Snakebites and ethnobotany in the northwest region of Colombia

Journal of Ethnopharmacology 71 (2000) 505 – 511 www.elsevier.com/locate/jethpharm

Snakebites and ethnobotany in the northwest region of Colombia Part II: Neutralization of lethal and enzymatic effects of Bothrops atrox venom R. Otero a,b,*, V. Nu´n˜ez a, S.L. Jime´nez a, R. Fonnegra a, R.G. Osorio a, M.E. Garcı´a a, A. Dı´az a a

b

Programa de Ofidismo, Facultad de Medicina, Uni6ersidad de Antioquia, A.A. 1226, Medellı´n, Colombia Departamento de Pediatrı´a, Facultad de Medicina, Uni6ersidad de Antioquia, A.A. 1226, Medellı´n, Colombia Received 2 September 1999; received in revised form 29 September 1999; accepted 23 November 1999

Abstract Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18 – 20 g). After preincubation of sublethal doses of every extract (0.5 – 4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD50) (99.3 mg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia ( Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45–80%; 10–30% survival rate in the control group receiving the venom alone; P B0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 mg= 1.5 i.m. LD50), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A2 activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 mg) in agarose-erythrocyte-egg yolk gels. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neutralization; B. atrox venom; Plant extracts; Colombia

* Corresponding author. Tel.: + 57-4-2631914; fax: + 57-4-2638282. E-mail address: [email protected] (R. Otero). 0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 9 7 - X

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ological importance in the region. (Otero et al., 1996 Otero-Patin˜o et al., 1998)

1. Introduction Antioquia and Choco´, north-western Colombia, have a large reserve of tropical rain forest and biodiversity. Two million people live in rural communities of this region and are highly exposed to snakebites. Unfortunately, antivenoms and medical resources are limited in such rural places, so patients cannot seek an adequate and rapid attention of those envenomations. As a consequence, 60% of snakebite victims are initially attended by traditional healers, who use aqueous or ethanolic extracts from at least 100 different plant species, administered per oral or cutaneous route (Otero et al., 1992a; unpublished observations; Otero et al., 1992b). Although plants are known to be the origin of several drugs used in modern medicine (Gowda, 1997), the validity of traditional treatments must rigorously be evaluated in well designed experiments. In this work, the ethanolic extracts of 74 different species of plants from Antioquia and Choco´, used by shamans of the region for snakebites, were evaluated for their neutralizing ability against lethal and enzymatic effects of Bothrops atrox venom, the snake species of major epidemi-

2. Materials and methods

2.1. Plants and extracts preparation The plants were collected in rural places from Antioquia and Choco´ after being indicated by traditional healers and then, they were identified and deposited in the Herbarium at the Universidad de Antioquia (HUA) in Medellı´n. After drying and crushing individual samples of the relevant part of each species used, they were percolated with 96% ethanol for 2 days. Extracts were concentrated to a semisolid paste using a BU8 CHI R-124 rotavapor (Flawil, Switzerland), lyophilized for 4 days and stored at − 20°C until used (Weniger, 1991).

2.2. Mice and 6enom Swiss Webster mice 18–20 g body weight were used for the studies of acute extract or venom toxicity and in the experiments of venom neutral-

Table 1 Plants active against lethal effect of Bothrops atrox venom: experiments with preincubation of extract and venoma Plant species

Extract (mg/mouse)

Mice survival rate (%)/time 6h

Bixa orellana L. Brownea rosademonte Berg. Citrus limon (L.) Burm.f. Costus lasius Loes Dracontium croatii Zhu. Heliconia curtispatha Petersen Pleopeltis percussa (Cav.) Hook & Grev. Renealmia alpinia (Rottb.) Maas Sida acuta Burm.f. Struthanthus orbicularis (H.B.K.) Blume Tabebuia rosea (Bertold.) DC. Trichomanes elegans L.C. Rich

