Fluorometric quantification of alkaloids in the homeopathic mother tinctures of Vinca minor L. and Fumaria officinalis L.

To assess the toxic potential of the alkaloids, a quantification method is necessary. An ion pair extraction method was used for quantitative fluorometric determination of vincamine, protopine and all contained alkaloids in the mother tinctures of Vinca minor and Fumaria officinalis. The non-fluorescent alkaloids were transformed into an ion pair with sodium-9,10-dimethoxy-anthracene-sulfonate and then fluorometrically determined and quantified in this study. The applicable ion pair was extracted in a suitable organic solvent, where dichloromethane has proven to be beneficial. Conditions for the ion pairing and fluorometric quantification are given. The recovery rate was used to investigate the quality of determinability and the influence of the mother tincture matrix. The method was applied to determine the concentration of protopine in the range 0.1 - 15 µg/ml and of vincamine in the range of 0.5 - 20 µg/ml. The limit of detection was < 0.3 µg/ml, and the limit of quantification < 0.9 µg/ml for both alkaloids.

SOD activity and extremophilicity: a screening of various plant species.

All aerobes are dependent on enzymatic and non-enzymatic antioxidants to withstand the presence of reactive oxygen species (ROS). Superoxide dismutase (SOD) is a part of the enzymatic antioxidant system. It is one of the most important antioxidant enzymes, enabling organisms to survive in an oxygen containing atmosphere. A disorder in the oxidative and antioxidative balance can be associated with the occurrence of diseases in human organisms. Little data exist on the relevance of SOD in plants. Moreover, it is not known whether there is any association between a plant's origin and its SOD activity. Our screening of 27 different plant species was intended to expose whether there is a connection. The highest SOD activities were found for extremophile plants. Especially the Crassulaceae Aeonium haworthii Salm-Dyck Ex Webb & Berthel. and Crassula multiflora Schönland & Baker F. were highly active. Nevertheless, we did not find unambiguous evidence for a correlation between extremophilicity and SOD activity.

Green tea reduces body fat via upregulation of neprilysin.

BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

The influence of toll-like receptor (TLR-) agonists on lysozyme activity, TNF-alpha secretion and intercellular adhesion in THP-1 cells.

Monocytes play a major role in modulating inflammation and can be found as circulating blood cells or, after extravasation, as differentiated tissue specific macrophages. The activation of TLRs (Toll-like receptors) modulates their action and contributes considerably to the eradication of pathogens. Aim of this study was to investigate the influence of TLR-agonists on TNF-alpha secretion and lysozyme activity in a comparative assay. To explore the importance of differentiation the monocytic cell line THP-1 was pre-treated with PMA and IFNy. Stimulation of undifferentiated cells with various bacterial antigens had almost no effect on TNFalpha secretion and lysozyme activity but influenced adhesion molecule expression clearly. Differentiated THP-1 cells were much more sensitive to further stimulation with TLR-agonists yet revealed an opposing effect on lysozyme activity and TNF-alpha secretion. The results suggest a coordinated activation of intercellular adhesion molecule expression and cytokine secretion in the differentiation process from monocytes to macrophages.

Necrosis factor-alpha (TNF-alpha) response in human hepatoma HepG2 cells treated with hepatotoxic agents.

The liver plays an essential role in xenobiotic metabolism including alcohol and drugs. Oxidative stress that usually occurs during the hepatic metabolism participates in the pathogenesis of liver disease. Inflammatory cytokines that exist in liver in both physiological and pathophysiological conditions may change the hepatic toxic response to hepatotoxicants. The human hepatoma cell line HepG2 is frequently used as in vitro model for biomedical studies. In this work, HepG2 cells were pre-incubated with or without TNF-alpha, and then treated with ethanol, acetaldehyde, acetaminophen and tert-butyl hydroperoxide, respectively. Cell viability was measured by MTT assay. The data showed that HepG2 cells were generally resistant to xenobiotic compounds, especially to alcohol and acetaldehyde, which may be partially caused by the absence of specific cytochrome P450 systems in these cells. TNF-alpha could sensitize the toxic response of HepG2 cells to those exogenous compounds, indicating the important role of TNF-alpha in the pathogenesis of alcohol, drugs and oxidant related liver diseases.

