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Snakebites and ethnobotany in the northwest region of Colombia
Journal of Ethnopharmacology 73 (2000) 233 – 241 www.elsevier.com/locate/jethpharm
Snakebites and ethnobotany in the northwest region of Colombia Part III: Neutralization of the haemorrhagic effect of Bothrops atrox venom R. Otero a,b,*, V. Nu´n˜ez a, J. Barona a, R. Fonnegra a, S.L. Jime´nez a, R.G. Osorio a, M. Saldarriaga 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 20 December 1999; received in revised form 22 July 2000; accepted 29 July 2000
Abstract Thirty-one of 75 extracts of plants used by traditional healers for snakebites, had moderate or high neutralizing ability against the haemorrhagic effect of Bothrops atrox venom from Antioquia and Choco´, north-western Colombia. After preincubation of several doses of every extract (7.8 – 4000 mg/mouse) with six minimum haemorrhagic doses (10 mg) of venom, 12 of them demonstrated 100% neutralizing capacity when the mixture was i.d. injected into mice (18–20 g). These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plants of Pleopeltis percussa (Polypodiaceae), Trichomanes elegans (Hymenophyllaceae) and Senna dariensis (Caesalpiniaceae); rhizomes of Heliconia curtispatha (Heliconiaceae); leaves and branches of Bixa orellana (Bixaceae), Philodendron tripartitum (Araceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the ripe fruits of Citrus limon (Rutaceae); leaves, branches and stem of Ficus nymphaeifolia (Moraceae). Extracts of another 19 species showed moderate neutralization (21 – 72%) at doses up to 4 mg/mouse, e.g. the whole plants of Aristolochia grandiflora (Aristolochiaceae), Columnea kalbreyeriana (Gesneriaceae), Sida acuta (Malvaceae), Selaginella articulata (Selaginellaceae) and Pseudoelephantopus spicatus (Asteraceae); rhizomes of Renealmia alpinia (Zingiberaceae); the stem of Strychnos xinguensis (Loganiaceae); leaves, branches and stems of Hyptis capitata (Lamiaceae), Ipomoea cairica (Convolvulaceae), Neurolaena lobata (Asteraceae), Ocimum micranthum (Lamiaceae), Piper pulchrum (Piperaceae), Siparuna thecaphora (Monimiaceae), Castilla elastica (Moraceae) and Allamanda cathartica (Apocynaceae); the macerated ripe fruits of Capsicum frutescens (Solanaceae); the unripe fruits of Crescentia cujete (Bignoniaceae); leaves and branches of Piper arboreum (Piperaceae) and Passiflora quadrangularis (Passifloraceae). When the extracts were independently administered by oral, i.p. or i.v. route either before or after an i.d. venom injection (10 mg), neutralization of haemorrhage dropped below 25% for all the extracts. Additionally, B. rosademonte and P. percussa extracts were able to inhibit the proteolytic activity of B. atrox venom on casein. * 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 ( 0 0 ) 0 0 3 2 1 - 4
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R. Otero et al. / Journal of Ethnopharmacology 73 (2000) 233–241
© 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neutralization; Haemorrhage; B. atrox venom; Plant extracts; Colombia
1. Introduction
2. Materials and methods
Pitvipers of the genus Bothrops are responsible for 90% of the snakebites in Latin America (Fan and Cardoso, 1995; Gutie´rrez, 1995). Bothrops atrox asper, a snake species widely distributed up to 1200 m altitude in Antioquia and Choco´, north-western Colombia, inflicts at least 50% of the bites in this region. Its venom induces conspicuous local and systemic effects such as swelling, haemorrhage, myonecrosis, haemostatic disorders and nephrotoxicity (Otero et al., 1992a,b, 1996; Otero-Patin˜o et al., 1998). For many centuries, traditional healers throughout the world have been using medicinal plants for the treatment of snakebites, some of those being indicated to alleviate swelling and haemorrhage (Houghton and Osibogun, 1993; Reyes and Jime´nez, 1995). The healers from the study region use at least 20 different aqueous or ethanolic extracts of plants as antihaemorrhagic therapy for Bothrops bites: H. curtispatha, B. rosademonte, Columnea pulcherrima Morton, C. kalbreyeriana, B. orellana, G. panamensis, Psychotria poeppigiana Muell-Ang., S. xinguensis, S. orbicularis, Costus guanaiensis Rusby. var. macrostrobilus (K. Sch.) Maas, N. lobata, Momordica charantia L., Quassia amara L., C. elastica, F. nymphaeifolia, Simaba cedron Planch., T. rosea, Odontocarya tenacissima Diels, O. micranthum and Episcia dianthiflora Moore & Wilson. Thus, extracts are administered either orally or by external application on the wound, or both (Otero et al., unpublished observations). In this work, the ethanolic extracts of 75 different plant species used by traditional healers from Antioquia and Choco´ for snakebites, were evaluated in their neutralizing ability against the haemorrhagic effect of B. atrox venom. Additionally, the neutralization of venom proteolytic activity was studied, looking for insights into the mechanism of action of plant extracts.
2.1. Plant and extract preparation Rural communities located near towns in the Departments of Antioquia (Vigı´a del Fuerte) and Choco´ (Bojaya´, Riosucio, Unguı´a, Nuquı´, Bahı´a Solano) were selected as study sites due to the prevalence of traditional medicine practices in the treatment of snakebites (Otero et al., 1992a). Interviews were held with traditional healers in all sites selected during two visits of 6 days duration each one in every place. Data on plant species, part used for snakebites and methods of extract preparation, were recorded. The plants (Table 1) were then collected together with the healers, 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, each was percolated with 96% ethanol for 2 days. Extracts were concentrated to a semisolid paste using a Bu¨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 wt. were used for the experiments of haemorrhagic activity of venom and extracts and venom neutralization. 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. Haemorrhagic acti6ity of the extracts and 6enom. The haemorrhagic activity was determined following the method of Kondo et al. (1960), modified by Gutie´rrez et al. (1981). Briefly, groups of four mice were injected by i.d. route in the
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Table 1 In vitro neutralization of B. atrox venom haemorrhagic effect by plants used in northwestern Colombia Family/species (voucher specimen)a
Part usedb
% Neutralizationc
