In Vitro. Investigation of the Protective Effects of Tannic Acid Against the Activities of Naja kaouthia. Venom

Abstract

The in vitro. venom neutralizing capacity of tannic acid against the activities of Naja kaouthia. (Naja naja kaouthia. Lesson [Elapidae]) venom was investigated. Tannic acid was found to be effective in neutralizing the activities of Naja kaouthia. (NK) venom. The lethal effect of four-times the LD₅₀ in mice and the necrotizing effect of one minimum necrotizing dose (1 MND) in rats of NK venom were fully inhibited by tannic acid at ≥ 431 µg/mouse, with the median effective dose (ED₅₀) of 334 µg/mouse and ≥ 30 µg/rat, respectively. The acetylcholinesterase activity of NK venom was almost completely neutralized (≥ 99%) by tannic acid at ≥ 1% w/v. It was evident that tannic acid was nontoxic and did not cause either a lethal effect in mice (at its maximum tested dose of 845 µg/mouse) or necrotic lesions in rats (at doses between 7.5 and 60 µg/rat).

Keywords:

• Local tissue necrosis
• Naja kaouthia. venom
• tannic acid

Introduction

Tannic acid and plant polyphenols have both been shown to exert various pharmacological effects in biological systems (Havsteen, 1983; Ratty & Das, 1988; Haslam, 1989; Ramanathan & Das, 1992). Their protective effects against the toxicity of snake venom activities have been reported (Duke, 1985; Houghton & Harvey, 1989; Mors et al., 1989; Kuppusamy & Das, 1993; Pithayanukul et al., 20042005). Furthermore, some researchers have also suggested the application of tannic acid and plant polyphenols as washing agents for the emergency treatment of snakebites (Okonogi et al., 1979).

Local tissue damage is a continuous process that goes on even after antivenom therapy. As such, the lack of sufficient protection for local tissue damage is considered as one of the important limitations associated with the aforementioned therapy (Yingprasertchai et al., 2003; Rucavado et al., 2004). The neutralization of snake venom by natural or synthetic inhibitors locally at the bite site might be useful in overcoming this limitation (Girish & Kemparaju, 2005). The retardation of diffused toxins at the bite site should minimize local tissue damage as well as prolong the survival time of the victims (Yingprasertchai et al., 2003). Among many venomous snakes in Thailand, severe local tissue necrosis results in 50% of the bites (Pochanugool et al., 1998). Because tannic acid is readily available, naturally occurring within the plant kingdom, and commercially available, the aim of this study was to evaluate the in vitro. protective effects of tannic acid against Naja kaouthia. (Naja naja kaouthia. Lesson [Elapidae]) venom activities. The results should be useful to some extent to indicate the potential of this natural inhibitor in neutralizing the Naja kaouthia. venom activities in vivo..

Materials and Methods

Chemicals

Tannic acid and other reagents were of USP grade and were obtained from local suppliers.

Animals and venom

Swiss albino mice of both sexes weighing between 18 and 20 g were used for the antilethal effect test. Male Sprague-Dawley rats weighing about 250–280 g were used for inhibition of the necrotizing effect test. The experiments were performed according to international and Mahidol University's guidelines on animal studies. Lyophilized Naja kaouthia. (NK) venom was provided by Queen Saovabha Memorial Institute of the Thai Red Cross Society in Bangkok, Thailand.

Tests for anti-snake venom activities

Inhibition of lethality

The median lethal dose (LD₅₀) of NK venom was determined according to the method described by Theakston and Reid (1983). The venom, in 0.2 mL of physiologic saline (normal saline solution; NSS), was injected into the tail vein of mice. Triplicate experiments were carried out in six mice for each venom dose. The LD₅₀ was calculated from the number of deaths occurring within 24 h of venom injection (Reed & Muench, 1938) with the confidence limit at 95% probability (Pizzi, 1950). Inhibition of lethality by tannic acid was determined against four times the LD₅₀ of NK venom by the in vitro. neutralization method. Different doses of tannic acid were preincubated with the venom at 37°C for 1 h and centrifuged at 18,110 × g. for 10 min before 0.2 mL of the supernatant was injected intravenously through the tail vein of mice. Six mice were used for each tested dose, and the experiments were performed in duplicate. Tannic acid, the venom, and physiologic saline alone were used as controls. The death and survival of mice were recorded for 24 h, and the median effective dose (ED₅₀) of tannic acid, producing 50% survival of the mice against four-times the LD₅₀ of NK venom, was calculated according to the method of Reed and Muench (1938).

Inhibition of necrotizing activity

The methods of Kondo et al. (1960) and Theakston and Reid (1983) were followed in order to determine the minimum necrotizing dose (MND), which is defined as the smallest amount of venom that can cause a necrotic lesion of 5 mm in diameter on the inner dorsal skin of rat after 72 h of intradermal injection. The inhibition of 1 MND of NK venom was determined by preincubating the venom with several concentrations of tannic acid at 37°C for 1 h. After centrifugation at 18,110 × g. for 10 min, the supernatant (0.1 mL) was injected intradermally into the shaved dorsal skin of rats. Triplicate experiments were carried out using three rats for each tested dose of tannic acid. After 72 h, the animals were sacrificed with an overdose of ether, their dorsal skin was removed, and the necrotic lesion on the inner surface of the skin was measured. Tannic acid, the venom, and physiologic saline alone were used as controls.

Inhibition of acetylcholinesterase activity

The physiologic saline solutions of tannic acid, with concentrations between 0.001 and 10% w/v, were preincubated with NK venom (0.1% w/v) at 37°C for 1 h. The supernatant was then assayed for acethylcholinesterase enzyme activity following the modified method of Ellman et al. (1961). The reaction rate was calculated according to the method of Ellman (1959).