1.0 2.0 4.0 2.0 4.0 2.0 2.0 2.0 2.0 2.0 4.0 2.0

24 h

48 h

V+E

V

V+E

V

V+E

V

90910 100 100 60 9 10 65 9 20 100 100 100 70920 60 910 100 100

30 9 10 10 209 10 30 9 10 15 910 10 25 9 10 10 10 9 10 30 9 10 20 920 15910

80 910 100 100 60910 50 9 10 100 100 100 60 9 20 609 10 100 100

10 9 10 10 15 9 10 309 10 10 10 15 910 10 10 910 20 9 10 15 9 10 10 910

80 910 100 100 60 9 10 45 910 100 100 100 60 9 20 60 100 100

10 9 10 10 15 9 10 30 9 10 10 10 15 910 10 10 910 20 910 15 9 10 10

a All the extracts were tested against 1.5 i.p. lethal dose 50% (LD50) (99.3 mg) of venom, and the mixture was injected i.p. into mice (18–20 g) after preincubation at 37°C during 30 min. Results are expressed as mean 9S.D of three determinations. V+E: groups of mice receiving the mixture of venom and extract. V: groups of mice receiving venom alone.

R. Otero et al. / Journal of Ethnopharmacology 71 (2000) 505–511 Table 2 Neutralizing potency of the extracts against lethal effect of Bothrops atrox venom from Antioquia and Choco´a Plant extract

ED50 (mg/mouse)b

ED100 (mg/mouse)c

Brownea rosademonte Berg. Citrus limon (L.) Burm.f. Heliconia curtispatha Petersen Pleopeltis percussa (Cav.) Hook & Grev. Renealmia alpinia (Rottb.) Maas Tabebuia rosea (Bertold.) DC. Trichomanes elegans L.C. Rich

0.14 (0.08–0.25)a 1.5 (0.8–2.7)c

0.25

0.46 (0.26–0.81)b 0.35 (0.20–0.62)b 0.84 (0.48–1.46)b 0.81 (0.45–1.45)b 0.95 (0.55–1.67)b

1.0

4.0

1.0 2.0 4.0 2.0

a Experiments performed by preincubating venom (1.5 i.p. lethal dose 50% (LD50)= 99.3 mg) and several doses of every extract. Then, mice (18–20 g) were injected i.p. (See Section 2). Results are expressed as mean of three experiments. Values in parentheses are 95% confidence limits. Values with different superscripts are significantly different (PB0.05). b Effective dose 50% (ED50) is the extract dose that protects 50% of mice population against lethal effect of venom. c Effective dose 100% (ED100) is the extract dose that protects 100% of mice population against lethal effect of venom.

ization. B. atrox venom was obtained by milking more than 40 specimens captured in the study region. Then, venom was centrifuged, supernatant lyophilized and stored at − 20°C until used.

2.3. Acute toxicity of the extracts and 6enom The lethal dose 50% (LD50) was determined by the Spearman–Karber method (WHO, 1981), using groups of four mice injected per i.p. route with variable doses of either every extract or B. atrox venom, dissolved in 0.5 ml phosphate buffered saline (PBS) pH 7.2. In another experiment, mice were injected into the right gastrocnemius muscle with variable doses of B. atrox venom dissolved in 0.1 ml PBS pH 7.2, in order to obtain the i.m. LD50 of venom. Deaths were registered within 48 h and results were the mean of three experiments.

507

All the extracts had a LD50 above 5.0 mg/ mouse. There were no deaths at doses below 2.5 mg for any extract. The LD50 values of B. atrox venom were 66.2 and 136 mg/mouse per i.p. and i.m. route, respectively.

