Pharmaceutical Biology

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In Vitro. Investigation of the Protective Effects of

Tannic Acid Against the Activities of Naja kaouthia.
Venom

Pimolpan Pithayanukul, Pakatip Ruenraroengsak, Rapepol Bavovada,

Narumol Pakmanee & Rutt Suttisri

To cite this article: Pimolpan Pithayanukul, Pakatip Ruenraroengsak, Rapepol Bavovada,

Narumol Pakmanee & Rutt Suttisri (2007) In�Vitro. Investigation of the Protective Effects of Tannic
Acid Against the Activities of Naja�kaouthia. Venom, Pharmaceutical Biology, 45:2, 94-97, DOI:
10.1080/13880200601112885

To link to this article: https://doi.org/10.1080/13880200601112885

Published online: 07 Oct 2008.

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Pharmaceutical Biology
2007, Vol. 45, No. 2, pp. 94–97

In Vitro Investigation of the Protective Effects of Tannic Acid

Against the Activities of Naja kaouthia Venom

Pimolpan Pithayanukul1, Pakatip Ruenraroengsak1, Rapepol Bavovada2, Narumol Pakmanee3, and Rutt Suttisri2
1
Faculty of Pharmacy, Mahidol University, Bangkok, Thailand; 2Faculty of Pharmaceutical Sciences, Chulalongkorn
University, Bangkok, Thailand; 3Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

Abstract
The in vitro venom neutralizing capacity of tannic acid acid and plant polyphenols as washing agents for the
against the activities of Naja kaouthia (Naja naja kaouthia emergency treatment of snakebites (Okonogi et al.,
Lesson [Elapidae]) venom was investigated. Tannic acid 1979).
was found to be effective in neutralizing the activities Local tissue damage is a continuous process that goes
of Naja kaouthia (NK) venom. The lethal effect of on even after antivenom therapy. As such, the lack of
four-times the LD50 in mice and the necrotizing effect sufficient protection for local tissue damage is considered
of one minimum necrotizing dose (1 MND) in rats of as one of the important limitations associated with the
NK venom were fully inhibited by tannic acid at aforementioned therapy (Yingprasertchai et al., 2003;
431 mg=mouse, with the median effective dose (ED50) Rucavado et al., 2004). The neutralization of snake
of 334 mg=mouse and 30 mg=rat, respectively. The ace- venom by natural or synthetic inhibitors locally at the
tylcholinesterase activity of NK venom was almost com- bite site might be useful in overcoming this limitation
pletely neutralized (99%) by tannic acid at 1% w=v. (Girish & Kemparaju, 2005). The retardation of diffused
It was evident that tannic acid was nontoxic and did toxins at the bite site should minimize local tissue dam-
not cause either a lethal effect in mice (at its maximum age as well as prolong the survival time of the victims
tested dose of 845 mg=mouse) or necrotic lesions in rats (Yingprasertchai et al., 2003). Among many venomous
(at doses between 7.5 and 60 mg=rat). 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
Keywords: Local tissue necrosis, Naja kaouthia venom,
within the plant kingdom, and commercially available,
tannic acid.
the aim of this study was to evaluate the in vitro protec-
tive effects of tannic acid against Naja kaouthia (Naja
naja kaouthia Lesson [Elapidae]) venom activities. The
Introduction results should be useful to some extent to indicate the
Tannic acid and plant polyphenols have both been potential of this natural inhibitor in neutralizing the Naja
shown to exert various pharmacological effects in bio- kaouthia venom activities in vivo.
logical systems (Havsteen, 1983; Ratty & Das, 1988;
Haslam, 1989; Ramanathan & Das, 1992). Their protec-
tive effects against the toxicity of snake venom activities Materials and Methods
have been reported (Duke, 1985; Houghton & Harvey,
Chemicals
1989; Mors et al., 1989; Kuppusamy & Das, 1993;
Pithayanukul et al., 2004, 2005). Furthermore, some Tannic acid and other reagents were of USP grade and
researchers have also suggested the application of tannic were obtained from local suppliers.