Gel electrophoresis of venom proteins

After the inhibition of lethality test, the venom proteins in the supernatants and the precipitates of the venom–tannic acid mixture were analyzed by gel electrophoresis (SDS-PAGE) and were compared with the following standard molecular weight markers: bovine serum albumin, ovalbumin, soybean trypsin inhibitor, lysozyme, and neurotoxin 3. The method of Laemmli (1970) was followed with a slight modification. The results were confirmed by using a gel scanner to measure the optical densities of the proteins at 280 nm.

Statistical analysis

The survival rates of the mice that received NK venom and tannic acid mixtures were compared with those of the controls (receiving either venom or tannic acid alone) at different time intervals. Analysis of variance (ANOVA) was used to determine the significance (p < 0.05) of the data obtained in all experiments.

Results

Inhibition of lethality

The LD₅₀ of NK venom was 9.32 µg/mouse. The lethal effect of four-times the LD₅₀ of the venom in mice could be completely inhibited (p < 0.05 compared with the venom control group) by tannic acid at ≥ 431 µg/mouse with its median effective dose (ED₅₀) of 334 µg/mouse (Table 1). Tannic acid, at its maximum tested dose of 845 µg/mouse, and physiologic saline solution did not cause lethality to mice. It was noted that although mice receiveing four-times the LD₅₀ of NK venom alone showed typical toxic signs of sedation, akinesia, dyspnea, abnormal respiration, and death within 30–60 min, all of the surviving mice in the tannin-treated groups showed none of these signs.

Table 1. Median effective dose (ED₅₀) of tannic acid against four-times the LD₅₀ of Naja kaouthia.

Tannic acid (µg/mouse)	% Death	ED50 (µg/mouse)
0 (Control, normal saline)	0	334.89 (267.24–419.66)**
845 (Control, tannic acid)	0
0 (Control, venom)	100
220	100
308	66.67
431	0
604	0
845	0

*From duplicate experiments, p < 0.05.

**At 95% probability.

Inhibition of necrotizing activity

The minimum necrotizing dose of NK venom was 30 µg/rat (p < 0.05). Tannic acid at 30 µg/rat was shown to be effective in completely protecting rats from the necrotizing activity of one minimum necrotizing dose of NK venom (Table 2). Both physiologic saline and tannic acid (7.5–60 µg/rat) did not induce necrotic lesion in rats.

Table 2. Percentage of rats that developed necrotic lesion after being injected with 1 MND (30 µg) of Naja kaouthia. venom preincubated with tannic acid

Tannic acid (µg/rat)	% Rats that developed necrotic lesion
0 (Control, normal saline)	0
0 (Control, venom)	100
7.5–60 (Control, tannic acid)	0
7.5	100
15	100
30	0
45	0
60	0

Inhibition of acetylcholinesterase activity

The acetylcholinesterase activity of the venom was considered as 100%. Tannic acid could inhibit acetylcholinesterase activity of 0.1% w/v of NK venom almost completely (≥ 99%, p < 0.05) at a concentration of ≥ 1% w/v (Fig. 1).

Figure 1 Inhibition of acetylcholinesterase activity of 0.1% (w/v) Naja kaouthia. venom by tannic acid.

Analysis of venom proteins

The SDS-PAGE electrophoretograms demonstrated that when tannic acid of the venom–tannic acid mixture was increased from 0.001% to 10% w/v, the venom proteins in the supernatant gradually disappeared but could still be detected in the precipitates. This indicated that the precipitation of the venom proteins from the venom–tannic acid mixture happened in a dose-dependent manner. The electrophoretogram results corresponded with the optical density of proteins in both the supernatant and the precipitates.

Discussion

Polyphenols, especially tannins, have been reported to possess the ability to form complexes with proteins (Haslam, 1989). The involvement of protein complex formation with tannic acid in the inhibition of NK venom activities could not therefore be ruled out. The binding of venom proteins with tannic acid, which leads to the precipitation of the venom proteins, was confirmed by the SDS-PAGE electrophoretograms and the optical density studies. As a result, the venom activities were inhibited. It was evident that tannic acid could protect the tested animals from death (at ≥ 431 µg/mouse) and necrosis (at ≥ 30 µg/rat) against four-times LD₅₀ and 1 MND of NK venom, respectively. The acetylcholinesterase activity of 0.1% w/v NK venom was almost completely (99%) inhibited by ≥ 1% w/v tannic acid. This could be due to the selective blockage of the nicotinic acetylcholine receptor site by tannic acid molecules and/or the nonselective complexation of the venom enzymes by tannic acid (Pithayanukul et al., 2005).

Tannic acid was found in this study to be nontoxic and did not cause lethality in mice (at the maximum dose of 845 µg/mouse) or induce necrotic lesions in rats (at doses between 7.5 and 60 µg/rat, or 0.03 and 0.24 mg/kg). It was noticed that the amounts of tannic acid used for the antinecrotizing experiments were very low while the lethal dosage of tannic acid was 75 g/kg after subcutaneous administration into mice (Robinson & Graessle, 1943). This nontoxic effect of tannic acid was in accordance with the findings of Kuppusamy and Das (1993), which demonstrated that the intravenous administration of 1 mg tannic acid did not cause lethality in mice.

Based on the current findings, it is evident that tannic acid may serve as a rich source for the development of therapeutics in the treatment of venom poisoning either as first aid or as supplements for traditional antivenoms, particularly on the inhibition of local tissue necrosis in which an antivenom is incapable (Ownby et al., 1984; Gene et al., 1985). The inhibition of NK venom activities by tannic acid may also prolong the survival time of snakebite victims before they are given appropriate treatment in a hospital. However, further in vivo. investigation would be necessary to confirm these in vitro. results.