Acanthaceae/Justicia secunda Vahl (RF 6408) Amaranthaceae/Achyranthes aspera L. (RF 6343) Apocynaceae/Allamanda cathartica L. (RF 6737) Araceae/Alocasia cucullata (Lour.) G. Don (RF 6239) Araceae/Dieffenbachia longispatha Engl. & Krause (RF 6450) Araceae/Dracontium croatii Zhu. (RF 6245) Araceae/Philodendron tripartitum (Jacq.) Schott. (RF6460) Aristolochiaceae/Aristolochia grandiflora Sw. (RF 6340) Aristolochiaceae/Aristolochia pilosa H. B. K. (RF 6454) Asteraceae/Adenostemma la6enia (L.) Ktze. (RF 6247) Asteraceae/Clibadium sil6estre (Aublet) Baill. (RF 6447) Asteraceae/Erechtites 6alerianaefolia (Wolf.) DC. (RF 6432) Asteraceae/Mikania guaco H.B.K. (RF 6412) Asteraceae/Neurolaena lobata (L.) K. & R. (RF 6406) Asteraceae/Pseudoelephantopus spicatus (Aubl.) Gleas (RF 6943) Bignoniaceae/Crescentia cujete L. (RF 6739) Bignoniaceae/Macfadyena unguiscati (L.) A. Gentry (RF 6734) Bignoniaceae/Tabebuia rosea (Bertold.) DC. (RF 6458) Bixaceae/Bixa orellana L. (RF 6485) Boraginaceae/Tournefortia cuspidata H. B. K. (RF 6420) Cactaceae/Pereskia bleo (H.B.K.) DC. (RO 4) Caesalpiniaceae/Brownea rosademonte Berg. (RF 6455) Caesalpiniaceae/Senna dariensis (Br. & R.) I. & B. (RF 6418) Convolvulaceae/Ipomoea cairica (L.) Sweet. (RF 6738) Costaceae/Costus guanaiensis Rusby. var. macrostrobilus (K. Sch.) Maas (RF 6251) Costaceae/Costus lasius Loes (RF 6244) Cucurbitaceae/Momordica charantia L. (RF 6407)
WP WP LF, RH WP RH LF, WP RT WP WP LF, WP LF, WP UF WP SB LF, LF, LF, SB WP LF, ST LF, LF, RF LF, WP LF, WP WP WP WP RH WP LF, LF, LF, ST LF, WP WP LF, LF, LF, LF, LF, WP LF, LF, LF,
0 992f 72 9 8b 18 95f 0 15 9 5f 100a 30 9 4e 13 95f 8 94f 12 94f 0 8 9 1f 44 9 5d 69 9 2b 21 93e 0 100a 100a 0 0 100a 100a 22 9 9e 0 18 9 5f 0
Euphorbiaceae/Phyllanthus acuminatus Vahl. (RF 6435) Fabaceae/Desmodium adscendens (Sw.) DC. (RF 6431) Gentianaceae/Irlbachia alata (Aubl.) Maas subsp. alata. (RF 6747) Gesneriaceae/Columnea kalbreyeriana Mast. (RF 6487) Gesneriaceae/Columnea pulcherrima Morton (RF 6417) Gesneriaceae/Episcia dianthiflora Moore & Wilson (RF 6461) Haemodoraceae/Xiphidium caeruleum Aublet. (RF 6731) Heliconiaceae/Heliconia curtispatha Petersen (RF 6486) Hymenophyllaceae/Trichomanes elegans L.C. Rich (RF 6744) Lamiaceae/Ocimum basilicum (L.) Willd. (RF 6730) Lamiaceae/Ocimum micranthum Willd. (RF 6940) Lamiaceae/Hyptis capitata Jacquin (RF 6732) Loganiaceae/Strychnos xinguensis Krukoff (RF 6729) Loranthaceae/Struthanthus orbicularis (H. B. K.) Blume (RF 6422) Malvaceae/Sida acuta Burm. F. (RF 6238) Menispermaceae/Odontocarya tenacissima Diels (RF 6445) Monimiaceae/Siparuna thecaphora (P. & E.) A. DC. (RF 6419) Moraceae/Castilla elastica Sesse´ (RF 6443) Moraceae/Ficus nymphaeifolia Miller (RF 6448) Passifloraceae/Passiflora quadrangularis L. (RF 6415) Phytolaccaceae/Peti6eria alliacea L. (RF 6241) Piperaceae/Peperomia elsana Trel. & Yun. (RF 6409) Piperaceae/Piper arboreum Aublet (RF 6413) Piperaceae/Piper auritum H.B.K. (RF 6426) Piperaceae/Piper hispidum Sw. (RF 6433)
BR, ST
BR
BR, ST BR, ST
BR BR, ST ST
BR, ST BR, ST BR, ST, BR BR
BR, ST BR, ST BR, ST BR
BR, ST BR, ST BR, ST BR BR BR BR, ST BR, ST
17 95f 19 9 2f 13 9 2f 33 97e 14 96f 19 9 8f 9 92f 100a 100a 6 93f 24 97e 24 9 3e 42 9 10d 100a 34 93e 0 50 913d 52 97d 100a 63 9 8c 14 9 4f 0 59 9 7c 10 94f 9 9 1f
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Table 1 (Continued) Family/species (voucher specimen)a
Part usedb
% Neutralizationc
Piperaceae/Piper longi6illosum T. & Y. (RF 6484) Piperaceae/Piper marginatum Jacq. (RF 6342) Piperaceae/Piper multipliner6ium C. DC. (RF 6434) Piperaceae/Piper peltatum L. [(= Pothomorphe peltata (L.) Miquel)] (RF 6441) Piperaceae/Piper pulchrum C. DC. (RF 6430) Piperaceae/Piper reticulatum L. (RF 6428) Piperaceae/Piper cf. Spoliatum Yun. (RF 6462) Piperaceae/Piper tricuspe DC. (RF 6237) Polypodiaceae/Pleopeltis percussa (Cav.) Hook & Grev. (RF 6410) Rubiaceae/Gonzalagunia panamensis (Cav.) Schumm. (RF 6405) Rubiaceae/Psychotria ipecacuanha (Brot.) Stokes. [( = Cephaelis ipecacuanha (Brot.) H. Rich)] (RF 6800) Rubiaceae/Psychotria poeppigiana Muell-Ang. (RF 6246) Rutaceae/Citrus limon (L.) Burm.f. (RF 6736) Scrophulariaceae/Lindernia diffusa (L.) Wettest (RF 6488) Scrophulariaceae/Scoparia dulcis L. (RF 6242) Selaginellaceae/Selaginella articulata (Kunze) Spring (RF 6457) Simaroubaceae/Quassia amara L. (RF 6442) Simaroubaceae/Simaba cedron Planch. (RF 6449) Solanaceae/Capsicum frutescens L. (RF 6345) Solanaceae/Solanum allophyllum (Miers.) Standl. (RF 6437) Solanaceae/Solanum nudum Dunal (RF 6429) Verbenaceae/Aegiphila panamensis Mold. (RF 6411) Zingiberaceae/Renealmia alpinia (Rottb.) Maas (RF 6456)
WP LF, LF, LF, LF, LF, LF, LF, WP LF, RT
0 3 9 3f 0 15 94f 44 9 16d 79 3f 0 0 100a 100a 3 9 2f
BR, BR, BR, BR, BR, BR, BR,
ST ST ST ST ST ST ST
BR, ST
LF, BR, ST 14 9 5f RF 100a WP 0 11 95f WP WP 38 9 6e WP, MR 0 WP, MCS 12 91f 58 9 6c RF LF, BR, ST 6 93f LF, BR, ST, FR 2 9 2f LF, BR, ST 0 RH 37 9 8e
a
Voucher specimen: RF, Ramiro Fonnegra and other (collectors); RO, Rafael Otero and other (collectors). Part used: LF, leaves; WP, the whole plant; BR, branches; ST, stem; RH, rhizomes; FR, fruits; UF, unripe fruits; RF, ripe fruits; RT, roots; SB, stem bark; MR, mainly roots; MCS, mainly crushed seeds. c Neutralization: variable doses of the extracts were preincubated with a fixed dose (10 mg = 6 MHD) of B. atrox venom at 37°C for 30 min (see Section 2). Results (mean 9 S.D. of three experiments) are the highest neutralization achieved at doses up to 4 mg/mouse. Values with different superscripts are significantly different (PB0.05). b
abdomen, with variable doses of either extract or B. atrox venom, dissolved in 0.1 ml phosphate buffered saline (PBS) pH 7.2. The control group received PBS alone in identical conditions. Two hours later, mice were sacrificed by inhalation of ether and the diameter of the haemorrhagic area was measured. The minimum haemorrhagic dose (MHD) was the lowest dose of venom that induced a haemorrhagic area of 10 mm diameter in 2 h (mean 9 S.D. of three experiments). Except for C. pulcherrima (Gesneriaceae) and I. cairica (Convolvulaceae), none of the extracts had haemorrhagic effect at doses up to 4 mg/mouse. The MHD of B. atrox venom was 1.6 9 0.6 mg/ mouse.
2.4. Neutralization of the haemorrhagic effect of 6enom 2.4.1. In 6itro experiments Several doses (7.8–4000 mg/mouse) of each extract were preincubated at 37°C during 30 min with 10 mg of B. atrox venom (6 MHD) dissolved in 0.1 ml PBS pH 7.2 and then, the mixture was injected i.d. to groups of four mice. A control group received the venom alone. The next steps were similar to those described above. The effective dose 50% (ED50) was the extract dose that reduced by 50% the diameter of the haemorrhagic area induced by the venom alone within 2 h (mean 9 S.D. of three experiments).
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ED50 was estimated by inverse regression from the fitted dose–response linear regression.