2.4. Neutralization of the lethal effect of 6enom 2.4.1. In 6itro experiments Sublethal doses (0.5–4.0 mg/mouse) of each extract were preincubated at 37°C during 30 min with 99.3 mg of B. atrox venom (1.5 i.p. LD50) dissolved in 0.5 ml PBS pH 7.2 and the mixture was injected i.p. to groups of ten mice. In all the experiments, a control group of ten mice received the venom alone. The experiments were repeated three times for every extract and mortality was registered at 6, 24 and 48 h. To obtain the effective doses 50 and 100% (ED50 –ED100) of the extracts with neutralizing capacity, the Spearman–Karber method (WHO, 1981) was followed. Groups of four mice were injected i.p. with a preincubated mixture of variable doses of the extract and fixed doses of venom (99.3 mg). 2.4.2. In 6i6o experiments The extracts that demonstrated in vitro neutralizing ability against the lethal effect of venom, were then independently administered to groups of ten mice at different times, doses and routes as following: 1. 1.0, 2.0 and 4.0 mg of each extract dissolved in 50 ml distilled water were administered per oral route 60 min before or 5 min after the venom injection (99.3 mg i.p. or 204 mg i.m. = 1.5LD50). 2. 0.5, 1.0 and 2.0 mg of each extract dissolved in 0.2 ml PBS pH 7.2, were injected i.v. 15 min before or 5 min after the venom injection (99.3 mg i.p. or 204 mg i.m.). 3. 0.5, 1.0 and 2.0 mg of each extract dissolved in 0.1 ml PBS pH 7.2, were injected into the left gastrocnemius muscle 60 min before or 5 min after the venom injection (204 mg i.m) into the right leg. 4. 1.0 and 2.0 mg of each extract dissolved in 0.1 ml PBS pH 7.2 were immediately injected by

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i.m. route (in situ) after the venom injection into the right leg (204 mg). 5. 1.0, 2.0 and 4.0 mg of each extract dissolved in 0.5 ml PBS pH 7.2 were injected i.p. either 60 min before or 5 min after the venom injection (204 mg i.m.) into the right gastrocnemius muscle. The mortality was estimated at 6, 24 and 48 hr after the venom injection for all groups. The experiments were repeated three times for each extract. There were also two control groups that received either the venom or the extract alone.

2.5. Neutralization of the indirect hemolytic acti6ity of 6enom The agarose-erythrocyte-egg yolk assay was used as described by Gutie´rrez et al. (1988). One minimum indirect hemolytic dose (MIHD) of B. atrox venom is the dose (2 mg) that induces a 20 mm diameter hemolysis halo after 20 h of incubation at 37°C (Otero et al., 1995). Each extract (25 – 1600 mg) was tested against one MIHD of venom and results were expressed as ED50, defined as the extract dose that reduced 50% the activity of the

venom alone. The experiments were perfomed by duplicate for all the extracts.

2.6. Statistical analysis The survival rate in the groups of mice that received venom and extract was compared for different times with that of controls (venom alone) using Gehan’s Wilcoxon test from the computer program STATISTICA 4.6 (StatSoft, Tulsa, OK). Differences were significant when aB 0.05. For the ED50 values, the significance of the differences observed was studied by analysis of variance (ANOVA). When the values were significantly different (PB0.05) by ANOVA, the significance of the differences was determined by the Newman– Keuls test.

3. Results

3.1. Neutralization of the lethal effect of 6enom 3.1.1. In 6itro experiments At the doses used, only seven extracts demon-

Table 3 Neutralization of Bothrops atrox venom lethal effect in experiments with independent administration of extract and venoma Plant extract

Mice survival rate (%)/time

Dose, route and time

6h

24 h

48 h

V+E

V

V+E

V

V+E

V

Bixa orellana L. 1.0 mg oral, 60 min before 1.0 mg i.p., 60 min before

959 10 75 9 20

70 9 20 709 15

50 910 50 915

10 910 15 910

40 910 40 910

10 910 10 910

Citrus limon (L.) Burm.f. 4.0 mg oral, 60 min before 4.0 mg i.p., 60 min before 1.0 mg i.v., 15 min before 4.0 mg i.p., 5 min after

70 9 30 95 9 10 809 30 859 5

65 9 15 759 15 759 10 509 20

35 910 55 95 40 910 65 910

10 910 10 910 15 95 1595

30 910 35 910 30 50

10 910 5 95 10 910 10 910

Tabebuia rosea (Bertold.) DC. 4.0 mg i.p., 60 min before 1.0 mg i.v., 15 min before

809 20 759 30

659 15 50930

45 9 15 40

10 9 10 10 9 5

30 30 9 5

595 10 9 5

Trichomanes elegans L.C. Rich 2.0 mg i.p., 60 min before

859 5

60

40

10

25 9 5

10

a All the extracts were tested against an i.m. venom injection (204 mg = 1.5 i.m. lethal dose 50% (LD50)). Results are expressed as mean9 S.D of three determinations. V+E: groups of mice receiving venom and extract; V: groups of mice receiving venom alone.

