METHODOLOGICAL CARE IN THE EVALUATION OF THE LD50 AND OF THE NEUTRALIZATION OF THE LETHAL EFFECT OF Crotalus durissus terrificus VENOM BY THE PLANT Peschiera fuchsiaefolia (APOCYNACEAE)

Original paper

METHODOLOGICAL CARE IN THE EVALUATION OF THE LD50 AND OF THE NEUTRALIZATION OF THE LETHAL EFFECT OF Crotalus durissus terrificus VENOM BY THE PLANT Peschiera fuchsiaefolia (APOCYNACEAE)

M. de F. C. BATINA, J. R. GIGLIO , S. V. SAMPAIO

1 Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences, 2 Department of Biochemistry, School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil.

ABSTRACT: In the present study, we demonstrate that the volumes in which a given protein mass of Crotalus durissus terrificus venom or of a lyophilized stabilized aqueous extract (LSAE) of Peschiera fuchsiaefolia, an antivenom agent injected intramuscularly, have a decisive influence on the results. The LD50 of C. d. terrificus venom injected i.m. in a final volume of 200µl (2µl/g) (saline solution, 0.9% NaCl) was 180µg/100g rat body weight (p<0.05, 161 to 202µg/100g body weight) and the LD50 of the venom injected i.m. in a volume of 50µl (0.5µl/g) was 120µg/100g body weight (p<0.05, 107 to 134 µg/100g body weight). The reduction of the final volume injected i.m. also required a reduced mass of LSAE necessary to neutralize the lethal effect of C. d. terrificus venom. The dose of 60mg LSAE/100g rat body weight in a final volume of 200µl administered i.m. 20 seconds after venom injection, and that of 40mg LSAE/100g body weight/200µl mixed and incubated with the venom for 1 h at 25ºC before i.m. injection were able to neutralize the lethal activity of 2LD50. However, the LSAE doses that neutralized the 2LD50 were reduced to 20mg LSAE/100g body weight in a final volume of 50µl when administered i.m. 20 seconds after venom injection and to 2.5mg LSAE/100g body weight/50µl when mixed and incubated for 1 h at 25ºC with the venom before i.m. injection. The LD50 of C. d. terrificus venom and the doses of P. fuchsiaefolia LSAE that neutralized the venom lethal activity were, therefore, significantly lower when the final volume injected i.m. was reduced.

KEY WORDS: LD50, Peschiera fuchsiaefolia, Crotalus durissus terrificus, snake venom antidotes.

INTRODUCTION

A number of plants are used in Brazilian folk medicine against envenomation, especially by snakebites(14). The following species have been studied: Curcuma longa against the action of the hemorrhagic activity of Bothrops jararaca venom and the lethal effect of Crotalus durissus terrificus venom(3); Eclipta prostrata against the lethal action of Crotalus durissus terrificus and Bothrops jararaca venoms, and the hemorrhagic and myotoxic actions of Bothrops venom(8,9); Apuleia leiocarpa, Bredemeyra floribunda, Silybum mariaum and Hibiscus esculentus against the lethality of Bothrops jararaca venom(10,12). Pereira et al. and Ruppelt et al.(11,15,16) studied the analgesic, anti-inflammatory, antiedematogenic and capillary permeability affecting activities of several plant species against Bothrops jararaca venom, among them Casearia sylvestris, Pothomorphe umbellata, Chiococa brachiata and Brunfelsia uniflora.

In field studies carried out in 1992 on the rural population of the urban community of Assis, SP, Brazil, we noted the use of the species Peschiera fuchsiaefolia (popularly called "leiteiro" or "leiteiro de vaca" in Brazil)(1). The local people use the ground root bark as a poultice at the site of crotalid and bothropic venom inoculations. The poultice is changed at 5 min intervals and it is said to change in color from black to yellow and then to white. If the root bark remains white, the popular belief is that the venom has been removed from the victim.

The aim of this paper is to demonstrate that the volumes in which a given protein mass of Crotalus durissus terrificus (South American rattlesnake) venom and of P. fuchsiaefolia extracts injected i.m. have a decisive effect on the value of the LD50 of the venom and of the lyophilized stabilized aqueous extract (LSAE) doses that neutralize the venom lethality.

MATERIAL AND METHODS

MATERIAL:C. d. terrificus venom was purchased from Butantan Institute, São Paulo, SP, Brazil, whereas P. fuchsiaefolia was collected in Assis, SP, Brazil. P. fuchsiaefolia may be found in the herbarium (SPFR) of the Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, University of São Paulo, under the number 03224. The root bark was removed and treated as follows: the bark was washed, dried in a ventilated oven at 40ºC, ground, extracted with distilled water for five days at 4ºC and filtered under reduced pressure. The aqueous extract was then lyophilized (LSAE) and stored at -10ºC. Male, albino Wistar rats weighing 90 to 110g were used. The animals were left at 24±1ºC, fed commercial ration and given water ad libitum.

