Matthias F. Melzig

Occurrence and properties of proteases in plant latices.

Proteases appear to play key roles in the regulation of biological processes in plants, such as the recognition of pathogens and pests and the induction of effective defence responses. On the other side these enzymes are able to activate protease-activated receptors (PARs) and in that way to act as agents with pharmacological and toxicological significance. An important source of plant proteases used in traditional medicine and industry is latex. Over 110 latices of different plant families are known to contain at least one proteolytic enzyme. Most of them belong to the cysteine or serine endopeptidases family and only one to the aspartatic endopeptidases family. This review focuses on the characterization of proteases found in latices of several plant families (Apocynaceae, Asclepiadaceae, Asteraceae, Caricaceae, Convolvulaceae, Euphorbiaceae, Moraceae), and summarizes the known chemical and biological properties of the isolated proteases as well as their importance in pharmacology and toxicology.

Antiplasmodial activity of naphthoquinones and one anthraquinone from Stereospermum kunthianum.

A lipophilic extract of the root bark of Stereospermum kunthianum revealed antiplasmodial activity in vitro. Bioassay-guided fractionation led to the isolation of four novel naphthoquinones (sterekunthals A and B, pyranokunthones A and B) and one novel anthraquinone (anthrakunthone) together with the known naphthoquinone pinnatal. The structures of the novel compounds were determined by comprehensive analyses of their 1D and 2D NMR data. The antiplasmodial activities and toxicity against the endothelial cell line ECV-304 of the isolated compounds have been assessed.

Saponins can perturb biologic membranes and reduce the surface tension of aqueous solutions: a correlation?

Saponins are secondary plant compounds. They have a triterpenoid or steroidal backbone. Sugars are attached to one or more points of this structure, forming chains that can be branched. This appearance leads to amphiphilic properties giving saponins the ability to interact with both lipophilic and hydrophilic structures. The surfactant behavior lets them lower the surface tension in aqueous solutions and form micelles when reaching the critical micelle concentration (cmc). It also lets them interact with biologic membrane layers that usually consist of phospholipids and cholesterol. This action may perturb the membrane and its function leading to membrane perforation or complete lysis. Thus saponins are also known for their cytotoxicity and membranolytic, respectively hemolytic features. In our studies we wanted to answer the question if there is a correlation between the unspecific detergent behavior when lowering the surface tension and the ability to perforate cell membranes and to act cytotoxic. Do saponins showing a considerable reduction in the surface tension also reveal an evident cytotoxicity or/and a marked cell membrane perforation? We tested a variety of saponins with distinct structures. The reduction in the surface tension and the cmc were analyzed on a tensiometer using the Wilhelmy plate method. The general cytotoxicity was determined in a cell model by DNA quantification. The cell membrane toxicity or membrane perforation was explored in a cell model by quantification of the leakage of the intracellular enzyme lactate dehydrogenase (LDH). The experiments revealed a correlation between the membrane toxicity and the reduction in surface tension.

Role of human gut microbiota metabolism in the anti-inflammatory effect of traditionally used ellagitannin-rich plant materials

ETHNOPHARMACOLOGICAL RELEVANCE Ellagitannin-rich plant materials are widely used in traditional medicine as effective, internally used anti-inflammatory agents. Due to the not well-established bioavailability of ellagitannins, the mechanisms of observed therapeutic effects following oral administration still remain unclear. The aim of the study was to evaluate if selected ellagitannin-rich plant materials could be the source of bioavailable gut microbiota metabolites, i.e. urolithins, together with determination of the anti-inflammatory activity of the metabolites produced on the THP-1 cell line derived macrophages model. MATERIALS AND METHODS The formation of urolithins was determined by ex vivo incubation of human fecal samples with aqueous extracts from selected plant materials. The anti-inflammatory activity study of metabolites was determined on PMA differentiated, IFN-γ and LPS stimulated, human THP-1 cell line-derived macrophages. RESULTS The formation of urolithin A, B and C by human gut microbiota was established for aqueous extracts from Filipendula ulmaria (L.) Maxim. herb (Ph. Eur.), Geranium pratense L. herb, Geranium robertianum L. herb, Geum urbanum L. root and rhizome, Lythrum salicaria L. herb (Ph. Eur.), Potentilla anserina L. herb, Potentilla erecta (L.) Raeusch rhizome (Ph. Eur.), Quercus robur L. bark (Ph. Eur.), Rubus idaeus L. leaf, Rubus fruticosus L. and pure ellagitannin vescalagin. Significant inhibition of TNF-α production was determined for all urolithins, while for the most potent urolithin A inhibition was observed at nanomolar concentrations (at 0.625 μM 29.2±6.4% of inhibition). Urolithin C was the only compound inhibiting IL-6 production (at 0.625 μM 13.9±2.2% of inhibition). CONCLUSIONS The data obtained clearly indicate that in the case of peroral use of the examined ellagitannin-rich plant materials the bioactivity of gut microbiota metabolites, i.e. urolithins, has to be taken under consideration.