Accepted: August 29, 2006

Address correspondence to: Pimolpan Pithayanukul, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. Tel.: 662 6448694;
Fax: 662-6900756; E-mail: pypph@mahidol.ac.th

DOI: 10.1080/13880200601112885 # 2007 Informa Healthcare

 Antivenom effect of tannic acid 95

Animals and venom sacrificed with an overdose of ether, their dorsal skin
was removed, and the necrotic lesion on the inner surface
Swiss albino mice of both sexes weighing between 18 and
of the skin was measured. Tannic acid, the venom, and
20 g were used for the antilethal effect test. Male
physiologic saline alone were used as controls.
Sprague-Dawley rats weighing about 250–280 g were
used for inhibition of the necrotizing effect test. The
experiments were performed according to international Inhibition of acetylcholinesterase activity
and Mahidol University’s guidelines on animal studies.
Lyophilized Naja kaouthia (NK) venom was provided The physiologic saline solutions of tannic acid, with con-
by Queen Saovabha Memorial Institute of the Thai centrations between 0.001 and 10% w=v, were preincu-
Red Cross Society in Bangkok, Thailand. bated with NK venom (0.1% w=v) at 37C for 1 h. The
supernatant was then assayed for acethylcholinesterase
enzyme activity following the modified method of Ell-
Tests for anti-snake venom activities man et al. (1961). The reaction rate was calculated
Inhibition of lethality according to the method of Ellman (1959).

The median lethal dose (LD50) of NK venom was

determined according to the method described by Gel electrophoresis of venom proteins
Theakston and Reid (1983). The venom, in 0.2 mL of
After the inhibition of lethality test, the venom proteins
physiologic saline (normal saline solution; NSS), was
in the supernatants and the precipitates of the venom–
injected into the tail vein of mice. Triplicate experiments
tannic acid mixture were analyzed by gel electrophoresis
were carried out in six mice for each venom dose. The
(SDS-PAGE) and were compared with the following
LD50 was calculated from the number of deaths occur-
standard molecular weight markers: bovine serum
ring within 24 h of venom injection (Reed & Muench,
albumin, ovalbumin, soybean trypsin inhibitor, lysozyme,
1938) with the confidence limit at 95% probability (Pizzi,
and neurotoxin 3. The method of Laemmli (1970) was
1950). Inhibition of lethality by tannic acid was determ-
followed with a slight modification. The results were
ined against four times the LD50 of NK venom by the in
confirmed by using a gel scanner to measure the optical
vitro neutralization method. Different doses of tannic
densities of the proteins at 280 nm.
acid were preincubated with the venom at 37C for 1 h
and centrifuged at 18,110  g for 10 min before 0.2 mL
of the supernatant was injected intravenously through Statistical analysis
the tail vein of mice. Six mice were used for each tested
dose, and the experiments were performed in duplicate. The survival rates of the mice that received NK venom
Tannic acid, the venom, and physiologic saline alone and tannic acid mixtures were compared with those of
were used as controls. The death and survival of mice the controls (receiving either venom or tannic acid alone)
were recorded for 24 h, and the median effective dose at different time intervals. Analysis of variance
(ED50) of tannic acid, producing 50% survival of (ANOVA) was used to determine the significance
the mice against four-times the LD50 of NK venom, (p < 0.05) of the data obtained in all experiments.
was calculated according to the method of Reed and
Muench (1938).

Results
Inhibition of necrotizing activity Inhibition of lethality
The methods of Kondo et al. (1960) and Theakston and The LD50 of NK venom was 9.32 mg=mouse. The lethal
Reid (1983) were followed in order to determine the effect of four-times the LD50 of the venom in mice could
minimum necrotizing dose (MND), which is defined as be completely inhibited (p < 0.05 compared with the
the smallest amount of venom that can cause a necrotic venom control group) by tannic acid at 431 mg=mouse
lesion of 5 mm in diameter on the inner dorsal skin of with its median effective dose (ED50) of 334 mg=mouse
rat after 72 h of intradermal injection. The inhibition of (Table 1). Tannic acid, at its maximum tested dose of
1 MND of NK venom was determined by preincubating 845 mg=mouse, and physiologic saline solution did not
the venom with several concentrations of tannic acid at cause lethality to mice. It was noted that although mice
37C for 1 h. After centrifugation at 18,110  g for receiveing four-times the LD50 of NK venom alone
10 min, the supernatant (0.1 mL) was injected intrader- showed typical toxic signs of sedation, akinesia, dyspnea,
mally into the shaved dorsal skin of rats. Triplicate abnormal respiration, and death within 30–60 min, all of
experiments were carried out using three rats for each the surviving mice in the tannin-treated groups showed
tested dose of tannic acid. After 72 h, the animals were none of these signs.
96 P. Pithayanukul et al.