R. Otero et al. / Journal of Ethnopharmacology 71 (2000) 505–511 Table 4 Neutralization of the indirect hemolytic activity of Bothrops atrox venom by extracts of plantsa Plant extract

Effective dose 50% (ED50) (mg)

Bixa orellana L. Brownea rosademonte Berg. Costus lasius Loes Gonzalagunia panamensis (Cav.) Schumm Heliconia curtispatha Petersen Piper arboreum Aublet Pleopeltis percussa (Cav.) Hook & Grev. Renealmia alpinia (Rottb.) Maas Senna dariensis (Br. & R.) I. & B. Struthanthus orbicularis (H.B.K.) Blume Strychnos xinguensis Krukoff Tabebuia rosea (Bertold.) DC. Trichomanes elegans L.C. Rich

44 46 827 1153 206 869 65 939 490 485 1113 576 90

a All the extracts were tested against one minimum indirect hemolytic dose (MIHD) (2 mg) of B. atrox venom, in agaroseerythrocyte-egg yolk gels. Experiments performed by duplicate.

strated 100% neutralization against 1.5 LD50 of B. atrox venom: the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia ( Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae) (Table 1). Additionally, other five extracts showed partial protection of mice, with a mean survival rate varying from 45 to 80% at 48 h (10–30% in the control group; P B 0.05): leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae) (Table 1). The neutralizing ability of B. orellana at doses above 1.0 mg/mouse and that of S. orbicularis, S. acuta and C. lasius extracts at doses above 2.0 mg/mouse, significantly diminished indicating higher toxicity in the mixture when compared

509

with the extracts alone (results not shown). The highest neutralizing potency against 1.5 i.p. LD50 of B. atrox venom was that of B. rosademonte extract (ED50 = 0.14 mg/mouse; ED100 = 0.25 mg/mouse) (Table 2).

3.1.2. In 6i6o experiments When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 mg = 1.5 i.m. LD50), C. limon, T. elegans, B. orellana and T. rosea extracts had a low but significant (P B0.05) protective effect (Table 3). C. limon and T. rosea extracts were also partially effective when they were administered i.v. 15 min before an i.m. venom injection, and the former protected 50% of mice when it was injected i.p. 5 min after the venom injection (Table 3). 3.2. Neutralization of the indirect hemolytic acti6ity of 6enom Thirteen of 74 extracts demonstrated neutralizing activity against one MIHD of B. atrox venom, being highest for B. orellana and B. rosademonte extracts (Table 4). The extracts of C. limon, D. croatii and S. acuta did not neutralize this effect of venom.

4. Discussion More than 700 plants have been reported as used in folk medicine in the world for snakebites (Houghton and Osibogun, 1993; Reyes and Jime´nez, 1995). Nevertheless, only a few have been evaluated in well controlled assays and about 30 of them have been effective against snake venoms, mainly from Elapidae and Viperidae families (Martz, 1992; Houghton and Osibogun, 1993; Alam et al., 1994). In experiments performed with preincubation of extract and venom (in vitro), the extract and constituents of Eclipta prostrata diminished the lethality and myotoxicity of Crotalus durissus terrificus venom (Mors et al., 1989), as well as the myotoxic and hemorrhagic effects of B. jararaca, B. jararacussu and Lachesis muta snake venoms (Melo et al.,