2.4.2. In 6i6o experiments The extracts that demonstrated 100% in vitro neutralizing ability against the haemorrhagic effect of venom, were then independently administered to groups of four mice at different times, doses and routes as follows: 1. 1.0, 2.0 or 4.0 mg of each extract dissolved in 50 ml distilled water were administered by oral route 60 min before the venom injection (10 mg i.d.). 2. 0.5, 1.0 or 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 (10 mg i.d.). 3. 1.0, 2.0 or 4.0 mg of each extract dissolved in 0.5 ml PBS pH 7.2, were injected i.p. 60 min before or 5 min after the venom injection (10 mg i.d.). Two hours later, mice were sacrificed by inhalation of ether and the haemorrhagic areas were measured. Results were expressed as percentage of neutralization (mean9 S.D. of three experiments), in relation to the haemorrhage induced by the venom alone (control group). 2.5. Neutralization of the proteolytic acti6ity of 6enom The proteolytic activity of B. atrox venom (10 – 250 mg/ml) was determined following the method of Lomonte and Gutie´rrez (1983) with minor modifications (Otero et al., 1992b), using 1% w/v casein as substrate. For the neutralization experiments, several doses (31.2 – 500 mg) of B. rosademonte or P. percussa extracts were preincubated for 30 min at 37°C with variable doses (10 – 250 mg/ml) of B. atrox venom. Casein (2 ml) in PBS pH 7.2 was then added to the mixture and newly incubated for 30 min at 37°C. The reaction was stopped by addition of 5% trichloroacetic acid. The mixture was centrifuged at 3000× g for 10 min and the absorbance of supernatant was measured at 280 nm. The experiments were repeated three times and results were expressed as mean9 S.E.
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2.6. Statistical analysis The mean values of venom activity neutralization by the extracts and its respective ED50 were compared by analysis of variance (ANOVA) from the computer program Statistica 4.6 (Statsoft, Tulsa, OK). When the F-test was significant (aB 0.05), the differences were determined by the Newman–Keuls test.
3. Results
3.1. Neutralization of the haemorrhagic effect of 6enom 3.1.1. In 6itro experiments Twelve of the 75 extracts demonstrated 100% neutralization against 6 MHD of B. atrox venom, i.e. the stem barks of B. rosademonte (Caesalpiniaceae) and T. rosea (Bignoniaceae); the whole plants of P. percussa (Polypodiaceae), T. elegans (Hymenophyllaceae) and S. dariensis ( Caesalpiniaceae); rhizomes of H. curtispatha (Heliconiaceae); leaves and branches of B. orellana (Bixaceae), P. tripartitum (Araceae), S. orbicularis (Loranthaceae) and G. panamensis (Rubiaceae); the ripe fruits of C. limon (Rutaceae); leaves, branches and stem of F. nymphaeifolia (Moraceae) (Tables 1 and 2). This neutralizing effect was dose-dependent for all the extracts (results not shown). The highest potency was that of B. rosademonte (ED50 = 169 3 mg/mouse; PB 0.05), and the lowest were those of F. nymphaeifolia, S. orbicularis, G. panamensis and T. rosea, with ED50 values ranging from 1171 to 1314 mg/mouse (Table 2). Extracts of another 19 species neutralized 21– 72% the haemorrhagic effect of B. atrox venom at doses up to 4 mg/mouse: the whole plants of A. grandiflora, C. kalbreyeriana, S. acuta, S. articulata and P. spicatus; rhizomes of R. alpinia; the stem of S. xinguensis; leaves, branches and stems of H. capitata, I. cairica, N. lobata, O. micranthum, P. pulchrum, S. thecaphora, C. elastica and A. cathartica; the macerated ripe fruits of C. frutescens; the unripe fruits of C. cujete; leaves and branches of P. arboreum and P. quadrangu-
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laris (Table 1). Low or negligible neutralization ( B 20%) was obtained with 27 extracts and no neutralization was observed in another 17. Columnea pulcherima and I. cairica, that induced mild haemorrhage at a dose of 4 mg, were otherwise partially effective to neutralize this effect of venom at a dose of 2 mg/mouse (Table 1).
3.1.2. In 6i6o experiments When the extracts were independently administered by oral, i.p. or i.v. route before an i.d. venom injection (10 mg), there was a significant drop in the neutralizing ability. This was below 25% for all of them and disappeared in some cases according to the extract, dose and route of administration (e.g. B. rosademonte 0.5 mg i.v. 15 min Table 2 Extracts of plants with total in vitro neutralizing capacity of B. atrox venom haemorrhagic effecta Plant species
Effective dose (mg/mouse) ED50
Brownea rosademonte Berg. Pleopeltis percussa (Cav.) Hook & Grev. Heliconia curtispatha Petersen Bixa orellana L. Trichomanes elegans L.C. Rich Citrus limon (L.) Burm. f. Philodendron tripartitum ( Jacq.) Schott. Ficus nymphaeifolia Miller Struthanthus orbicularis (H.B.K.) Blume Gonzalagunia panamensis (Cav.) Schumm Tabebuia rosea (Bertold.) DC. Senna dariensis (Br. & R.) I. & B.
1693b 1169 9c
ED100 62.5 500
198968cd 2609 28d 275935d 5309 127de 8109 113de
2000 2000 2000 2000 2000
11719 434ef 11829 280ef
4000 4000
12009 12f
4000
13149 160f B500*
4000 1000
a Variable doses of the extracts were preincubated with a fixed dose (10 mg/mouse=6 MHD) of B. atrox venom at 37°C during 30 min. Then, the mixtures were i.d. injected in the abdominal skin to groups of four mice (18–20 g). Two hours later, mice were sacrificed with ether and the haemorrhagic areas were measured. All the results are expressed as mean 9 S.D. of three experiments. Values with different superscripts (b–f) are significantly different (PB0.05). ED50, effective dose 50%; ED100, effective dose 100%. * Insufficient material.
before= 149 2%; B. orellana 0.5 mg i.p. 60 min before= 19%; F. nymphaeifolia 4 mg oral 60 min before= 239 5%). The rapid i.p. or i.v. injection of the extracts 5 min after an i.d. venom injection (10 mg), also had similar results (e.g. B. rosademonte 0.5 mg i.v. 5 min after = 13%; B. orellana 0.5 mg i.p. 5 min after= 139 1%; F. nymphaeifolia 2 mg i.v. 5 min after= 149 1%).
3.2. Neutralization of the proteolytic acti6ity of 6enom B. atrox venom had proteolytic activity on casein in a dose-dependent manner (Fig. 1). Both extracts tested demonstrated significant inhibition of the proteolytic effect of venom (PB 0.001), being higher (100%) when a low concentration of the latter (10 mg/ml) was used (Fig. 1). At high concentration of venom (250 mg/ml), B. rosademonte extract was more effective than that of P. percussa to neutralize this enzymatic activity of venom (PB 0.001). Extracts also demonstrated neutralizing ability in a dose-dependent manner (results not shown).
4. Discussion Haemorrhage is one of the most relevant signs of local and systemic envenoming by viperid and crotalid snakes. Various types of bleeding distant from the bite site are gingival haemorrhage, epistaxis, haemoptysis, haematuria, uterine bleeding, soft tissues haematomas and rarely intrathoracic or intrabdominal bleeding (Otero et al., 1992a; Kamiguti et al., 1996). Severe anemia and shock may arise as complications and haemorrhage in the central nervous system causes death and sequelae (Otero et al., 1992a). For this reason, one of the corner-stones of the treatment of those envenomations must be the inhibition of haemorrhage as indicative of venom neutralization (Otero et al., 1996; Otero-Patin˜o et al., 1998). During the last 20 years, other authors have reported the presence of antihaemorrhagic activity in several plant extracts and their constituents. Eclipta prostrata extract and its compound wedelolactone, in vitro neutralized B. jararaca, B.
