Dora Benjumea

Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom.

A hemorrhagic metalloproteinase, named Batx-I, was isolated from the venom of Bothrops atrox specimens (from Southeastern Colombian region) by a combination of CM-Sephadex C25 ion-exchange and Affi-gel Blue affinity chromatographies. This enzyme accounts for about 45% of venom proteins, and it has an ESI-MS isotope-averaged molecular mass of 23296.2 Da and a blocked N-terminus. Two internal fragments sequenced by mass spectrometric analysis showed similarity to other SVMPs from Bothrops venoms. To investigate the possible participation of Batx-I in the envenomation pathophysiology, proteolytic, fibrinogenolytic, hemorrhagic, and other biological activities were evaluated. The minimal hemorrhagic dose obtained was 17 microg/20 g body weight. The enzyme showed proteolytic activity on azocasein, comparable with activity of BaP1. This activity was inhibited by EDTA and 1, 10 o-phenanthroline but not by aprotinin, pepstatin A or PMSF. Fibrinogenolytic activity was analyzed by SDS-PAGE, revealing a preference for degrading the A alpha- and B beta-chains, although partial degradation of the gamma-chain was also detected. The protein lacks coagulant and defibrinating activity. The CK levels obtained, clearly reflects a myotoxic activity induced by Batx-I. The hemorrhagic and fibrinogenolytic activities exhibited by the isolated PI-SVMP may play a role in the hemorrhagic and blood-clotting disorders observed in patients bitten by B. atrox in Colombia.

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.

Inhibition of the toxic effects of Bothrops asper venom by pinostrobin, a flavanone isolated from Renealmia alpinia (Rottb.) MAAS

ETHNOPHARMACOLOGICAL RELEVANCE Renealmia alpinia has been traditionally used to treat snakebites by indigenous Embera-Katíos tribes belonging to the regions of Antioquia and Chocó, Colombia, and it has been shown to inhibit the enzymatic and biological activities of Bothrops venoms and their purified phospholipase A2 (PLA2) toxins. In addition to its common local usage against snakebites, Renealmia alpinia is commonly used to treat pain. To evaluate the inhibitory ability of pinostrobin, the main compound in the dichloromethane extract of Renealmia alpinia, on the toxic effects of Bothrops asper venom through in vitro and in vivo models and to evaluate its activity against pain and edema. MATERIALS AND METHODS Pinostrobin was isolated from the dichloromethane extract of Renealmia alpinia leaves. The protective properties of the extract and of pinostrobin against the indirect hemolytic, coagulant and proteolytic effects of Bothrops asper venom were evaluated in vitro, and the anti-hemorrhagic and anti-inflammatory activity were evaluated in vivo. RESULTS Renealmia alpinia extract significantly inhibited the proteolytic activity and indirect hemolytic activity of Bothrops asper venom at a venom:extract ratio of 1:20. Moreover, the present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage due to hemorrhagic effects, and the compound is also responsible for the analgesic and anti-inflammatory activity of the extract from Renealmia alpinia. This is the first report to describe pinostrobin in the species Renealmia alpinia and its properties in vitro against Bothrops asper venom. CONCLUSION Our studies of the activity of Renealmia alpinia against the venom of Bothrops asper have confirmed that this species possesses inhibitory effects against Bothrops asper venom in both in vitro and in vivo models and that these effects may be due to pinostrobin, supporting the traditional usage of the plant. Additionally, pinostrobin may be responsible for the anti-hemorrhagic and analgesic activity (peripheral analgesic activity) of Renealmia alpinia.

Traditional use of the genus Renealmia and Renealmia alpinia (Rottb.) Maas (Zingiberaceae)-a review in the treatment of snakebites.

Renealmia alpinia (R. alpinia) typically occurs in the tropical rainforests of Mexico to Peru, Brazil through the Antilles, Guyana, Suriname, Colombia and Venezuela. It has traditionally been used against snakebite in Colombia. In addition to the common local use for pain, R. alpinia has been used as an antipyretic and antiemetic to treat wounds, malignant ulcers, epilepsy and fungal infections. The species of the Zingiberaceae family are famous for their use as spices and herbs. This review provides information on the traditional use of plants in the Zingiberaceae family, Renealmia genus, and specifically R. alpinia. We conducted a narrative review in English and Spanish in electronic databases, such as ScienceDirect, Napralert, PubMed, ScFinder, SciElo, during August 2011 to March 2013. We were interested in their applications in ethnomedicine and their chemical components, providing a major and methodical revision. We found 120 sources, 98 of which were selected as they contained essential information. This study has stimulated the development of a new work, which aims to isolate and evaluate metabolites from R. alpinia leads toward the development of a phytotherapeutic product, which might be accessible to the population. Studies on the toxicity and safety of R. alpinia are insufficient to provide information on the ethnomedical use of this species. Thus, the present review summarizes information about R. alpinia and proposes possible scope of future research to fill gaps identified in this narrative review.

Inhibitory effect of pinostrobin from Renealmia alpinia, on the enzymatic and biological activities of a PLA2

Pinostrobin is a flavanone isolated from Renealmia alpinia, a plant used in folk medicine to treat snakebites. We tested the inhibitory ability of pinostrobin on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The compound displayed IC50 values of 1.76mM and 1.85mM (95% Confidence intervals: 1.34-2.18 and 1.21-2.45) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. When mice were injected with PLA2 preincubated with 0.4, 2.0 and 4.0mM of pinostrobin, myotoxic activity induced by the PLA2 was inhibited up to 87%. Nevertheless, these values decreased up to 56% when the pinostrobin was injected into muscle after PLA2. Pinostrobin inhibited edema-forming and anticoagulant activities of the PLA2. In order to have insights on the mode of action of pinostrobin, intrinsic fluorescence and ultraviolet studies were performed. Results suggest that pinostrobin interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that pinostrobin forms hydrogen bonds with residues His48 and Asp49 of PLA2, besides, a π-π stacking interactions with those of residues Phe5 and Trp31, and rings C of flavanone and Tyr52 of the toxin.

Interactions of Desmethoxyyangonin, a Secondary Metabolite from Renealmia alpinia, with Human Monoamine Oxidase-A and Oxidase-B

Renealmia alpinia (Zingiberaceae), a medicinal plant of tropical rainforests, is used to treat snakebites and other injuries and also as a febrifuge, analgesic, antiemetic, antiulcer, and anticonvulsant. The dichloromethane extract of R. alpinia leaves showed potent inhibition of human monoamine oxidases- (MAOs-) A and B. Phytochemical studies yielded six known compounds, including pinostrobin 1, 4′-methyl ether sakuranetin 2, sakuranetin 3, pinostrobin chalcone 4, yashabushidiol A 5, and desmethoxyyangonin 6. Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A, with Ki values of 31 and 922 nM, respectively. Kinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversible binding of desmethoxyyangonin 6 with MAO-A and MAO-B. The binding interactions of compound 6 in the active site of the MAO-A and MAO-B isoenzymes, investigated through molecular modeling algorithms, confirmed preferential binding of desmethoxyyangonin 6 with MAO-B compared to MAO-A. Selective reversible inhibitors of MAO-B, like desmethoxyyangonin 6, may have important therapeutic significance for the treatment of neurodegenerative disorders, such as Parkinsons disease and Alzheimers disease.