DETERMINATION OF THE LD50 (i.m.) OF C. d. terrificus VENOM: The i.m. LD50 was determined as described by Litchfield and Wilcoxon(7). The venom was dispersed in 0.9% (w/v) NaCl (saline) and cleared by centrifugation at 3,000 rpm for 5 min at room temperature. An aliquot of the solution was assayed for total protein by the microbiuret method of Itzhaki and Gill(5). This solution was then diluted to lower concentrations using 1.125 as a constant dilution factor. The resulting solutions were injected in volumes of 2µl/g and 0.5µl/g into the right hind thigh of the rats which were divided at random into groups of 6. The rats were observed during the first 12 h for dead animal counts and then for 20 days to confirm the stabilization of the number of live animals per group.

NEUTRALIZATION OF THE LETHALITY OF C. d. terrificus VENOM BY P. fuchsiaefolia: For the neutralization of 2LD50, the procedure of Mors et al.(8) was followed with modifications. The LSAE was dispersed in saline, centrifuged for 5 min and the clear supernatant injected i.m. The rats were distributed at random into groups of 6 and treated as follows: Protocol I - the first group was injected with 2LD50 of C. d. terrificus venom and 20 seconds later with saline; the second group received saline instead of the venom and 20 seconds later the highest dose of LSAE. The remaining groups were injected with increasing doses of LSAE 20 seconds after 2LD50. We carried out this protocol first using a final volume of 200µl/100g for the venom and 40 to 60mg LSAE, and later using a volume of 50µl/100g for the venom and 5 to 20mg LSAE. Protocol II - the first group was injected with a mixture of 2LD50 + saline; the second group received saline + the highest dose of LSAE, and the remaining groups were injected with increasing doses of LSAE + 2LD50. The final volume of the mixtures was 400µl/100g (200µl/100g for the venom and 20 to 40mg LSAE/200µl/100g) and 100µl/100g (50µl/100g for the venom and 1.25 to 5.0mg LSAE/50µl/100g). The mixtures were incubated for 60 min at room temperature before being injected i.m. One group from each set received the same volume of saline. The rats were observed during the first 24 h for dead animal counts and then for 20 days to ratify the stabilization of the number of live animals per group.

EVANS BLUE ASSAY: One hundred mg of Evans Blue was dispersed into 4ml of saline + 4ml distilled water, sonified for 20 minutes at room temperature to speed up solubilization and then filtered through Whatman number 1 filter paper. The resulting solution was injected i.m. in the right hind thigh of rats (1µl, 2µl and 4µl/g body weight). After 10, 20 and 30 min, the animals were sacrificed by deep anesthesia with ethyl ether, and the muscular and subcutaneous regions around the injected region were macroscopically examined in order to observe if the venom and the plant extract could be injected in these volumes without any extravasation into the subcutaneous region.

RESULTS

The 12 h LD50 of C. d. terrificus venom injected i.m. was 180µg protein/100g rat body weight in a volume of 200µl (p<0.05, 161 to 202µg/100g rat body weight/200µl) (Table 1) and 120µg protein/100g rat body weight in the volume of 50µl (p<0.05, 107 to 134µg/100g rat body weight/50µl) (Table 2). The LSAE of P. fuchsiaefolia had no lethal effect on control animals, since all of them survived the experiment. Table 3 shows that both the dose of 360µg venom protein/100g (2LD50/200µl) injected 20 seconds before the 200µl volume of saline and the dose of 240µg venom protein/100g (2LD50/50µl) injected 20 seconds before the 50µl volume of saline killed 83.3% of the rats within 4 hours and 100% of the rats within 6 hours (Protocol I). The dose of 60mg LSAE/100g rat body weight in a final volume of 200µl injected i.m. 20 seconds after the venom (200µl, i.m.) neutralized the 2LD50 lethality by the i.m. route(2). When the final volume of 4µl was reduced to 1µl/g rat body weight, the LSAE dose that neutralized 2LD50 was reduced to 20mg/100g body weight per 50µl (Protocol I, Table 3).

TABLE 1.
Fifty percent lethal dose (LD50) of C. d. terrificus venom injected i.m. in a volume of 2µl/g body weight (12 hours).

LD₅₀ = 180mg/100g rat body weight/200ml saline (p<0.05: 161 to 202mg/100g rat body weight/ 200ml saline).

TABLE 2.
Fifty percent lethal dose (LD50) of C. d. terrificus venom injected i.m. in a volume of 0.5µl/g body weight (12 hours).