Gniditrin is the main diterpenoid constituent in the bark of Daphne mezereum L.

The bark of Daphne mezereum L. is known as toxic drug due to the presence of diterpene esters. The phytochemical analysis of the bark used for preparation of homeopathic mother tinctures showed that gniditrin was the main diterpene constituent, only in the fruits of D. mezereum mezerein could be detected. The complete NMR data of gniditrin are published for the first time.

Stimulation of lysozyme release by selected microbial preparations.

Lysozyme is an important factor of innate immunity and a unique enzybiotic in that it exerts not only antibacterial activity, but also antiviral, anti-inflammatory, anticancer, and immunomodulatory activities. The purpose of the present study was to investigate whether in vitro exposure to microbial preparations can affect the release and production of lysozyme activity in human monocytic cell line THP-1. Lysozyme activity levels in cell culture fluids were measured using highly sensitive fluorescence-based lysozyme activity assay. Different preparations of bacteria and ascomycetes stimulated lysozyme release result in a higher lysozyme activity after one hour exposure. The demonstrated ability of selected microbial preparations to enhance the release of lysozyme activity can present a new mechanism contributing to explaining biological characteristics of microbial preparations, including their antibacterial and immune-stimulating properties.

Forskolin and derivatives as tools for studying the role of cAMP.

Forskolin (7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one) is the first main labdane diterpenoid isolated from the roots of the Indian Plectranthus barbatus ANDREWS and one of the most extensively studied constituents of this plant. The unique character of forskolin as a general direct, rapid and reversible activator of adenylyl cyclase not only underlies its wide range of pharmacological effects but also renders it as a valuable tool in the study of the role of cAMP. The purpose of this review is to provide data presenting the utility of forskolin--as a cAMP activator--for studying the function of cAMP from different biological viewpoints as follows: 1) Investigation on the role of cAMP in various cellular processes in different organs such as gastrointestinal tract, respiratory tract, reproductive organs, endocrine system, urinary system, olfactory system, nervous system, platelet aggregating system, skin, bones, eyes, and smooth muscles. 2) Studies on the role of cAMP activation and inhibition to understand the pathogenesis (e.g. thyroid autoimmune disorders, leukocyte signal transduction defect in depression, acute malaria infection, secretory dysfunction in inflammatory diseases) as well as its possibly beneficial role for curing diseases such as the regulation of coronary microvascular NO production after heart failure, the attenuation of the development or progression of fibrosis in the heart and lungs, the augmentation of myo-protective effects of ischemic preconditioning especially in the failing hearts after myocardial infarction, the stimulation of the regeneration of injured retinal ganglion cells, the curing of glaucoma and inflammatory diseases, the reducing of cyst formation early in the polycystic kidney disease, and the management of autoimmune disorders by enhancing Fas-mediated apoptosis. 3) Studies on the role of cAMP in the mechanism of actions of a number of drugs and substances such as the effect of the protoberberine alkaloid palmatine on the active ion transport across rat colonic epithelium, the inhibitory effect of retinoic acid on HIV-1-induced podocyte proliferation, the whitening activity of luteolin, the effect of cilostazol on nitric oxide production, an effect that is involved in capillary-like tube formation in human aortic endothelial cells, the apoptotic effect of bullatacin, the effects of paraoxon and chlorpyrifos oxon on nervous system. Moreover, cAMP was found to play a role in acute and chronic exposure to ethanol, in morphine dependence and withdrawal and in behavioral sensitization to cocaine as well as in the protection against cisplatin-induced oxidative injuries.

Research on an in vitro cell system for testing the neurotoxicity of kynurenine pathway metabolites.

