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.

Snakebite epidemiology and health-seeking behavior in Akonolinga health district, Cameroon: Cross-sectional study

“…The application of the very popular black-stone has been proven ineffective because insufficiently specific to venom proteins, although the absorbing capacity of some types of these "snake stones" has been observed [11,12]. Regarding any benefit of herbal traditional treatments, extensive research suggested encouraging local effects of many plants [13][14][15][16][17] but never when they are used in the raw manner in which they are used in communities [18].…”

Medicinal Plants for the Treatment of Local Tissue Damage Induced by Snake Venoms: An Overview from Traditional Use to Pharmacological Evidence

“…Although scientifically valid and even recommended by WHO for assessing antiophidic antivenoms [ 97 ], this preincubation approach makes a scenario unlikely to be possible in the field, where the medicine would be delivered after the snakebite. In fact, a recent study evaluated the inhibitory action of the medicinal plant Bellucia dichotoma (Melastomataceae) against Bothrops atrox snake venom using different protocols: preincubation, pretreatment, and posttreatment [ 98 ]. The authors observed that while the extract was greatly active when preincubated, this inhibitory activity was drastically reduced or even lost when the extract was injected independently of venom, simulating traditional use.…”

“…The authors observed that the extract has great amounts of tannins, which are compounds known to precipitate proteins. So, it was concluded that the “pseudo-inhibition” observed after preincubation may be due to the presence of these compounds, suggesting that the preincubation protocol overestimates inhibitory potential of medicinal plants, and for this reason, this kind of approach must be analyzed with caution for estimation of inhibitory potential of medicinal plants [ 13 , 98 ]. In this sense, many recent studies have been done using protocols of pre- and/or posttreatment, to ensure the potentiality of antiophidic plants, and for most of them, positive results have been found [ 96 , 98 – 102 ].…”

Catechin and epicatechin as an adjuvant in the therapy of hemostasis disorders induced by snake venoms

“…A second possible mechanism is based on the ability of phenolic compounds to chelate metal ions, such as zinc and calcium, which are essential cofactors of enzymatic toxins. [49][50][51] The regions involved in this mechanism of action are usually found between the 5-OH positions and the 4-oxo group, or between the 3′-and 4′-OH hydroxyls of the compounds. [52] However, the property of phenolic compounds enzymatic toxins, [53] resulting in a decrease in their activities.…”