The inhibitory effect of Camellia sinensis extracts against the neuromuscular blockade of Crotalus durissus terrificus venom

Abstract

In geographically isolated populations where intensive medical care or serum therapy is not easily accessible snake envenomation is a major cause for concern. The aim of the present study was to test Camellia sinensis extracts, theaflavin and epigallocatechin (two of the main C. sinensis components) against the irreversible neuromuscular blockade induced by Crotalus durissus terrificus venom in mouse phrenic-nerve diaphragm preparations. A quantitative histological study was also performed. The venom (20µg/ml) completely decreased twitch tension after 70min and 5µg/ml venom abolished 50% of twitch amplitude after 60min. C. sinensis extract induced intense facilitatory effect in the preparation activity at 0.2mg/ml and slightly facilitatory effect at 0.05mg/ml. Both 0.05mg/ml C. sinensis extract and 0.05mg/ml commercial theaflavin maintained partial muscular activity in presence of 5µg/ml venom. The histological data confirms that Cs is able to protect the muscle from the myotoxic activity of the venom. Commercial epigallocatechin gallate did not show pre-synaptic nor post-synaptic activities. C. sinensis extract was able to protect the mouse phrenic-nerve diaphragm against the irreversible neuromuscular blockade induced by C. durissus terrificus venom.

Keywords: Anticrotalic action; catechins; green tea; snake venom; theaflavins.

Figure 1.

Thin Layer Chromatography performed by using ethyl acetate:methanol:water (100:13.5:10) solvent/Developer: NP/PEG. Phytochemical standards: 1 - Theaflavin; 3-Epigallocatechin gallate. 2, 4-Camellia sinensis extract [1; Theaflavin, 2; Camellia sinensis extract, 3; Epigallocatechin gallate, 4; Camellia sinensis extract].

Figure 2.

Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Concentration-response curve of Crotalus durissus terrificus (Cdt) venom. Each point represents the mean ± SEM. * = p<0.05 in comparison with Tyrode control.

Figure 3.

Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Concentration-response curve of Camellia sinensis (Cs) extract. Each point represents the mean ± SEM. * = p<0.05 in comparison with Tyrode control.

Figure 4.

(A) Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Each point represents the mean ± SEM. * = p<0.05 in comparison with Cdt venom.

Figure 5.

Diaphragm muscles (cross-sections of 2µm thick). A. Muscle incubated with 5µg/ml Cdt venom shows 59.5 ±1% of fibers exhibiting myonecrotic states characterized by edema (e), delta lesion (arrow), sarcolemmal disruption with nuclei (n) dispersion, and “ghost” cells (g) visualized by spaces optically empty. Area with extensive myonecrosis assumes a hyaline aspect. B. Muscle incubated with 5µg/ml Cdt venom plus hydroalcoholic extract shows fibers maintaining its characteristic polygonal profile, major quantity of cells due minor edema resulting in a slower percentage of damage (31.4 ±0.8 %) compared to venom alone (p<0.05). Bar = 50µm.