Quinolinic acid (QUIN), kynurenine acid (KYNA) and 3-hydroxykynurenine (3-HK) - metabolites of the kynurenine pathway are considered to be associated with many central nervous system diseases. However, in neuroscience research in order to test neurotoxicity or neuroprotection against these compounds only primary cell models are available. In this investigation we aimed to develop a simple, rapid and accurate cellular in vitro model using immortalized human neuroblastoma cell lines, namely SK-N-SH and SH-SY5Y differentiated by treatment with various agents. In order to alter the cell response to the neurotoxins, tumor necrosis factor-alpha and retinoic acid (RA) as differentiating agents and modulation of the cellular metabolism through changing the sugar composition from galactose to glucose in media were used. Our results indicated that although RA-differentiation of both cell lines induced the expression of neuronal features, cell vulnerability after exposure to control neurotoxicants (salsolinol, 6-hydroxydopamine) and 3-HK was decreased in comparison to untreated cells and was not influenced after exposure to QUIN and KYNA. Interestingly, the same observations were done in cells grown in galactose containing media.

Liquid-chromatographic profiling of Saponinum album (Merck).

Saponinum album (Merck) is a complex composite of triterpene saponins. It was shown that Saponinum album (Merck) dramatically enhances the toxicity of the N-glycosylase saporin from the seeds of Saponaria officinalis L. as well as the toxicity of a saporin based anti-tumor toxin. This study was intended to chromatographically profile the saponins present in Saponinum album (Merck) in order to identify saponins that determine the cytotoxicity enhancing properties of Saponinum album (Merck) on saporin. For this purpose a liquid-chromatographic profiling (HPLC) followed by ESI-TOF-MS analysis and evaluation of cytotoxicity enhancer effects of saponins from Saponinum album (Merck) was performed. This is the first study describing a liquid-chromatographic profiling of saponins from Saponinum album (Merck). Ten different saponins were isolated. There was a lot of variation observed in the cytotoxicity enhancing properties of different isolated saponins, 8 out of 10 isolated saponins showed an enhancer effect on the toxicity of saporin. Based on these results it was concluded that the cytotoxicity enhancer effect of Saponinum album (Merck) is not attributable to a single, activity determining saponin.

Proteolytic activity in latex of the genus Euphorbia--a chemotaxonomic marker?

Eighteen species of the genus Euphorbia are known to have proteolytic enzymes in their latices, 9 of them are characterized by the type of endopeptidases (Cysteine-, Serine-, Metallo- or Aspartatic-endopeptidase) which are responsible for the activity, and all nine are serine endopeptidases. In our study we examined the latices of 64 different species of the genus Euphorbia concerning proteolytic activity and serine protease activity, five of them are mentioned in the literature to be proteolytic active and four are known to contain at least one serine endopeptidase. All tested samples were able to degrade labelled casein, the activity of six latices were completely inhibited by specific serine protease inhibitors, 15 samples were not influenced, and in 43 latices a remaining activity was measured, indicating that other types of endopeptidases seem to be involved.

Sialidases in biological systems.

Sialidases are very common in biological systems. They are found particularly in diverse virus families and bacteria, but also in protozoa, some invertebrates and mammalian. The enzymes differ in their biochemical properties, e.g., kinetics, binding affinity or substrate preference. Nevertheless, they have conserved domains and structural similarities. Their functions offer a wide spread spectrum in nutrition, pathomechanisms and communication in mammalian. In pathogen organisms sialidases give insight in coevolution and interaction between pathogen or commensale and host. As well the enzymes are of interest in drug targeting, like neuraminidase inhibitors of influenza viruses.

Extracts of Cynomorium songaricum protect human neuroblastoma cells from beta-amyloid25-35 and superoxide anion induced injury.