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Fig. 1. Neutralization of proteolytic activity of B. atrox venom on casein. Venom (10 – 250 mg/ml) was preincubated at 37°C for 30 min with the extract A (B. rosademonte) or the extract B (P. percussa), 500 mg/ml. The mixture was then added to the substrate (1% w/v casein) and newly incubated for 30 min at 37°C. In all the experiments, the proteolytic activity of the venom alone was tested (control). Results (absorbances at 280 nm) are the mean 9 S.E. of three determinations.
jararacussu and Lachesis muta snake venoms haemorrhagic effect (Melo et al., 1994). A tannin extracted from Diospyros kaki neutralized the haemorrhage induced by Agkistrodon halys snake venom (Okonogi et al., 1979). The root extracts of Hemidesmus indicus, Pluchea indica, Vitex negundo, Emblica officinalis and Aristolochia indica in vitro and in vivo were able to neutralize the haemorrhagic effect of Vipera russellii, Echis carinatus, Naja naja and Ophiophagus hannah snake venoms; and the compound HI-RVIF isolated from the root extract of H. indicus and administered i.v. to mice, significantly neutralized lethal, haemorrhagic and defibrinogenating activities of V. russellii venom (Alam et al., 1994; Alam and Gomez, 1995). Our results demonstrated that 31 of the 75 plant extracts tested had in vitro moderate or high neutralizing ability against the haemorrhagic effect of B. atrox venom from Antioquia and Choco´, 12 of them being 100% effective (Tables 1 and 2). The extract of B. rosademonte, which had the highest neutralizing ability in this work (ED50 =1693 mg/mouse) (Table 2), also had the
highest potency (ED50 = 140 mg/mouse) to neutralize the lethal effect of venom (1.5 i.p. LD50 = 99.3 mg) in a previous study performed with in vitro experiments (Otero et al., unpublished observations). Twelve of the 31 plants have been reported by traditional healers from the region as having antihaemorrhagic properties, e.g. B. orellana, B. rosademonte, C. elastica, C. kalbreyeriana, F. nymphaeifolia, G. panamensis, H. curtispatha, N. lobata,O. micranthum, S. orbicularis, S. xinguensis and T. rosea. Snake toxins responsible for haemorrhage are a large group of metalloproteins with catalytic activity. They degrade capillary basal lamina components and they are also cytotoxic on endothelial cells opening gaps through which erythrocytes escape (Moreira et al., 1994; Kamiguti et al., 1996). As some plant constituents have the ability to bind snake venom proteins and inhibit their catalytic activity in a dose-dependent manner, an antiproteolytic mechanism has been proposed to explain the antihaemorrhagic effect of the plant extracts (Melo et al., 1994; Melo and Ownby, 1999). Our results also demonstrated that B.
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rosademonte and P. percussa extracts neutralized the proteolytic activity of B. atrox venom. Venom phospholipases A2 enzymes have also been implicated in the haemorrhage pathogenesis of snakebites (Gowda, 1997; Melo and Ownby, 1999). A recent study demonstrated that B. rosademonte, P. percussa, H. curtispatha, B. orellana, T. elegans, S. orbicularis, G. panamensis, T. rosea, S. dariensis, R. alpinia, P. arboreum, S. xinguensis and C. lasius had antiphospholipase A2 activity (Otero et al., unpublished observations). Nevertheless, C. limon that was devoid of antiphospholipase A2 activity in that study, has indeed antihaemorrhagic effect as was demonstrated herein (Tables 1 and 2). Other mechanisms of metalloprotein inhibition may be possible such as the presence of metal-chelator substances in the extracts, e.g. in B. orellana (Duke, 1998). The significant drop in the neutralization of the haemorrhagic effect when the extracts were independently administered either before or after an i.d. venom injection, may be in relation to the short time (4–6 min) required for erythrocyte extravasation from capillaries and small venules after tissue venom exposure (Lomonte et al., 1994). Furthermore, the kinetic of the absorption, distribution, metabolism and excretion of the extracts, as well as their concentration into the tissue where venom was injected, are still unknown. On the other hand, an antivenom administered i.v. immediately after an i.m. venom injection does not totally neutralize the haemorrhagic effect of B. asper venom (Gutie´rrez et al., 1981). Venom metalloproteinases have been implicated in systemic bleeding by their capacity to inhibit platelet aggregation, e.g. jararhagin from Bothrops jararaca affects the platelet collagen receptor a2b1 integrin and degrades the adhesive plasma protein von Willebrand factor (Kamiguti et al., 1996). Thus, these observations could explain the recognized feature that patients do not present systemic bleeding sooner than 30 – 60 min after the bite, the time required for the establishment of haemostatic alterations (Otero et al., 1992a). Additional experimental work will be necessary to test the ability of the extracts for the in
vivo neutralization of systemic bleeding induced by B. atrox venom, so validating the information obtained from traditional healers.
Acknowledgements We thank the staff of the hospitals, local governmental authorities, the communities in the region of study, the Direccio´n Seccional de Salud del Choco´, Ministerio del Medio Ambiente and the personnel of the Utrı´a and Katı´os National Parks for their cooperation. 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 we also express our thanks. Thanks go to Elizabeth Cadavid and Janeth Garcı´a for preparing the manuscript. This project was supported by funds from the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologı´a Francisco Jose´ de Caldas (Colciencias) and from the Universidad de Antioquia.
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, Paris. 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. Duke, J., 1998. Phytochemical and Ethnobotanical Databases. http://www.ars-grin.gov/cgi-bin/duke/ethnobotuse.pl Fan, H.W., Cardoso, J.L.C., 1995. Clinical toxicology of snakebite in South America. In: Meier, J., White, J. (Eds.), Handbook of Clinical Toxicology of Animal, Venoms and Poisons. CRC Press, Boca Raton, FL. Gowda, T.V., 1997. Interaction of snake venom phospholipases A2 with plant isolates. In: Kini, R.M. (Ed.), Venom Phospholipase A2 Enzymes. Wiley, Chichester, UK.
R. Otero et al. / Journal of Ethnopharmacology 73 (2000) 233–241 Gutie´rrez, J.M., 1995. Clinical toxicology of snakebite in Central America. In: Meier, J., White, J. (Eds.), Handbook of Clinical Toxicology of Animal, Venoms and Poisons. CRC Press, Boca Raton, FL. Gutie´rrez, J.M., Chaves, F., Bolan˜os, R., Cerdas, L., Rojas, E., Arroyo, O., Portilla, E., 1981. Neutralizacio´n de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente. Toxicon 19, 493–500. Houghton, P.J., Osibogun, I.M., 1993. Flowering plants used against snakebite. Journal of Ethnopharmacology 39, 1– 29. Kamiguti, A.S., Hay, C.R.M., Theakston, R.D.G, Zuzel, M., 1996. Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases. Toxicon 34, 627–642. Kondo, H., Kondo, S., Ikesawa, H., Murata, R., Ohsaka, A., 1960. Studies on the quantitative method for determination of haemorrhagic activity of Habu snake venom. Japanese Journal of Medical Science and Biology 13, 43–51. Lomonte, B., Gutie´rrez, J.M., 1983. La actividad proteolı´tica de los venenos de serpientes de Costa Rica sobre la caseı´na. Revista de Biologı´a Tropical 31, 37–40. Lomonte, B., Lundgren, J., Johansson, B., Bagge, U., 1994. The dynamics of local tissue damage induced by Bothrops asper snake venom and myotoxin II on the mouse cremaster muscle: an intravital and electron microscopic study. Toxicon 32, 41 – 55. 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. Moreira, L., Borkow, G., Ovadia, M., Gutie´rrez, J.M., 1994. Pathological changes induced by Ba H1, a hemorrhagic
.
241
proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels. Toxicon 32, 977 – 987. Okonogi, T., Hattori, Z., Ogiso, A., Mitsui, S., 1979. Detoxification by persimmon tannin of snake venoms and bacterial toxins. Toxicon 17, 524 – 527. 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., Osorio, R.G., Valderrama, R., Giraldo, C.A., 1992b. Efectos farmacolo´gicos y enzima´ticos de los venenos de serpientes de Antioquia y Choco´ (Colombia). Toxicon 30, 611 – 620. 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. 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.