LD₅₀ = 120mg/110g rat body weight/50ml saline (p<0.05: 107 to 134mg/100g rat body weight/50ml saline).

TABLE 3.
Doses of P. fuchsiaefolia extract that neutralized the lethality of C. d. terrificus venom in a final volume of 4µl and 1µl/g rat body weight.

(a) The extracts were injected 20 seconds after i.m. venom injection. (b) The extract was incubated with the venom for 60 minutes at room temperature before being injected i.m.

When the 2LD50/200µl and 50µl of saline were incubated with 200µl and 50µl of saline for a total final volume of 400µl and 100µl of saline, rat mortality was 66.7% within 8 hours and 100% within 12 hours (Protocol II, Table 3). The dose of 40 mg LSAE/100g rat body weight/200µl mixed and incubated for 1 hour at 25ºC with the venom (200µl) before being injected i.m. neutralized the 2LD50 lethal activity(2). However, the LSAE dose that neutralized the mortality of 2LD50 was reduced to 2.5mg LSAE/100g rat body weight/50µl when mixed and incubated with the venom (50µl) for 1 hour before being injected i.m. (Protocol II, Table 3).

DISCUSSION

This work was carried out in order to investigate the influence of the injected volume on the LD50 of C. d. terrificus venom and on the antivenom action of P. fuchsiaefolia. The venom was injected intramuscularly because C. d. terrificus has solenoglyph teeth that reach the victim in the muscular and/or subcutaneous region(6). In addition, considering the folkloric belief that the plant is laid on the snakebite site, the extract of P. fuchsiaefolia was injected into the same site of the snakebite.

Literature data showed that venom doses of 5 to 21.5µg were injected into the thigh of mice (18 to 22g) for the calculation of i.m. LD50 in a volume of 10µl/g(13,17). Based on these data, the tests were started with a final volume of 4µl/g rat body weight. The results were not reproducible under these conditions. Thus, tests with Evans blue solution (initially 2 and 4µl/g rat body weight) were performed. The extravasation of the Evans blue solution into the subcutaneous region clearly indicated that the injected volume was too large. The experiments were repeated with a final volume of 1µl/g rat body weight which caused no extravasation in any of the animals tested. Using this volume, reproducible results were obtained and a significant reduction of the LD50 and of the extract doses that neutralized the lethality of the venom was observed.

Our data show that the i.m. LD50 of C. d. terrificus venom is volume dependent (Table 1 and Table 2), and therefore, the same weight of venom may produce different effects as a function of the injected volume. This observation was first made by Vital Brazil(18) when he assembled in a single table the s.c. LD50 and LD100 of the Elapidae and Viperidae venoms, showing that these values were subject to several variables such as: injected volume, injected site, animal breed and age and even the number of utilized injected animals. We can add one more observation to the ones above mentioned, namely: the composition of the venom pool which is now known to depend on its geographical origin, age, feeding and other factors regarding the snakes themselves(19). Gombarosvts et al.(4) made biochemical, pharmacological and chromatograpic comparisons of individual venoms of C. d. terrificus from several cities of the states of Rio de Janeiro and Minas Gerais. Phospholipase A2, proteolytic and platelet aggregating activities were significantly different due to different proportions of crotoxin, crotamine, convulxin and giroxin in each sample. In the present work, a single pool of venom was used and all variables except the injected volume were kept constant.

Extravasation of venom and extract solutions into the subcutaneous region during injection in a volume of 4µl/g rat body weight explains the increase in their effective mass at this larger volume. Table 3shows that the stabilization of the ratio of dead rats to total number of animals per group occurred within 48 hours for the total volume of 4 µl/g rat body weight and 24 hours for the total volume of 1µg/g rat body weight. The increased time for the venom action in the presence of the LSAE extract can also be attributed to extravasation of both solutions into the subcutaneous region.

ACKNOWLEDGEMENTS

The authors are indebted to Profa. Dra. Diones A. Dias, Prof. Dr. Wiliam A. do Prado and Prof. Dr. João J. Lachat for their assistance and to Fernanda Araújo, João J. Franco, Tatiane de C. Izidoro and Vânia B. de Rezende for their collaboration. Research supported by CAPES, FAPESP and CNPq.

19 WILLEMSE, GT. Individual variations in snake venom. Comp. Biochem. Physiol., 1978, 61B, 553-7

Received 01 June 1996

Accepted 26 July 1996

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• ¹⁹
  19 WILLEMSE, GT. Individual variations in snake venom. Comp. Biochem. Physiol., 1978, 61B, 553-7  

CORRESPONDENCE TO:

J. R. GIGLIO - Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo, CEP 14049-900, Ribeirão Preto, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997

History

• Received
  01 June 1996
• Accepted
  26 July 1996