In Traditional Chinese Medicine a number of herbs are used to alleviate age-related diseases including memory impairment and dementia, among them stems of Cynomorium songaricum, Cynomoriaceae. In this study, we evaluated the protective effect of different extracts of aerial parts of C. songaricum on amyloid-beta peptide (Abeta) and hypoxanthine/xanthine oxidase induced cell death in SK-N-SH neuroblastoma cells. Abeta (20 microM) as well as superoxide anions generated by the hypoxanthine/xanthine oxidase system both reduced cell viability to about 60%. The methanolic extract of C. songaricum attenuated Abeta induced cell death at concentrations of 100 and 10 microg/ml, an even stronger effect was observed for the ethyl acetate fraction obtained from the crude methanolic extract. On the other hand, the dichloromethane as well as water fractions showed no protective effects. In order to further analyze the protective mode of action, the ability of extracts to protect against superoxide anions induced cell death was also evaluated. In this system, cell viability could again be restored by methanol and ethyl acetate extracts, the latter showingsignificant protective effects even at concentrations as low as 0.1 microg/ml.

Enhancement of cytotoxicity of ribosome-inactivating-protein type I by saponinum album is not based on stimulation of phagocytosis.

Saponinum album, a mixture of triterpenoic saponins derived from Gypsophila species, led to an increased internalization of agrostin, a ribosome-inactivating-protein (RIP) type I in U-937 cells differentiated with interferon-gamma or phorbol myristate acetate. Treatment with agrostin only showed no cytotoxicity. It was hypothesized that saponinum album stimulated phagocytosis and by that the uptake of agrostin. For this purpose phagocytosis experiments with Alexa-Fluor-488-labelled 1-microm amino-latex beads and FITC-labelled Escherichia coli (K-12 strain) were performed. The results indicated no stimulation of phagocytosis by treatment with saponinum album.

Dual inhibition of metallopeptidases ACE and NEP by extracts, and iridoids from Ligustrum vulgare L.

AIM OF THE STUDY: In order to confirm the traditional use of Ligustrum vulgare L. (common privet, Oleaceae) we investigated the inhibitory activity of different extracts from leaves (LlE), flowers (LflE) and fruits (LfrE) on metallopeptidases ACE and NEP. MATERIALS AND METHODS: Powdered plant materials were first extracted with water and then with ethyl acetate and n-butanol saturated with water. The metallopeptidases activity was determined using in vitro fluorimetric assays. RESULTS: At a concentration of 100microg/ml the ethyl acetate extracts showed the highest activity. The bio-guided fractionation of the leaves extract led to the isolation of two iridoids which were identified by (1)H, (13)C and HETCOR NMR spectroscopy as oleuropein and ligstroside aglycones. Both compounds are dual ACE/NEP inhibitors with IC(50) of 20 and 25microM for ACE and IC(50) of 35 and 75microM for NEP, respectively. Secoirydoids glycosides, tyrozol and hydroxytyrozol, as well as, flavonoids present in the ethyl acetate extracts showed little or no inhibitory activity. CONCLUSIONS: Our results partially support the diuretic and hypotensive activities of common privet.

Antispasmodic activity of essential oil from Lippia dulcis Trev.

AIM OF THIS STUDY: To investigate the essential oil of Lippia dulcis Trev. (Verbenaceae) that is traditionally used in the treatment of cough, colds, bronchitis, asthma, and colic in Middle America for antispasmodic activity. MATERIALS AND METHODS: We used a porcine bronchial bioassay to study contractile responses to carbachol and histamine in the absence or presence of the essential oil. RESULTS: The essential oil showed anti-histaminergic and anti-cholinergic activities at 100 microg/ml. CONCLUSIONS: The anti-histaminergic and anti-cholinergic activities of the essential oil of Lippia dulcis support the rational use of the plant or plant extracts to treat bronchospasm.

Screening of some Yemeni medicinal plants for inhibitory activity against peptidases.