Snakebites and ethnobotany in the northwest region of Colombia Part III: Neutralization of the haemorrhagic effect of Bothrops atrox venom R. Otero a,b,*, V. Nu´n˜ez a, J. Barona a, R. Fonnegra a, S.L. Jime´nez a, R.G. Osorio a, M. Saldarriaga 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 20 December 1999; received in revised form 22 July 2000; accepted 29 July 2000
Abstract Thirty-one of 75 extracts of plants used by traditional healers for snakebites, had moderate or high neutralizing ability against the haemorrhagic effect of Bothrops atrox venom from Antioquia and Choco´, north-western Colombia. After preincubation of several doses of every extract (7.8 – 4000 mg/mouse) with six minimum haemorrhagic doses (10 mg) of venom, 12 of them demonstrated 100% neutralizing capacity when the mixture was i.d. injected into mice (18–20 g). These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plants of Pleopeltis percussa (Polypodiaceae), Trichomanes elegans (Hymenophyllaceae) and Senna dariensis (Caesalpiniaceae); rhizomes of Heliconia curtispatha (Heliconiaceae); leaves and branches of Bixa orellana (Bixaceae), Philodendron tripartitum (Araceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the ripe fruits of Citrus limon (Rutaceae); leaves, branches and stem of Ficus nymphaeifolia (Moraceae). Extracts of another 19 species showed moderate neutralization (21 – 72%) at doses up to 4 mg/mouse, e.g. the whole plants of Aristolochia grandiflora (Aristolochiaceae), Columnea kalbreyeriana (Gesneriaceae), Sida acuta (Malvaceae), Selaginella articulata (Selaginellaceae) and Pseudoelephantopus spicatus (Asteraceae); rhizomes of Renealmia alpinia (Zingiberaceae); the stem of Strychnos xinguensis (Loganiaceae); leaves, branches and stems of Hyptis capitata (Lamiaceae), Ipomoea cairica (Convolvulaceae), Neurolaena lobata (Asteraceae), Ocimum micranthum (Lamiaceae), Piper pulchrum (Piperaceae), Siparuna thecaphora (Monimiaceae), Castilla elastica (Moraceae) and Allamanda cathartica (Apocynaceae); the macerated ripe fruits of Capsicum frutescens (Solanaceae); the unripe fruits of Crescentia cujete (Bignoniaceae); leaves and branches of Piper arboreum (Piperaceae) and Passiflora quadrangularis (Passifloraceae). When the extracts were independently administered by oral, i.p. or i.v. route either before or after an i.d. venom injection (10 mg), neutralization of haemorrhage dropped below 25% for all the extracts. Additionally, B. rosademonte and P. percussa extracts were able to inhibit the proteolytic activity of B. atrox venom on casein. * 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 ( 0 0 ) 0 0 3 2 1 - 4
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© 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neutralization; Haemorrhage; B. atrox venom; Plant extracts; Colombia
1. Introduction
2. Materials and methods
Pitvipers of the genus Bothrops are responsible for 90% of the snakebites in Latin America (Fan and Cardoso, 1995; Gutie´rrez, 1995). Bothrops atrox asper, a snake species widely distributed up to 1200 m altitude in Antioquia and Choco´, north-western Colombia, inflicts at least 50% of the bites in this region. Its venom induces conspicuous local and systemic effects such as swelling, haemorrhage, myonecrosis, haemostatic disorders and nephrotoxicity (Otero et al., 1992a,b, 1996; Otero-Patin˜o et al., 1998). For many centuries, traditional healers throughout the world have been using medicinal plants for the treatment of snakebites, some of those being indicated to alleviate swelling and haemorrhage (Houghton and Osibogun, 1993; Reyes and Jime´nez, 1995). The healers from the study region use at least 20 different aqueous or ethanolic extracts of plants as antihaemorrhagic therapy for Bothrops bites: H. curtispatha, B. rosademonte, Columnea pulcherrima Morton, C. kalbreyeriana, B. orellana, G. panamensis, Psychotria poeppigiana Muell-Ang., S. xinguensis, S. orbicularis, Costus guanaiensis Rusby. var. macrostrobilus (K. Sch.) Maas, N. lobata, Momordica charantia L., Quassia amara L., C. elastica, F. nymphaeifolia, Simaba cedron Planch., T. rosea, Odontocarya tenacissima Diels, O. micranthum and Episcia dianthiflora Moore & Wilson. Thus, extracts are administered either orally or by external application on the wound, or both (Otero et al., unpublished observations). In this work, the ethanolic extracts of 75 different plant species used by traditional healers from Antioquia and Choco´ for snakebites, were evaluated in their neutralizing ability against the haemorrhagic effect of B. atrox venom. Additionally, the neutralization of venom proteolytic activity was studied, looking for insights into the mechanism of action of plant extracts.
2.1. Plant and extract preparation Rural communities located near towns in the Departments of Antioquia (Vigı´a del Fuerte) and Choco´ (Bojaya´, Riosucio, Unguı´a, Nuquı´, Bahı´a Solano) were selected as study sites due to the prevalence of traditional medicine practices in the treatment of snakebites (Otero et al., 1992a). Interviews were held with traditional healers in all sites selected during two visits of 6 days duration each one in every place. Data on plant species, part used for snakebites and methods of extract preparation, were recorded. The plants (Table 1) were then collected together with the healers, 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, each was percolated with 96% ethanol for 2 days. Extracts were concentrated to a semisolid paste using a Bu¨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 wt. were used for the experiments of haemorrhagic activity of venom and extracts and venom neutralization. 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. Haemorrhagic acti6ity of the extracts and 6enom. The haemorrhagic activity was determined following the method of Kondo et al. (1960), modified by Gutie´rrez et al. (1981). Briefly, groups of four mice were injected by i.d. route in the
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Table 1 In vitro neutralization of B. atrox venom haemorrhagic effect by plants used in northwestern Colombia Family/species (voucher specimen)a
Part usedb
% Neutralizationc
