title: A comparison of the ability of Bellucia dichotoma Cogn. (Melastomataceae) extract to inhibit the local effects of Bothrops atrox venom when pre-incubated and when used according to traditional methods.
abstract: Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A₂ activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A₂ activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²⁺ ions and thus inhibiting hemorrhagin and PLA₂ activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA.
authors.affiliation: Programa Multi-Institucional de Pós-Graduação em Biotecnologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil; Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil. Electronic address: mouraovm@yahoo.com.br.
authors.forename: Valéria
authors.initials: V
authors.lastname: Mourão de Moura
authors.affiliation: Programa de Pós-Graduação em Recursos Naturais da Amazônia and Laboratório de Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará, Santarém, PA 68035-110, Brazil.
authors.forename: Adrielle N
authors.initials: AN
authors.lastname: Serra Bezerra
authors.affiliation: Programa de Pós-Graduação em Recursos Naturais da Amazônia and Laboratório de Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará, Santarém, PA 68035-110, Brazil.
authors.forename: Rosa Helena
authors.initials: RH
authors.lastname: Veras Mourão
authors.affiliation: Programa Multi-Institucional de Pós-Graduação em Biotecnologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil; Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil.
authors.forename: Juliana L
authors.initials: JL
authors.lastname: Varjão Lameiras
authors.affiliation: Programa de Pós-Graduação em Recursos Naturais da Amazônia and Laboratório de Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará, Santarém, PA 68035-110, Brazil.
authors.forename: Juliana D
authors.initials: JD
authors.lastname: Almeida Raposo
authors.affiliation: Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Luckwu de Sousa
authors.affiliation: Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil.
authors.forename: Antônio Luiz
authors.initials: AL
authors.lastname: Boechat
authors.affiliation: Programa de Pós-Graduação em Recursos Naturais da Amazônia and Laboratório de Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará, Santarém, PA 68035-110, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Bezerra de Oliveira
authors.affiliation: Laboratório de Pesquisas Zoológicas, Faculdades Integradas do Tapajós, Santarém, PA 68010-200, Brazil.
authors.forename: Hipocrátes
authors.initials: H
authors.lastname: de Menezes Chalkidis
authors.affiliation: Programa Multi-Institucional de Pós-Graduação em Biotecnologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil; Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, AM 69077-000, Brazil.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Dos-Santos
mesh_terms: D000818:Animals; D000997:Antivenins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D017837:Bothrops; D001938:Brazil; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000201:enzymology / Q000633:toxicity; D004487:Edema / Q000209:etiology / Q000517:prevention & control; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D020834:Ethnopharmacology; D054501:Group II Phospholipases A2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006470:Hemorrhage / Q000209:etiology / Q000517:prevention & control; D006490:Hemostatics / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D008297:Male; D008519:Medicine, Traditional; D035322:Melastomataceae / Q000737:chemistry*; D051379:Mice; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000633:toxicity; D024301:Plant Bark / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D030162:Reptilian Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012867:Skin / Q000187:drug effects; D012909:Snake Bites / Q000188:drug therapy* / Q000503:physiopathology; D013634:Tannins / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D000997:Antivenins; D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D006490:Hemostatics; D009498:Neurotoxins; D010936:Plant Extracts; D030162:Reptilian Proteins; D013634:Tannins; D054501:Group II Phospholipases A2
keywords: Anti-snake bite plants; Bellucia dichotoma; Bothrops atrox; Snake-bite accidents