Chemistry and pharmacology of saponins: special focus on cytotoxic properties

Correspondence: Alexander weng institut fur Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charite Universitatsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany Tel +49 30 8445 3097 Fax +49 30 8445 4152 email alexander.weng@charite.de Abstract: Saponins are bioactive compounds produced mainly by plants but also by some marine organisms and insects. In the recent past, there has been unforeseen interest in the clinical utilization of saponins as chemotherapeutic agents. The research on saponins in various forms as a treatment for cancer has generated a lot of potential. The advent of nanotechnology and the cytotoxicity enhancing properties of saponins are some of the highlights of the current decade. This review gives an updated overview of the clinical potential that saponins hold as cytotoxic agents, and covers the literature for 1957–2011, with the main focus on research conducted in the last decade. It is conceivable that saponins hold a lot of therapeutic potential and could be a lead for identification of synthetic or semisynthetic molecules for the treatment of cancer via membrane-mediated or transport-mediated pathways.

The Saponin-Mediated Enhanced Uptake of Targeted Saporin-Based Drugs is Strongly Dependent on the Saponin Structure

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane–permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a nonpermeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC50) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Polyphenolic Compounds as Pancreatic Lipase Inhibitors

Obesity and its associated diseases such as diabetes mellitus and coronary heart diseases are a major challenge for our society. An important target for the treatment of obesity includes the development of inhibitors of nutrient digestion and absorption. Inhibition of pancreatic lipase and the associated reduction of lipid absorption is an attractive approach for the discovery of potent agents. Currently, the only clinically approved pharmacologic agent as pancreatic lipase inhibitor is Orlistat. However, its usage is compromised by unpleasant gastrointestinal adverse reactions (oily stools, oily spotting, flatulence). The use of botanical materials as a potential source of new drugs is of increasing importance and application. Natural products that are interesting for obesity treatment are generally considered to have less toxic and side effects than totally synthetic drugs. One of the most important sources of potential pancreatic lipase inhibitors represents the class of polyphenols. This article summarizes most studied subclasses of polyphenols including flavonoids, hydroxycinnamic acids, hydroxybenzoic acids and lignans with pancreatic lipase inhibitory effects. A structural comparison of potent inhibitors shows an increased inhibitory effect depending on number and position of phenolic hydroxyl groups, degree of polymerization and elimination of glycosylation during digestion.

Anti-inflammatory and spasmolytic activity of extracts from Droserae herba

An ethanolic extract of Drosera madagascariensis inhibited human neutrophil elastase with an IC50 of 9.4 microg/ml. The naphthoquinones present in the extract were not responsible for this effect, but flavonoids like quercetin (IC50 0.8 microg/ml), hyperoside (IC50 0.15 microg/ml) and isoquercitrin (IC50 0.7 microg/ml) contributed to inhibition of the enzyme. In guinea-pig ileum the extract (0.5-1 mg/ml) induced a spasmolytic effect via affecting cholinergic M3 receptors and histamine H1 receptors, respectively. At contractile prostanoid receptors of guinea-pig trachea the Drosera extract was not effective.

Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success.

Inhibition of Tumor Growth by Targeted Toxins in Mice is Dramatically Improved by Saponinum Album in a Synergistic Way

The application of targeted toxins in cancer therapy remains a challenge due to the severe side effects as a consequence of the high systemic doses required. Here, we describe the combined application of a glycosylated triterpenoid (Spn) and epidermal growth factor receptor (EGFR)-targeted chimeric toxins (SA2E). The cytotoxicity of SA2E on murine TSA tumor cells transfected with human EGFR was enhanced 20,000-fold by low nonpermeabilizing Spn concentrations in a synergistic manner. Subcutaneous application of Spn and SA2E in BALB/c mice bearing a solid TSA cells transfected with epidermal growth factor receptor tumor resulted in 94% tumor volume reduction with a 50-fold lower chimeric toxin concentration compared with pure SA2E treatment. Side effects as monitored by observable complications, body weight, blood parameters; histologic analyses and antibody responses were only moderate and usually reversible.