Acanthaceae/Justicia secunda Vahl (RF 6408) Amaranthaceae/Achyranthes aspera L. (RF 6343) Apocynaceae/Allamanda cathartica L. (RF 6737) Araceae/Alocasia cucullata (Lour.) G. Don (RF 6239) Araceae/Dieffenbachia longispatha Engl. & Krause (RF 6450) Araceae/Dracontium croatii Zhu. (RF 6245) Araceae/Philodendron tripartitum (Jacq.) Schott. (RF6460) Aristolochiaceae/Aristolochia grandiflora Sw. (RF 6340) Aristolochiaceae/Aristolochia pilosa H. B. K. (RF 6454) Asteraceae/Adenostemma la6enia (L.) Ktze. (RF 6247) Asteraceae/Clibadium sil6estre (Aublet) Baill. (RF 6447) Asteraceae/Erechtites 6alerianaefolia (Wolf.) DC. (RF 6432) Asteraceae/Mikania guaco H.B.K. (RF 6412) Asteraceae/Neurolaena lobata (L.) K. & R. (RF 6406) Asteraceae/Pseudoelephantopus spicatus (Aubl.) Gleas (RF 6943) Bignoniaceae/Crescentia cujete L. (RF 6739) Bignoniaceae/Macfadyena unguiscati (L.) A. Gentry (RF 6734) Bignoniaceae/Tabebuia rosea (Bertold.) DC. (RF 6458) Bixaceae/Bixa orellana L. (RF 6485) Boraginaceae/Tournefortia cuspidata H. B. K. (RF 6420) Cactaceae/Pereskia bleo (H.B.K.) DC. (RO 4) Caesalpiniaceae/Brownea rosademonte Berg. (RF 6455) Caesalpiniaceae/Senna dariensis (Br. & R.) I. & B. (RF 6418) Convolvulaceae/Ipomoea cairica (L.) Sweet. (RF 6738) Costaceae/Costus guanaiensis Rusby. var. macrostrobilus (K. Sch.) Maas (RF 6251) Costaceae/Costus lasius Loes (RF 6244) Cucurbitaceae/Momordica charantia L. (RF 6407)
WP WP LF, RH WP RH LF, WP RT WP WP LF, WP LF, WP UF WP SB LF, LF, LF, SB WP LF, ST LF, LF, RF LF, WP LF, WP WP WP WP RH WP LF, LF, LF, ST LF, WP WP LF, LF, LF, LF, LF, WP LF, LF, LF,
0 992f 72 9 8b 18 95f 0 15 9 5f 100a 30 9 4e 13 95f 8 94f 12 94f 0 8 9 1f 44 9 5d 69 9 2b 21 93e 0 100a 100a 0 0 100a 100a 22 9 9e 0 18 9 5f 0
Euphorbiaceae/Phyllanthus acuminatus Vahl. (RF 6435) Fabaceae/Desmodium adscendens (Sw.) DC. (RF 6431) Gentianaceae/Irlbachia alata (Aubl.) Maas subsp. alata. (RF 6747) Gesneriaceae/Columnea kalbreyeriana Mast. (RF 6487) Gesneriaceae/Columnea pulcherrima Morton (RF 6417) Gesneriaceae/Episcia dianthiflora Moore & Wilson (RF 6461) Haemodoraceae/Xiphidium caeruleum Aublet. (RF 6731) Heliconiaceae/Heliconia curtispatha Petersen (RF 6486) Hymenophyllaceae/Trichomanes elegans L.C. Rich (RF 6744) Lamiaceae/Ocimum basilicum (L.) Willd. (RF 6730) Lamiaceae/Ocimum micranthum Willd. (RF 6940) Lamiaceae/Hyptis capitata Jacquin (RF 6732) Loganiaceae/Strychnos xinguensis Krukoff (RF 6729) Loranthaceae/Struthanthus orbicularis (H. B. K.) Blume (RF 6422) Malvaceae/Sida acuta Burm. F. (RF 6238) Menispermaceae/Odontocarya tenacissima Diels (RF 6445) Monimiaceae/Siparuna thecaphora (P. & E.) A. DC. (RF 6419) Moraceae/Castilla elastica Sesse´ (RF 6443) Moraceae/Ficus nymphaeifolia Miller (RF 6448) Passifloraceae/Passiflora quadrangularis L. (RF 6415) Phytolaccaceae/Peti6eria alliacea L. (RF 6241) Piperaceae/Peperomia elsana Trel. & Yun. (RF 6409) Piperaceae/Piper arboreum Aublet (RF 6413) Piperaceae/Piper auritum H.B.K. (RF 6426) Piperaceae/Piper hispidum Sw. (RF 6433)
BR, ST
BR
BR, ST BR, ST
BR BR, ST ST
BR, ST BR, ST BR, ST, BR BR
BR, ST BR, ST BR, ST BR
BR, ST BR, ST BR, ST BR BR BR BR, ST BR, ST
17 95f 19 9 2f 13 9 2f 33 97e 14 96f 19 9 8f 9 92f 100a 100a 6 93f 24 97e 24 9 3e 42 9 10d 100a 34 93e 0 50 913d 52 97d 100a 63 9 8c 14 9 4f 0 59 9 7c 10 94f 9 9 1f
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Table 1 (Continued) Family/species (voucher specimen)a
Part usedb
% Neutralizationc
Piperaceae/Piper longi6illosum T. & Y. (RF 6484) Piperaceae/Piper marginatum Jacq. (RF 6342) Piperaceae/Piper multipliner6ium C. DC. (RF 6434) Piperaceae/Piper peltatum L. [(= Pothomorphe peltata (L.) Miquel)] (RF 6441) Piperaceae/Piper pulchrum C. DC. (RF 6430) Piperaceae/Piper reticulatum L. (RF 6428) Piperaceae/Piper cf. Spoliatum Yun. (RF 6462) Piperaceae/Piper tricuspe DC. (RF 6237) Polypodiaceae/Pleopeltis percussa (Cav.) Hook & Grev. (RF 6410) Rubiaceae/Gonzalagunia panamensis (Cav.) Schumm. (RF 6405) Rubiaceae/Psychotria ipecacuanha (Brot.) Stokes. [( = Cephaelis ipecacuanha (Brot.) H. Rich)] (RF 6800) Rubiaceae/Psychotria poeppigiana Muell-Ang. (RF 6246) Rutaceae/Citrus limon (L.) Burm.f. (RF 6736) Scrophulariaceae/Lindernia diffusa (L.) Wettest (RF 6488) Scrophulariaceae/Scoparia dulcis L. (RF 6242) Selaginellaceae/Selaginella articulata (Kunze) Spring (RF 6457) Simaroubaceae/Quassia amara L. (RF 6442) Simaroubaceae/Simaba cedron Planch. (RF 6449) Solanaceae/Capsicum frutescens L. (RF 6345) Solanaceae/Solanum allophyllum (Miers.) Standl. (RF 6437) Solanaceae/Solanum nudum Dunal (RF 6429) Verbenaceae/Aegiphila panamensis Mold. (RF 6411) Zingiberaceae/Renealmia alpinia (Rottb.) Maas (RF 6456)
WP LF, LF, LF, LF, LF, LF, LF, WP LF, RT
0 3 9 3f 0 15 94f 44 9 16d 79 3f 0 0 100a 100a 3 9 2f
BR, BR, BR, BR, BR, BR, BR,
ST ST ST ST ST ST ST
BR, ST
LF, BR, ST 14 9 5f RF 100a WP 0 11 95f WP WP 38 9 6e WP, MR 0 WP, MCS 12 91f 58 9 6c RF LF, BR, ST 6 93f LF, BR, ST, FR 2 9 2f LF, BR, ST 0 RH 37 9 8e
a
Voucher specimen: RF, Ramiro Fonnegra and other (collectors); RO, Rafael Otero and other (collectors). Part used: LF, leaves; WP, the whole plant; BR, branches; ST, stem; RH, rhizomes; FR, fruits; UF, unripe fruits; RF, ripe fruits; RT, roots; SB, stem bark; MR, mainly roots; MCS, mainly crushed seeds. c Neutralization: variable doses of the extracts were preincubated with a fixed dose (10 mg = 6 MHD) of B. atrox venom at 37°C for 30 min (see Section 2). Results (mean 9 S.D. of three experiments) are the highest neutralization achieved at doses up to 4 mg/mouse. Values with different superscripts are significantly different (PB0.05). b
abdomen, with variable doses of either extract or B. atrox venom, dissolved in 0.1 ml phosphate buffered saline (PBS) pH 7.2. The control group received PBS alone in identical conditions. Two hours later, mice were sacrificed by inhalation of ether and the diameter of the haemorrhagic area was measured. The minimum haemorrhagic dose (MHD) was the lowest dose of venom that induced a haemorrhagic area of 10 mm diameter in 2 h (mean 9 S.D. of three experiments). Except for C. pulcherrima (Gesneriaceae) and I. cairica (Convolvulaceae), none of the extracts had haemorrhagic effect at doses up to 4 mg/mouse. The MHD of B. atrox venom was 1.6 9 0.6 mg/ mouse.
2.4. Neutralization of the haemorrhagic effect of 6enom 2.4.1. In 6itro experiments Several doses (7.8–4000 mg/mouse) of each extract were preincubated at 37°C during 30 min with 10 mg of B. atrox venom (6 MHD) dissolved in 0.1 ml PBS pH 7.2 and then, the mixture was injected i.d. to groups of four mice. A control group received the venom alone. The next steps were similar to those described above. The effective dose 50% (ED50) was the extract dose that reduced by 50% the diameter of the haemorrhagic area induced by the venom alone within 2 h (mean 9 S.D. of three experiments).
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ED50 was estimated by inverse regression from the fitted dose–response linear regression.
