Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055.

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Solid-phase extraction of galloyl- and caffeoylquinic acids from natural sources (Galphimia glauca and Arnicae flos) using pure zirconium silicate and bismuth citrate powders as sorbents inside micro spin columns.

Galloyl- and caffeoylquinic acids are among the most important pharmacological active groups of natural compounds. This study describes a pre-step in isolation of some selected representatives of these groups from biological samples. A selective solid-phase extraction (SPE) method for these compounds may help assign classes and isomer designations within complex mixtures. Pure zirconium silicate and bismuth citrate powders (325 mesh) were employed as two new sorbents for optimized SPE of phenolic acids. These sorbents possess electrostatic interaction sites which accounts for additional interactions for carbon acid moieties as compared to hydrophilic and hydrophobic sorbents alone. Based on this principle, a selective SPE method for 1,3,4,5-tetragalloylquinic acid (an anti-HIV and anti-asthamatic agent) as a starting compound was developed and then deployed upon other phenolic acids with success. The recoveries and selectivities of both sorbents were compared to most commonly applied and commercially available sorbents by using high performance liquid chromatography. The nature of interaction between the carrier sorbent and the acidic target molecules was investigated by studying hydrophilic (silica), hydrophobic (C18), mixed-mode (ionic and hydrophobic: Oasis(®) MAX) and predominantly electrostatic (zirconium silicate) materials. The newly developed zirconium silicate and bismuth citrate stationary phases revealed promising results for the selective extraction of galloyl- and caffeoylquinic acids from natural sources. It was observed that zirconium silicate exhibited maximum recovery and selectivity for tetragalloylquinic acid (84%), chlorogenic acid (82%) and dicaffeoylquinic acid (94%) among all the tested sorbents.

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS.

INTRODUCTION: Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. OBJECTIVE: This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. METHODOLOGY: The specific focus of the study is on thin-layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and microwave-assisted water extracts from Quercus. RESULTS: TLC analysis proved the presence of mono-, di- and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC-MS confirmed data obtained via TLC for mono- to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf-MELDI-MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf-MELDI-MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. CONCLUSION: Evaluation of all three techniques employed, clearly proved the heightened performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC-MS is suitable for quantitative analysis.

Identification of Verbena officinalis based on ITS sequence analysis and RAPD-derived molecular markers.

Verbenae herba is a widely used drug and consists of the aerial parts of Verbena officinalis (Verbenaceae). Until now, the identification has been performed based on morphological and phytochemical analyses, which are not reliable enough to distinguish Verbena officinalis from other relevant species of the genus Verbena. Hence, impurities and adulterants, negatively influencing the therapeutic effect of the drug, may remain undetected. In an attempt to generate an accurate authentication method we used two different DNA-based approaches: comparison of ITS sequences and molecular markers (RAPD). Both approaches generally enabled discrimination of V. officinalis from the rest of the genus despite the intraspecific variation existing within V. officinalis. The application of the two independent methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, however, a SCAR marker and primers for HRM were derived from the RAPD results. The SCAR marker could distinguish V. officinalis from all other verbena species except its closest relative V. hastata, while discrimination of V. officinalis even from V. hastata was unproblematic with HRM.

Additive antimicrobial [corrected] effects of the active components of the essential oil of Thymus vulgaris--chemotype carvacrol.

Herbal remedies are multicomponent mixtures by their nature as well as by pharmaceutical definition. Being a multicomponent mixture is not only a crucial property of herbal remedies, it also represents a precondition for interactions such as synergism or antagonism. Until now, only a few phytomedicines are accurately described concerning the interactions of their active components. The aim of this study was to search for interactions within such a naturally given multi-component mixture and to discuss the pharmaceutical and clinical impacts. The thyme oil chosen for the examination belongs to the essential oils with the most pronounced antimicrobial activity. Antibiotic activity of thyme oil and single active components were tested against six different strains of microorganisms. The checkerboard assay was used to search for interactions. The time-kill assay was used to verify the observed effects and to get information about the temporal resolution of the antimicrobial activity. The degree of the detected interactions corresponded with the demarcating FICI measure of 0.5, which separates the additive from the over-additive (synergistic) effects. Therefore, the observed effect was called a "borderline case of synergism" or, respectively, "partial synergism". Partial synergism was observed only in the presence of Klebsiella pneumoniae. Additive antimicrobial activity was observed for the combination of the two monosubstances carvacrol plus linalool and thymol plus linalool as well as with the combination of the two essential oils of the carvacrol and linalool chemotypes. An increase of the carvacrol oil concentration from one to two times the MIC resulted in a considerable acceleration of the kill-rate. Thyme oil is composed of several different components that show antimicrobial activity (at least: carvacrol, thymol and linalool). The antimicrobial activity of thyme oil is partly based on additive effects, which might especially enhance the rapidity of the antimicrobial action. In addition, a mixture of several active ingredients that varies in its composition from year to year and from lot to lot as is the case with herbal remedies may be more stable concerning the antimicrobial activity than mixtures containing just a single active component.