Extracts of different polarities (dichloromethane, methanol, and aqueous extracts) from 5 Yemeni medicinal plants (Aspilia helianthoides, leaves; Ceropegia rupicola, whole plant; Kniphofia sumarae, whole plant; Pavetta longiflora, leaves; and Plectranthus cf barbatus, leaves) were screened for their inhibitory effects against angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and aminopeptidase N (APN) activities. Four extracts (methanol extracts of Ceropegia rupicola, Kniphofia sumarae, and Plectranthus cf barbatus, and the aqueous extract of Pavetta longiflora) were found able to inhibit the enzymatic activity of NEP. Significant reduction in the activity of NEP (p < 0.01) was observed at a concentration of 50 microg/ml, and above of all tested extracts. The most active extract was the methanolic extract of Ceropegia rupicola with IC50 of 111 microg/ml. Only the methanolic extract of Aspilia helianthoides was found to exhibit inhibitory effect against the ACE activity with IC50 = 133 microg/ml. None of the tested plant extracts was found active against the aminopeptidase N activity.

Quantitative determination of cucurbitacin E and cucurbitacin I in homoeopathic mother tincture of Gratiola officinalis L. by HPLC.

"Hedgehyssop" Gratiola officinalis L. (Scrophulariaceae) is found as an ingredient in homeopathic remedies. Among the active compounds found in G. officinalis, the cucurbitacines constitute a group of triterpenoid substances which are well-known for their bitterness and toxicity. Due to the toxicity of the cucurbitacin's aglycones it becomes necessary to determine the content of the aglycones and glycosides that may be responsible for the pharmacological activity and toxicity in homeopathic tinctures according to the European Pharmacopeia guidelines. In this context a HPLC method was developed for the identification and determination of cucurbitacin E and I in homeopathic mother tinctures. To evaluate the total concentration of the aglycones, cucurbitacin E and I formed after hydrolysis we determined the concentration of both compounds after enzymatic hydrolysis with beta-glucosidase in vitro. Reversed-phase HPLC with a Eurospher C18 column with precolumn and acetonitrile-water gradient system as the mobile phase proved to be suitable for direct determination of both aglycons, cucurbitacin E and I in Gratiola-mother tinctures. The contents of cucurbitacin E, cucurbitacin I, cucurbitacin E glycoside and cucurbitacin I glycoside were found as 0.0065%, 0.0031%, 0.0011% and 0.0006%, respectively in Gratiola-mother tincture prepared according to method 2a HAB.

Determination of lysozyme activity by a fluorescence technique in comparison with the classical turbidity assay.

The aim of this comparable study was to evaluate two different methods for the assay of lysozyme activity. Lysozyme activity was assayed by turbidimetric and fluorescence-based methods with hen egg white lysozyme as standard. The standard activity of each assay was calibrated in units of activity per mg under the experimental conditions (37 degrees C) so that direct comparison between these two assays could be made. The turbidimetric assay was performed using a 0.36 mg/ml Micrococcus lysodeikticus suspension and a microtiter plate reader capable of analyzing enzyme kinetics at 450 nm, and the linearity, in the range of 2.3-23.8 units/ml, presented a correlation coefficient (R2) as high as 0.9967. The fluorescence-based assay was performed with EnzChek kit using a suspension of Micrococcus lysodeikticus labeled with fluorescein and a fluorescence microplate reader. The linearity in the range of 0.47-5.28 units/ml, presented a correlation coefficient (R2) as high as 0.9881. Thus, the turbidimetric assay provides a simple rapid accurate and specific method for the determination of lysozyme activity but with relatively low sensitivity in comparison with the fluorescence-based method which was demonstrated to be a high sensitivity assay, and hence a reliable applicable technique to determine lysozyme activity at low levels e.g. in cell culture systems.

Enhancement of cytotoxicity of lectins by Saponinum album.

Saponinum album, a mixture of saponins with an aldehyde function bound at C4 from Gypsophila species, increased the cytotoxicity of lectins like agrostin and saporin by enhancing its penetration through the cell membrane. The effect was attenuated by latrunculin, an inhibitor of endocytosis, and by bafilomycin, an inhibitor of transport from early to late endosomes and lysosomes. In contrast, the effect was not influenced by brefeldin A that causes dissolution of the Golgi stacks and by the addition of different monosaccharides. The toxicity of other peptidic toxins was only slightly enhanced by the saponins indicating a specific interaction between lectins and saponins present in Caryophyllaceae.