2.4.2. In 6i6o experiments The extracts that demonstrated 100% in vitro neutralizing ability against the haemorrhagic effect of venom, were then independently administered to groups of four mice at different times, doses and routes as follows: 1. 1.0, 2.0 or 4.0 mg of each extract dissolved in 50 ml distilled water were administered by oral route 60 min before the venom injection (10 mg i.d.). 2. 0.5, 1.0 or 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 (10 mg i.d.). 3. 1.0, 2.0 or 4.0 mg of each extract dissolved in 0.5 ml PBS pH 7.2, were injected i.p. 60 min before or 5 min after the venom injection (10 mg i.d.). Two hours later, mice were sacrificed by inhalation of ether and the haemorrhagic areas were measured. Results were expressed as percentage of neutralization (mean9 S.D. of three experiments), in relation to the haemorrhage induced by the venom alone (control group). 2.5. Neutralization of the proteolytic acti6ity of 6enom The proteolytic activity of B. atrox venom (10 – 250 mg/ml) was determined following the method of Lomonte and Gutie´rrez (1983) with minor modifications (Otero et al., 1992b), using 1% w/v casein as substrate. For the neutralization experiments, several doses (31.2 – 500 mg) of B. rosademonte or P. percussa extracts were preincubated for 30 min at 37°C with variable doses (10 – 250 mg/ml) of B. atrox venom. Casein (2 ml) in PBS pH 7.2 was then added to the mixture and newly incubated for 30 min at 37°C. The reaction was stopped by addition of 5% trichloroacetic acid. The mixture was centrifuged at 3000× g for 10 min and the absorbance of supernatant was measured at 280 nm. The experiments were repeated three times and results were expressed as mean9 S.E.
237
2.6. Statistical analysis The mean values of venom activity neutralization by the extracts and its respective ED50 were compared by analysis of variance (ANOVA) from the computer program Statistica 4.6 (Statsoft, Tulsa, OK). When the F-test was significant (aB 0.05), the differences were determined by the Newman–Keuls test.
3. Results
3.1. Neutralization of the haemorrhagic effect of 6enom 3.1.1. In 6itro experiments Twelve of the 75 extracts demonstrated 100% neutralization against 6 MHD of B. atrox venom, i.e. the stem barks of B. rosademonte (Caesalpiniaceae) and T. rosea (Bignoniaceae); the whole plants of P. percussa (Polypodiaceae), T. elegans (Hymenophyllaceae) and S. dariensis ( Caesalpiniaceae); rhizomes of H. curtispatha (Heliconiaceae); leaves and branches of B. orellana (Bixaceae), P. tripartitum (Araceae), S. orbicularis (Loranthaceae) and G. panamensis (Rubiaceae); the ripe fruits of C. limon (Rutaceae); leaves, branches and stem of F. nymphaeifolia (Moraceae) (Tables 1 and 2). This neutralizing effect was dose-dependent for all the extracts (results not shown). The highest potency was that of B. rosademonte (ED50 = 169 3 mg/mouse; PB 0.05), and the lowest were those of F. nymphaeifolia, S. orbicularis, G. panamensis and T. rosea, with ED50 values ranging from 1171 to 1314 mg/mouse (Table 2). Extracts of another 19 species neutralized 21– 72% the haemorrhagic effect of B. atrox venom at doses up to 4 mg/mouse: the whole plants of A. grandiflora, C. kalbreyeriana, S. acuta, S. articulata and P. spicatus; rhizomes of R. alpinia; the stem of S. xinguensis; leaves, branches and stems of H. capitata, I. cairica, N. lobata, O. micranthum, P. pulchrum, S. thecaphora, C. elastica and A. cathartica; the macerated ripe fruits of C. frutescens; the unripe fruits of C. cujete; leaves and branches of P. arboreum and P. quadrangu-
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laris (Table 1). Low or negligible neutralization ( B 20%) was obtained with 27 extracts and no neutralization was observed in another 17. Columnea pulcherima and I. cairica, that induced mild haemorrhage at a dose of 4 mg, were otherwise partially effective to neutralize this effect of venom at a dose of 2 mg/mouse (Table 1).
3.1.2. In 6i6o experiments When the extracts were independently administered by oral, i.p. or i.v. route before an i.d. venom injection (10 mg), there was a significant drop in the neutralizing ability. This was below 25% for all of them and disappeared in some cases according to the extract, dose and route of administration (e.g. B. rosademonte 0.5 mg i.v. 15 min Table 2 Extracts of plants with total in vitro neutralizing capacity of B. atrox venom haemorrhagic effecta Plant species
Effective dose (mg/mouse) ED50
Brownea rosademonte Berg. Pleopeltis percussa (Cav.) Hook & Grev. Heliconia curtispatha Petersen Bixa orellana L. Trichomanes elegans L.C. Rich Citrus limon (L.) Burm. f. Philodendron tripartitum ( Jacq.) Schott. Ficus nymphaeifolia Miller Struthanthus orbicularis (H.B.K.) Blume Gonzalagunia panamensis (Cav.) Schumm Tabebuia rosea (Bertold.) DC. Senna dariensis (Br. & R.) I. & B.
1693b 1169 9c
ED100 62.5 500
198968cd 2609 28d 275935d 5309 127de 8109 113de
2000 2000 2000 2000 2000
11719 434ef 11829 280ef
4000 4000
12009 12f
4000
13149 160f B500*
4000 1000
a Variable doses of the extracts were preincubated with a fixed dose (10 mg/mouse=6 MHD) of B. atrox venom at 37°C during 30 min. Then, the mixtures were i.d. injected in the abdominal skin to groups of four mice (18–20 g). Two hours later, mice were sacrificed with ether and the haemorrhagic areas were measured. All the results are expressed as mean 9 S.D. of three experiments. Values with different superscripts (b–f) are significantly different (PB0.05). ED50, effective dose 50%; ED100, effective dose 100%. * Insufficient material.
before= 149 2%; B. orellana 0.5 mg i.p. 60 min before= 19%; F. nymphaeifolia 4 mg oral 60 min before= 239 5%). The rapid i.p. or i.v. injection of the extracts 5 min after an i.d. venom injection (10 mg), also had similar results (e.g. B. rosademonte 0.5 mg i.v. 5 min after = 13%; B. orellana 0.5 mg i.p. 5 min after= 139 1%; F. nymphaeifolia 2 mg i.v. 5 min after= 149 1%).
3.2. Neutralization of the proteolytic acti6ity of 6enom B. atrox venom had proteolytic activity on casein in a dose-dependent manner (Fig. 1). Both extracts tested demonstrated significant inhibition of the proteolytic effect of venom (PB 0.001), being higher (100%) when a low concentration of the latter (10 mg/ml) was used (Fig. 1). At high concentration of venom (250 mg/ml), B. rosademonte extract was more effective than that of P. percussa to neutralize this enzymatic activity of venom (PB 0.001). Extracts also demonstrated neutralizing ability in a dose-dependent manner (results not shown).
4. Discussion Haemorrhage is one of the most relevant signs of local and systemic envenoming by viperid and crotalid snakes. Various types of bleeding distant from the bite site are gingival haemorrhage, epistaxis, haemoptysis, haematuria, uterine bleeding, soft tissues haematomas and rarely intrathoracic or intrabdominal bleeding (Otero et al., 1992a; Kamiguti et al., 1996). Severe anemia and shock may arise as complications and haemorrhage in the central nervous system causes death and sequelae (Otero et al., 1992a). For this reason, one of the corner-stones of the treatment of those envenomations must be the inhibition of haemorrhage as indicative of venom neutralization (Otero et al., 1996; Otero-Patin˜o et al., 1998). During the last 20 years, other authors have reported the presence of antihaemorrhagic activity in several plant extracts and their constituents. Eclipta prostrata extract and its compound wedelolactone, in vitro neutralized B. jararaca, B.