Table 1. Median effective dose (ED50) of tannic acid against

four-times the LD50 of Naja kaouthia

Tannic acid (mg=mouse) % Death ED50 (mg=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 Figure 1. Inhibition of acetylcholinesterase activity of 0.1% (w=v)

Naja kaouthia venom by tannic acid.
The minimum necrotizing dose of NK venom was
30 mg=rat (p < 0.05). Tannic acid at 30 mg=rat was shown
to be effective in completely protecting rats from the the precipitation of the venom proteins from the
necrotizing activity of one minimum necrotizing dose venom–tannic acid mixture happened in a dose-
of NK venom (Table 2). Both physiologic saline and dependent manner. The electrophoretogram results cor-
tannic acid (7.5–60 mg=rat) did not induce necrotic lesion responded with the optical density of proteins in both
in rats. the supernatant and the precipitates.

Inhibition of acetylcholinesterase activity

The acetylcholinesterase activity of the venom was con-
Discussion
sidered as 100%. Tannic acid could inhibit acetylcholi- Polyphenols, especially tannins, have been reported to
nesterase activity of 0.1% w=v of NK venom almost possess the ability to form complexes with proteins
completely (99%, p < 0.05) at a concentration of (Haslam, 1989). The involvement of protein complex for-
1% w=v (Fig. 1). mation 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
Analysis of venom proteins
precipitation of the venom proteins, was confirmed by
The SDS-PAGE electrophoretograms demonstrated that the SDS-PAGE electrophoretograms and the optical
when tannic acid of the venom–tannic acid mixture was density studies. As a result, the venom activities were
increased from 0.001% to 10% w=v, the venom proteins inhibited. It was evident that tannic acid could protect
in the supernatant gradually disappeared but could the tested animals from death (at 431 mg=mouse) and
still be detected in the precipitates. This indicated that necrosis (at 30 mg=rat) against four-times LD50 and 1
MND of NK venom, respectively. The acetylcholinester-
ase activity of 0.1% w=v NK venom was almost com-
Table 2. Percentage of rats that developed necrotic lesion after pletely (99%) inhibited by 1% w=v tannic acid. This
being injected with 1 MND (30 mg) of Naja kaouthia venom could be due to the selective blockage of the nicotinic
preincubated with tannic acid. acetylcholine receptor site by tannic acid molecules
and=or the nonselective complexation of the venom
% Rats that developed
enzymes by tannic acid (Pithayanukul et al., 2005).
Tannic acid (mg=rat) necrotic lesion
Tannic acid was found in this study to be nontoxic
0 (Control, normal saline) 0 and did not cause lethality in mice (at the maximum
0 (Control, venom) 100 dose of 845 mg=mouse) or induce necrotic lesions in rats
7.5–60 (Control, tannic acid) 0 (at doses between 7.5 and 60 mg=rat, or 0.03 and
7.5 100 0.24 mg=kg). It was noticed that the amounts of tannic
15 100 acid used for the antinecrotizing experiments were very
30 0
low while the lethal dosage of tannic acid was 75 g=kg
45 0
after subcutaneous administration into mice (Robinson
60 0
& Graessle, 1943). This nontoxic effect of tannic acid
 Antivenom effect of tannic acid 97

was in accordance with the findings of Kuppusamy and Laemmli UK (1970): Cleavage of structural proteins during
Das (1993), which demonstrated that the intravenous the assembly of the head of bacteriophage T4. Nature
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