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1994). The root extracts of Hemidesmus indicus, Pluchea indica, Vitex negundo, Emblica officinalis and Aristolochia indica neutralized the lethal and hemorrhagic effects of Vipera russellii, Echis carinatus, Naja naja and Ophiophagus hannah venoms (Alam et al., 1994; Alam and Gomez, 1995). In this work, seven plants (B. rosademonte, R. alpinia, H. curtispatha, P. percussa, C. limon, T. elegans and T. rosea) whose ethanolic extracts completely in vitro neutralized the lethal effect of B. atrox venom from Antioquia and Choco´ are reported. Five other extracts (C. lasius, S. orbicularis, B. orellana, D. croatii, S. acuta) partially neutralized the venom. All of them there were not been previously reported as effective against snake venoms, likewise contributing to increase the scientific knowledge of anti-snakebite treatments of our folk medicine. The extract of Alocasia cucullata, previously reported with neutralizing capacity against lethal effect of N. naja and O. hannah venoms, but ineffective against Agkistrodon halys venom (Martz, 1992), had no neutralizing ability against the lethal effect of B. atrox venom in this work. In experiments performed with independent administration of extract and venom (in vivo), other authors demonstrated that the extracts of Phyllanthus klotzschianus, Casearia syl6estris and Apuleia leiocarpa 100% neutralized the lethal effect of B. jararaca venom when mice were oral pretreated with the extracts (Martz, 1992; Pereira et al., 1994). The extract of Diodia scandens i.p. injected into mice neutralized the lethal effect of E. carinatus venom and that of Andographis paniculata showed a life-prolonging effect against N. naja venom (Martz, 1992). The compound HI-RVIF, isolated from the root extract of H. indicus and administered by i.v. route, significantly neutralized lethal, hemorrhagic and defibrinogenating activities of V. russellii venom (Alam et al., 1994). In this study, four plant extracts (B. orellana, C. limon, T. rosea, T. elegans) were partially effective when mice were pretreated either by i.p. injection or by oral administration of the extracts; C. limon extract also protected 50% of mice when it was administered i.p. five min after an i.m. venom

injection. Except for T. elegans, the other three plants are very common flora in the study region and would be good tools to search for alternative remedies for snakebites. Phospholipase A2 (PLA2) enzymes are important constituents of snake venoms. They are responsible for several pharmacological effects such as neurotoxic, myotoxic, cardiotoxic, anticoagulant, hemorrhagic, hemolytic, edema-inducing, convulsant, hypotensive and platelet aggregation activities (Gowda, 1997). On the other hand, some constituents of plants have the ability to bind proteins and inhibit their enzymatic activity as well as the catalytic activity of snake venoms PLA2 (Gowda, 1997; Melo and Ownby, 1999). The results demonstrated that 13 of the 74 plant extracts tested had in vitro neutralizing effect against the indirect hemolytic activity of B. atrox venom, indicating inhibition of venom PLA2 enzymes.

Acknowledgements We thank the staff of the Hospitals, local governmental authorities and the communities of the study region; to the traditional healers: Emiliano Palacio, Emiliano Palacio Jr., Basiliso Alvarez and Alberto Chaverra from Bojaya´; Dora Blando´n, Zoilo Sa´nchez and Rosaura Gutie´rrez from Vigı´a del Fuerte; Arnulfo Arango, Leandro Palomeque and Juan de Dios Rojas from Nuquı´; Menedesmo Valoy, Francisco Valoy, Gabriel Conde and Isidro Alvarado from Bahı´a Solano; Anı´bal Padilla, Hilario Ramı´rez, Angel Padilla, Julio Galvis, Darı´o Ma´rquez and Angel Salazar from Unguı´a; to the Direccio´n Seccional de Salud del Choco´, Ministerio del Medio Ambiente and the personnel of the Utrı´a and Katı´os National Natural Parks for their collaboration; to Jorge Enrique Asprilla for the laboratory support and Elizabeth Cadavid for preparing the manuscript; the financial support of the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologı´a Francisco Jose´ de Caldas (Colciencias) and the Universidad de Antioquia.