Proanthocyanidins: target compounds as antibacterial agents.

Grape seeds accumulate in huge quantities as byproduct during wine production and are therefore a cheap source for pharmacologically active agents. However, studies prove poor antibacterial activity, and results of analyses are sometimes contradictory. The aim of this study was, thus, to determine the antibacterial activity of grape seed extracts with special focus on the chromatographic characterization of active fractions. In the course of these investigations, extraction protocols were optimized so that microwave-assisted extraction (MAE) guaranteed highest preconcentration efficiency. Proanthocyanidins, monomeric flavonoid aglycones, as well as some of their glycosides could be identified within yielded extracts via high-performance liquid chromatography-mass spectrometry (HPLC-MS). By that means the coherence number of possible isomers of procyanidins was approximated by a newly developed equation. As far as antibacterial activity determined via screening tests is concerned, the extracts generally have been found to be positively responsive toward 10 different gram-positive and gram-negative bacteria strains. After fractionation of the raw extracts, proanthocyanidins P2, P3, P4 and gallate esters P2G and P3G (P = proanthocyanidin consisting of catechin and epicatechin units, n = oligomerization degree, G = gallate ester) were determined as active antibacterial agents toward 10 different pathogens. Only moderate activity was found for monomeric flavonoid fractions.

Quality assessment and quantitative analysis of flavonoids from tea samples of different origins by HPLC-DAD-ESI-MS.

Components of green tea ( Camellia sinensis) have been of considerable interest in recent years because of their potential utility as pharmaceutical agents, particularly for their antioxidant and anticarcinogenic activity. Responding to the increasing scientific validation of numerous health benefits of tea, a comprehensive approach was adopted to carry out analysis for the quality assessment of flavonoids in tea samples of different origins. For this purpose, extraction, separation, and mass spectrometric parameters were optimized. Extraction methods evaluated include reflux extraction, a modified accelerated solvent extraction (ASE), namely, Aquasolv extraction, and microwave-assisted extraction (MAE) using different percentages of solvents. Separation was performed by a specifically developed reversed phase high-performance liquid chromatography (RP-HPLC) method using different C18 and C8 stationary phases. Optimization of extraction techniques clearly proved the performance of MAE, which delivered highest yields in a very short time. Additionally, the comparison with Aquasolv extraction provided new insights, as variations in quantified amounts of target compounds between the extracts could be explained on the basis of thermal degradation and epimerization phenomena. Especially the epimerization phenomenon for catechin/epicatechin oligomers, that is, of procyanidins P 2 and P 3, was observed for the first time. Finally, an optimized extraction and separation system was used for qualitative and quantitative investigations of compounds from different green tea samples from Ceylon (cultivated under biologically controlled conditions), Japan, India, and China as well as from one black tea sample from India.

Identification of carbohydrates by matrix-free material-enhanced laser desorption/ionisation mass spectrometry.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is a sensitive mass spectrometric technique which utilises acidic materials as matrices for laser energy absorption, desorption and ionisation of analytes. These matrix materials produce background signals particularly in the low-mass range and make the detection and identification of small molecules difficult and nearly impossible. To overcome this problem this paper introduces matrix-free material-enhanced laser desorption/ionisation mass spectrometry (mf-MELDI-MS) for the screening and analysis of small molecules such as carbohydrates. For this purpose, 4,4'-azo-dianiline was immobilised on silica gel enabling the absorption of laser energy sufficient for successful desorption and ionisation of low molecular weight compounds. The particle and pore sizes, the solvent system for suspension and the sample preparation procedures have been optimised. The newly synthesised MELDI material delivered excellent spectra with regard to signal-to-noise ratio and detection sensitivity. Finally, wheat straw degradation products and Salix alba L. plant extracts were analysed proving the high performance and excellent behaviour of the introduced material.

Willow bark extract (BNO1455) and its fractions suppress growth and induce apoptosis in human colon and lung cancer cells.