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Fig. 1. Neutralization of proteolytic activity of B. atrox venom on casein. Venom (10 – 250 mg/ml) was preincubated at 37°C for 30 min with the extract A (B. rosademonte) or the extract B (P. percussa), 500 mg/ml. The mixture was then added to the substrate (1% w/v casein) and newly incubated for 30 min at 37°C. In all the experiments, the proteolytic activity of the venom alone was tested (control). Results (absorbances at 280 nm) are the mean 9 S.E. of three determinations.
jararacussu and Lachesis muta snake venoms haemorrhagic effect (Melo et al., 1994). A tannin extracted from Diospyros kaki neutralized the haemorrhage induced by Agkistrodon halys snake venom (Okonogi et al., 1979). The root extracts of Hemidesmus indicus, Pluchea indica, Vitex negundo, Emblica officinalis and Aristolochia indica in vitro and in vivo were able to neutralize the haemorrhagic effect of Vipera russellii, Echis carinatus, Naja naja and Ophiophagus hannah snake venoms; and the compound HI-RVIF isolated from the root extract of H. indicus and administered i.v. to mice, significantly neutralized lethal, haemorrhagic and defibrinogenating activities of V. russellii venom (Alam et al., 1994; Alam and Gomez, 1995). Our results demonstrated that 31 of the 75 plant extracts tested had in vitro moderate or high neutralizing ability against the haemorrhagic effect of B. atrox venom from Antioquia and Choco´, 12 of them being 100% effective (Tables 1 and 2). The extract of B. rosademonte, which had the highest neutralizing ability in this work (ED50 =1693 mg/mouse) (Table 2), also had the
highest potency (ED50 = 140 mg/mouse) to neutralize the lethal effect of venom (1.5 i.p. LD50 = 99.3 mg) in a previous study performed with in vitro experiments (Otero et al., unpublished observations). Twelve of the 31 plants have been reported by traditional healers from the region as having antihaemorrhagic properties, e.g. B. orellana, B. rosademonte, C. elastica, C. kalbreyeriana, F. nymphaeifolia, G. panamensis, H. curtispatha, N. lobata,O. micranthum, S. orbicularis, S. xinguensis and T. rosea. Snake toxins responsible for haemorrhage are a large group of metalloproteins with catalytic activity. They degrade capillary basal lamina components and they are also cytotoxic on endothelial cells opening gaps through which erythrocytes escape (Moreira et al., 1994; Kamiguti et al., 1996). As some plant constituents have the ability to bind snake venom proteins and inhibit their catalytic activity in a dose-dependent manner, an antiproteolytic mechanism has been proposed to explain the antihaemorrhagic effect of the plant extracts (Melo et al., 1994; Melo and Ownby, 1999). Our results also demonstrated that B.
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rosademonte and P. percussa extracts neutralized the proteolytic activity of B. atrox venom. Venom phospholipases A2 enzymes have also been implicated in the haemorrhage pathogenesis of snakebites (Gowda, 1997; Melo and Ownby, 1999). A recent study demonstrated that B. rosademonte, P. percussa, H. curtispatha, B. orellana, T. elegans, S. orbicularis, G. panamensis, T. rosea, S. dariensis, R. alpinia, P. arboreum, S. xinguensis and C. lasius had antiphospholipase A2 activity (Otero et al., unpublished observations). Nevertheless, C. limon that was devoid of antiphospholipase A2 activity in that study, has indeed antihaemorrhagic effect as was demonstrated herein (Tables 1 and 2). Other mechanisms of metalloprotein inhibition may be possible such as the presence of metal-chelator substances in the extracts, e.g. in B. orellana (Duke, 1998). The significant drop in the neutralization of the haemorrhagic effect when the extracts were independently administered either before or after an i.d. venom injection, may be in relation to the short time (4–6 min) required for erythrocyte extravasation from capillaries and small venules after tissue venom exposure (Lomonte et al., 1994). Furthermore, the kinetic of the absorption, distribution, metabolism and excretion of the extracts, as well as their concentration into the tissue where venom was injected, are still unknown. On the other hand, an antivenom administered i.v. immediately after an i.m. venom injection does not totally neutralize the haemorrhagic effect of B. asper venom (Gutie´rrez et al., 1981). Venom metalloproteinases have been implicated in systemic bleeding by their capacity to inhibit platelet aggregation, e.g. jararhagin from Bothrops jararaca affects the platelet collagen receptor a2b1 integrin and degrades the adhesive plasma protein von Willebrand factor (Kamiguti et al., 1996). Thus, these observations could explain the recognized feature that patients do not present systemic bleeding sooner than 30 – 60 min after the bite, the time required for the establishment of haemostatic alterations (Otero et al., 1992a). Additional experimental work will be necessary to test the ability of the extracts for the in
vivo neutralization of systemic bleeding induced by B. atrox venom, so validating the information obtained from traditional healers.
Acknowledgements We thank the staff of the hospitals, local governmental authorities, the communities in the region of study, the Direccio´n Seccional de Salud del Choco´, Ministerio del Medio Ambiente and the personnel of the Utrı´a and Katı´os National Parks for their cooperation. 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 we also express our thanks. Thanks go to Elizabeth Cadavid and Janeth Garcı´a for preparing the manuscript. This project was supported by funds from the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologı´a Francisco Jose´ de Caldas (Colciencias) and from the Universidad de Antioquia.
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, Paris. 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. Duke, J., 1998. Phytochemical and Ethnobotanical Databases. http://www.ars-grin.gov/cgi-bin/duke/ethnobotuse.pl Fan, H.W., Cardoso, J.L.C., 1995. Clinical toxicology of snakebite in South America. In: Meier, J., White, J. (Eds.), Handbook of Clinical Toxicology of Animal, Venoms and Poisons. CRC Press, Boca Raton, FL. Gowda, T.V., 1997. Interaction of snake venom phospholipases A2 with plant isolates. In: Kini, R.M. (Ed.), Venom Phospholipase A2 Enzymes. Wiley, Chichester, UK.
R. Otero et al. / Journal of Ethnopharmacology 73 (2000) 233–241 Gutie´rrez, J.M., 1995. Clinical toxicology of snakebite in Central America. In: Meier, J., White, J. (Eds.), Handbook of Clinical Toxicology of Animal, Venoms and Poisons. CRC Press, Boca Raton, FL. Gutie´rrez, J.M., Chaves, F., Bolan˜os, R., Cerdas, L., Rojas, E., Arroyo, O., Portilla, E., 1981. Neutralizacio´n de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente. Toxicon 19, 493–500. Houghton, P.J., Osibogun, I.M., 1993. Flowering plants used against snakebite. Journal of Ethnopharmacology 39, 1– 29. Kamiguti, A.S., Hay, C.R.M., Theakston, R.D.G, Zuzel, M., 1996. Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases. Toxicon 34, 627–642. Kondo, H., Kondo, S., Ikesawa, H., Murata, R., Ohsaka, A., 1960. Studies on the quantitative method for determination of haemorrhagic activity of Habu snake venom. Japanese Journal of Medical Science and Biology 13, 43–51. Lomonte, B., Gutie´rrez, J.M., 1983. La actividad proteolı´tica de los venenos de serpientes de Costa Rica sobre la caseı´na. Revista de Biologı´a Tropical 31, 37–40. Lomonte, B., Lundgren, J., Johansson, B., Bagge, U., 1994. The dynamics of local tissue damage induced by Bothrops asper snake venom and myotoxin II on the mouse cremaster muscle: an intravital and electron microscopic study. Toxicon 32, 41 – 55. 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. Moreira, L., Borkow, G., Ovadia, M., Gutie´rrez, J.M., 1994. Pathological changes induced by Ba H1, a hemorrhagic
.
241
proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels. Toxicon 32, 977 – 987. Okonogi, T., Hattori, Z., Ogiso, A., Mitsui, S., 1979. Detoxification by persimmon tannin of snake venoms and bacterial toxins. Toxicon 17, 524 – 527. 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., Osorio, R.G., Valderrama, R., Giraldo, C.A., 1992b. Efectos farmacolo´gicos y enzima´ticos de los venenos de serpientes de Antioquia y Choco´ (Colombia). Toxicon 30, 611 – 620. 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. 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.