R. Otero et al. / Journal of Ethnopharmacology 71 (2000) 505–511

References Alam, M.I., Gomez, A., 1995. Indian medicinal plants active against Elapidae and Viperidae snake venoms. In: Abstracts First International Congress on Envenomations and their Treatments. Institut Pasteur, Parı´s. Alam, M.I., Auddy, B., Gomez, A., 1994. Isolation, purification and partial characterization of viper venom inhibiting factor from the root extract of the indian medicinal plant sarsaparilla (Hemidesmus indicus R. Br.). Toxicon 32, 1551 – 1557. Gowda, T.V., 1997. Interaction of snake venom phospholipases A2 with plant isolates. In: Kini, R.M. (Ed.), Venom Phospholipase A2 Enzymes. Wiley, Chichester. Gutie´rrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in 6itro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411 – 413. Houghton, P.J., Osibogun, I.M., 1993. Flowering plants used against snakebite. Journal of Ethnopharmacology 39, 1– 29. Martz, W., 1992. Plants with a reputation against snakebite. Toxicon 30, 1131 – 1142. Melo, P.A., Ownby, C.L., 1999. Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins. Toxicon 37, 199–215. Melo, P.A., Nascimento, M.C., Mors, W.B., Suarez-Kurtz, G., 1994. Inhibition of the myotoxic and hemorrhagic activities of crotalid venoms by Eclipta prostrata (Asteraceae) extracts and constituents. Toxicon 32, 595–603. Mors, W.B., Do Nascimento, M.C., Parente, J.P., Da Silva, M.H., Melo, P.A., Suarez-Kurtz, G., 1989. Neutralization of lethal and myotoxic activities of South American rattlesnake venom by extracts and constituents of the plant Eclipta prostrata (Asteraceae). Toxicon 27, 1003–1009. Otero, R., Valderrama, R., Osorio, R.G., Posada, L.E., 1992b. Programa de atencio´n primaria del accidente ofı´dico. Una propuesta para Colombia. Iatreia 5, 96–102.

.

511

Otero, R., To´bon, G.S., Go´mez, L.F., Osorio, R.G., Valderrama, R., Hoyos, D., Urreta, J.E., Molina, S., Arboleda, J.J., 1992a. Accidente ofı´dico en Antioquia y Choco´. Aspectos clı´nicos y epidemiolo´gicos (marzo de 1989 – febrero de 1990). Acta Me´dica Colombiana 17, 229 – 249. Otero, R., Nu´n˜ez, V., Osorio, R.G., Gutie´rrez, J.M., Giraldo, C.A., Posada, L.E., 1995. Ability of six Latin American antivenoms to neutralize the venom of mapana´ equis (Bothrops atrox) from Antioquia and Choco´ (Colombia). Toxicon 33, 809 – 815. Otero, R., Gutie´rrez, J.M., Nu´n˜ez, V., Robles, A., Estrada, R., Segura, E., Toro, M.F., Garcı´a, M.E., Dı´az, A., Ramirez, E.C., Go´mez, G., Castan˜eda, J., Moreno, M.E., 1996. A randomized double-blind clinical trial of two antivenoms in patients bitten by Bothrops atrox in Colombia. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 696 – 700. Otero-Patin˜o, R., Cardoso, J.L.C., Higashi, H.G., Nu´n˜ez, V., Dı´az, A., Toro, M.F., Garcı´a, M.E., Sierra, A., Garcı´a, L.F., Moreno, A.M., Medina, M.C., Castan˜eda, N., SilvaDı´az, J.F., Murcia, M., Ca´rdenas, S.Y., Dias Da Silva, W.D., 1998. A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba´, Colombia. American Journal of Tropical Medicine and Hygiene 58, 183 – 189. Pereira, N.A., Pereira, B.M., Do Nascimento, M.C., Parente, J.P., Mors, W.B., 1994. Pharmacological screening of plants recommended by folk medicine as snake venom antidotes; IV. Protection against jararaca venom by isolated constituents. Planta Medica 60, 99 – 100. Reyes, R., Jime´nez, M., 1995. Quı´mica de las plantas alexı´teras. Interciencia 20, 257 – 264. Weniger, B., 1991. Theory and instrumentation involved with extraction, control, quality insurance and registration of natural products. In: First International Advanced Course on Technology and Control of Drugs, Perugia, pp. 31 – 40. World Health Organization, 1981. Progress in the Characterization of Venoms and Standardization of Antivenoms. WHO Offset Publication 58, 1 – 44.