and detection of various classes of natural and synthetic compounds. Since the availability of commercially manufactured high-performance instruments, CE represents an interesting alternative to high-pressure liquid chromatography (HPLC), mainly because of its speed and high separation efficiency. In this overview a short description of the basic and widely used CE methods will be given and the applicability of these methods for the analysis of natural products will be discussed. Due to the growing number of publications dealing with CE or CE/MS of secondary plant metabolites, an exhaustive overview of all current applications cannot be given in this contribution. Therefore, the focus of this mini-review will be on the advances and new aspects of recently published CE methods in natural products analysis.

Structure-activity relationship and studies on the molecular mechanism of leishmanicidal N,C-coupled arylisoquinolinium salts.

Alternative drugs against leishmaniasis are desperately needed. Antimonials, the main chemotherapeutic tool, cause serious side effects and promote chemoresistance. We previously demonstrated that representatives of N,C-linked arylisoquinolines are promising leishmanicidal drug candidates. We now performed structure-activity relationship studies varying the aryl portion of our lead substrate. The new series of compounds show an enhanced selectivity against Leishmania major in comparison to their major host cell, the macrophage. Our results suggest that the arylisoquinolinium salts decrease the macrophage infection rate acting directly on the intracellular parasites. However, the activity of the 4'-i-propyl derivative might also involve the modulation of cytokine and nitric oxide production by host macrophages. Additionally, this isoquinoline acts synergistically with amphotericin B and does not interact with drug-metabolizing cytochrome P450 enzymes involved in the metabolism of antileishmanial drugs. The results demonstrate that the newly synthesized structurally simplified N,C-coupled arylisoquinolinium salts are promising candidates to be considered as leishmanicidal pharmacophores.

Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries.

The highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-methyl- and 10-methoxystrictosidines. Based on the addition of strictosidine to a crude strictosidine glucosidase preparation from Catharanthus cells, a combined chemoenzymatic approach to generating large alkaloid libraries for future pharmacological screenings is presented.

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction.

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.