BACKGROUND: Recently, there have been extensive efforts to evaluate the chemopreventive role of substances present in natural products. The aim of this study was to examine the effects of the main groups of compounds (salicylalcohol derivates, flavonoids, proanthocyanidins), and salicin isolated from willow bark extract BNO 1455 on proliferation and apoptosis in human colon and cancer cells. METHODS: We used human colon cyclooxygenase-2 (COX-2)-positive HT 29 and (COX-2)-negative HCT 116 or lung COX-2 proficient A 549 and low COX-2 expressing SW2 cells. After treatment for 72 h with various concentrations of single substances and acetylsalicylic acid (ASA) as control, inhibition of cell growth and cytotoxicity were measured by colorimetric WST-1 assay and propidium iodide uptake by flow cytometry, respectively. Apoptotic cells were identified by annexin V adhesion using flow cytometry. RESULTS: Studies on dose-dependent effects of BNO 1455 and its fractions showed anti-proliferative activity of all compounds with 50% maximal growth inhibitory concentrations (GI(50)) between 33.3 and 103.3 microg/ml for flavonoids and proanthocyanidins fractions and 50.0-243.0 microg/ml for salicylalcohol derivates and extract. Apoptosis induction was confirmed by annexin V adherence and analysis of cell morphology based on light scattering characteristics using flow cytometry in all cell lines at GI(50). CONCLUSIONS: We showed that willow bark extract BNO 1455 an its fractions inhibit the cell growth and promote apoptosis in human colon and lung cancer cell lines irrespective of their COX-selectivity.

Isolation and characterisation of a cysteine protease (phytolacain G), from Phytolacca americana roots.

Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl- L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed "lectin-like" activities such as haemagglutination and mitogenic effects towards human lymphocytes.

Cultivation of Cimicifuga racemosa (L.) nuttal and quality of CR extract BNO 1055.

OBJECTIVES: For Cimicifuga racemosa, well-founded investigations concerning multiplication, germination of seeds and field cultivation have not yet been published. Defined origins or varieties with certain agronomic properties and a specific pattern of active compounds are not commercially available. Special challenges are found with regard to growing of young plantlets from seeds. Comprehensive investigations have been started to find optimal conditions for all steps of the whole process to establish cultivation for Cimicifuga. Aim is to get defined varieties or sources with desirable agronomic characteristics and specific reproducible compound patterns in order to reach homogeneous plant raw material. METHODS: For analytical tests, validated HPLC and TLC methods were used. RESULTS: Results from germination experiments with different temperature regimens show that the time for germination can be shortened from about 20 months to about 6 months. Gibberellic acid had positive influence on the development of the embryo. Content of triterpenglycosides and phenolic compounds was highest in May and June and decreased then from July until September. The quality of the ethanolic extract BNO 1055 (contained in Klimadynon(R) and Menofem(R)) differs from that of an isopropanolic extract. Comparison was carried out by means of TLC pattern of triterpenglycosides and phenolic compounds. CONCLUSION: Extensive systematic research on cultivation parameters with regard to all stages from the seeds to the herbal drug enables commercial field cultivation of Cimicifuga. Controlled cultivation (according to good agricultural practice or GAP) ensures the availability of homogenous standardized raw material. For pharmacological and clinical studies, standardized extracts and finished herbal medicinal products are required. Results of these studies are never transferable to other products and therefore valid only for the tested extracts/products.

Systemic availability and pharmacokinetics of thymol in humans.

Essential oil compounds such as found in thyme extract are established for the therapy of chronic and acute bronchitis. Various pharmacodynamic activities for thyme extract and the essential thyme oil, respectively, have been demonstrated in vitro, but availability of these compounds in the respective target organs has not been proven. Thus, investigation of absorption, distribution, metabolism, and excretion are necessary to provide the link between in vitro effects and in vivo studies. To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites by headspace solid-phase microextraction prior to GC analysis and flame ionization detection. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1+/-24.5 ng ml(-1) and were reached after 2.0+/-0.8 hours. The mean terminal elimination half-life was 10.2 hours. Thymol sulfate was detectable up to 41 hours after administration. Urinary excretion could be followed over 24 hours. The amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2%+/-4.5% of the dose.

Determination of thymol in human plasma by automated headspace solid-phase microextraction-gas chromatographic analysis.

A reliable and sensitive method was developed for determination of thymol in human plasma by automated headspace solid-phase microextraction (SPME). After enzymatic cleavage of thymol sulfate thymol was extracted by a 65 microm polydimethylsiloxane-divinylbenzene crimped fiber (Supelco) after addition of sodium chloride and phosphoric acid (85%). Desorption of the fiber was performed in the injection port of a gas chromatograph at 220 degrees C (HP 5890; 50 m x 0.2 mm I.D., 0.2 microm HP Innowax capillary column; flame ionization detection). Fibers were used repeatedly up to 40 analysis. The recovery was 5% after 35 min of extraction. The calibration curve was linear in the range of 8.1-203.5 ng ml(-1) with a limit of quantitation (LOQ) of 8.1 ng ml(-1). The within-day and between-day precision and accuracy were < or = 20% at the LOQ and <15% at higher concentrations according to international guidelines for validation of bioanalytical methods. After administration of a thymol-containing herbal extract only thymol sulfate, no free thymol, could be detected in human plasma, thus analysis of thymol was after enzymatic cleavage of thymol sulfate. It is concluded that the newly developed automated method can be used in clinical trials on bioavailability and pharmacokinetics of thymol-containing herbal medicinal products.