Anticoagulant activity of two novel polysaccharides from flowers of Apocynum venetum L.

Two novel polysaccharides were obtained from flowers of Apocynum venetum L., and named as Vp2a-II and Vp3. Their average molecular weights were 7 kDa and 9 kDa, respectively. Methods of monosaccharide analysis, acid hydrolysis and methylation analysis, Fourier transform-infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to identify the structure of Vp2a-II and Vp3. Vp2a-II was composed of →6)-ß-d-Glcp-(1 → 6)-α-d-Galp-(1→ residues. Vp3 was composed of α-d-GlcpA-(3 → α-d-GalpA residues. The anticoagulant activity was evaluated by activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) assays in vitro. Results indicated that Vp3 exhibited the anticoagulant activity.

Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen.

Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: comparison of wild and in vitro propagated plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. MATERIALS AND METHODS: We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. RESULTS: Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. CONCLUSIONS: Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases.

Adjunctive and alternative approaches to current reperfusion therapy.

BACKGROUND AND PURPOSE: Current ischemic stroke reperfusion therapy consists of intravenous thrombolysis given in eligible patients after review of a noncontrast CT scan and a time-based window of opportunity. Rapid clot lysis has a strong association with clinical improvement but remains incomplete in many patients. This review appraises novel adjunctive or alternative approaches to current reperfusion strategies being tested in all trial phases. Summary of Review- Alternative approaches to current reperfusion therapy can be separated into 4 main categories: (1) combinatory approaches with other drugs or devices; (2) novel systemic thrombolytic agents; (3) endovascular medical or mechanical reperfusion treatments; and (4) noninvasive or minimally invasive methods to augment cerebral blood flow and alleviate intracranial blood flow steal. CONCLUSIONS: Reperfusion treatments must be provided as fast as possible in patients most likely to benefit. Patients who fail to rapidly reperfuse may benefit from other strategies that maintain collateral flow or protect tissue at risk.

Molecular docking studies and anti-snake venom metalloproteinase activity of Thai mango seed kernel extract.

Snakebite envenomations cause severe local tissue necrosis and the venom metalloproteinases are thought to be the key toxins involved. In this study, the ethanolic extract from seed kernels of Thai mango (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloylglucopyranose) exhibited potent and dose-dependent inhibitory effects on the caseinolytic and fibrinogenolytic activities of Malayan pit viper and Thai cobra venoms in in vitro tests. molecular docking studies revealed that the binding orientations of the phenolic principles were in the binding pockets of snake venom metalloproteinases (SVMPs). The phenolic principles could form hydrogen bonds with the three histidine residues in the conserved zinc-binding motif and could chelate the Zn(2+) atom of the SVMPs, which could potentially result in inhibition of the venom enzymatic activities and thereby inhibit tissue necrosis.

The antifibrogenic effect of (-)-epigallocatechin gallate results from the induction of de novo synthesis of glutathione in passaged rat hepatic stellate cells.

Hepatic stellate cells (HSC) are the major players during hepatic fibrogenesis. Overproduction of extracellular matrix (ECM) is a characteristic of activated HSC. Transforming growth factor-beta (TGF-beta) is the most potent fibrogenic cytokine while connective tissue growth factor (CTGF) mediates the production of TGF-beta-induced ECM in activated HSC. HSC activation and hepatic fibrogenesis are stimulated by oxidative stress. Glutathione (GSH) is the most important intracellular antioxidant. The aim of this study is to explore the mechanisms of (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, in the inhibition of ECM gene expression in activated HSC. It is hypothesized that EGCG inhibits ECM gene expression in activated HSC by interrupting TGF-beta signaling through attenuating oxidative stress. It is found that EGCG interrupts TGF-beta signaling in activated HSC by suppressing gene expression of type I and II TGF-beta receptors. EGCG inhibits CTGF gene expression, leading to the reduction in the abundance of ECM, including alphaI(I) procollagen. Exogenous CTGF dose dependently eliminates the antifibrogenic effect. EGCG attenuates oxidative stress in passaged HSC by scavenging reactive oxygen species and reducing lipid peroxidation. De novo synthesis of GSH is a prerequisite for EGCG to interrupt TGF-beta signaling and to reduce the abundance of alphaI(I) procollagen in activated HSC in vitro. Taken together, our results demonstrate that the interruption of TGF-beta signaling by EGCG results in the suppression of gene expression of CTGF and ECM in activated HSC in vitro. In addition, our results, for the first time, demonstrate that the antioxidant property of EGCG derived from de novo synthesis of intracellular GSH plays a critical role in its antifibrogenic effect. These results provide novel insights into the mechanisms of EGCG as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.

Sequential determination of ligands binding to discrete components in heterogeneous mixtures by iterative panning and blocking (IPAB).

Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.

Isolation of translactone-containing triterpenes with thrombin inhibitory activities from the leaves of Lantana camara.

Methanolic extracts prepared from the leaves of Lantana camara have been found to inhibit human thrombin. An assay, in which thrombin activity is measured as a function of clot formation from fibrinogen, was used to guide the fractionation and purification of five principal active constituents (1-5), which were all characterized as 5,5-trans-fused cyclic lactone-containing euphane triterpenes.