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

• Published in: Toxicon (Oxford) Vol. 85; pp. 59 - 68
• Main Authors: Mourão de Moura, Valéria, Serra Bezerra, Adrielle N., Veras Mourão, Rosa Helena, Varjão Lameiras, Juliana L., Almeida Raposo, Juliana D., Luckwu de Sousa, Rafael, Boechat, Antônio Luiz, Bezerra de Oliveira, Ricardo, de Menezes Chalkidis, Hipocrátes, Dos-Santos, Maria Cristina
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-07-2014
• Subjects: Animals; Anti-snake bite plants; Antivenins - chemistry; Antivenins - isolation & purification; Antivenins - pharmacology; Antivenins - therapeutic use; Bellucia dichotoma; Bothrops; Bothrops atrox; Brazil; Crotalid Venoms - antagonists & inhibitors; Crotalid Venoms - enzymology; Crotalid Venoms - toxicity; Edema - etiology; Edema - prevention & control; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - isolation & purification; Enzyme Inhibitors - pharmacology; Enzyme Inhibitors - therapeutic use; Ethnopharmacology; Group II Phospholipases A2 - antagonists & inhibitors; Group II Phospholipases A2 - metabolism; Hemorrhage - etiology; Hemorrhage - prevention & control; Hemostatics - chemistry; Hemostatics - isolation & purification; Hemostatics - pharmacology; Hemostatics - therapeutic use; Male; Medicine, Traditional; Melastomataceae - chemistry; Mice; Neurotoxins - antagonists & inhibitors; Neurotoxins - toxicity; Plant Bark - chemistry; Plant Extracts - chemistry; Plant Extracts - isolation & purification; Plant Extracts - pharmacology; Plant Extracts - therapeutic use; Reptilian Proteins - antagonists & inhibitors; Reptilian Proteins - metabolism; Skin - drug effects; Snake Bites - drug therapy; Snake Bites - physiopathology; Snake-bite accidents; Tannins - analysis; Tannins - pharmacology; Tannins - therapeutic use; Brazil; Anti-snake bite plants; Snake-bite accidents; Bellucia dichotoma; Bothrops atrox
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2014.04.009

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.
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•Bellucia dichotoma extract was tested with Bothrops atrox venom.•Methodologies based on pre-incubation and traditional use were compared.•Pre-incubation was more effective but overestimated the anti-snake bite activity.•Bellucia dichotoma extract enhanced the effect of Bothrops antivenom.