IRELAND Journal of Ethnopharmacology 42 (1994) 199-203

Preliminary results on the protective effect of (-)-edunol, a pterocarpan from Brongniartia podalyrioides (Leguminosae),

against Bothrops atrox venom in mice?

Ricardo Reyes-Chilpa”, Federico G6mez-Garibay”, Leovigildo Quijano*a, Gil Alfonso Magos-Guerrerob, Tirso Rios”

“Institute de Quimica. “Facultad de Medicina. Universidad National Autdnoma de MPxico. Ciudud Universitaria. Coyoach. 04510.

Mkxico. D. F.

(Received 2 August 1993: revision received 24 January 1994; accepted 24 March 1994)

Abstract

(-)-Edunol a prenylated pterocarpan was isolated from the roots of two Mexican ‘snakeweeds’, Brongniartia podalyrioides and B. intermedia (Leguminosae). Edunol (3.1 mg/kg. i.p.) reduced the expected mortality of mice previously treated by the same route with the LDSo of the venom of the serpent Bothrops atros. The molecular struc- ture and properties of edunol are similar to those previously reported for cabenegrins A-l and A-II.

Key words: Snake venom; Pterocarpan; Leguminosae

1. Introduction

Genus Brongniurtiu (Leguminosae, Papiliono- ideae) comprises approximately 65 species, almost all of them endemic to Mexico (Sousa and Delgado, 1993). Several species have been used by some ethnic groups and peasants in the treatment of snakebites, against lice, fleas, mange, and as a fish poison (Table 1). In the State of Sinaloa, Mexico, the bark and leaves of B. glabrata Hook

& Am., known as ‘hierba de la vibora’ - snake- weed - have been used as a remedy against rattle- snake bites (Standley, 1922). The roots of B. goldmanii Rose, ‘vara prieta’ - dark stick - are used against venom (Shapiro 286, MEXU). Other species such as B. podalyrioides Kunth and B.

intermedia Moric. are also known as ‘hierba de la vibora’ (Gonz$ez Ortega, 1929; Martinez, 1979), but their popular uses have not been documented. According to oral testimonies that we have recent- ly obtained at Cosali. Sinaloa, Brongniartia spp. are used specially to treat cattle. The roots are ground and applied as a poultice on the snakebite. The genus Brongniartiu has been taxonomically revised (Rydberg, 1923; Dorado 1992), but it is practically unknown from the chemical and phar-

l Corresponding author.

IContribution No. 1218 of lnstituto de Quimica. UNAM.

Presented in part as an oral communication at the International

Meeting ‘Etnobotanica 92’. Cordoba. Spain. 20-26 September

1992.

0378-8741/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0378-8741(94)01131-I

200 R. Reyes-Chilpu Ed ul. /J. Erhnuphurmucul. 42 ( 1994) 199-203

Table I Ethnobotanical data on genus Brungniurliu in Mexico

Species State

(common name) (ethnia)

References

B gluhrutu Sinaloa Standley. 1922

(hierba de la vibora)

‘Bark and leaves used in Sinaloa as a remedy for rattlesnake

bites’

B goldmunii Sinaloa

(vara prieta)

The root against venom

Shupiro 286,

MEXU. 1976

B. pulmerii Sonora Pennington. 1980

(pal0 piojo) (Pimas)

The grounded bark soaked in water for preparing a lotion

against fleas (domestic animals) and head lice (humans)

B. pudulyioides

(cahuiriquilla)

Michoacan Soto Nutiez. 1987

Soto 1153. MEXU,

1978

The boiled branches topically or as a washing for curing

mange, infusion of the leaves and branches for killing head lice

Brongniurriu sp. Chihuahua Pennington. 1967

(Tepehuanos)

The plant soaked in water as a lotion against fleas. the ground-

ed plant is a fish poison

macological point of view. The only toxicological study available dates from the beginning of the century. A Brongniartiu sp. (garbancillo) from the State of Queretaro, Mexico, was investigated along with other legumes believed to be toxic to cattle. Nevertheless, the root and leaves reduced to powder and their hydroalcoholic extracts injected or by oral administration were inocuous to domestic pigeons and rabbits (Armendaris, 1906).

We have undertaken the chemical analysis of Brongniartia podalyrioides, a small shrub known for its pressumed insecticidal and acaricidal pro- perties (Soto Nmiez, 1987). From the roots, we isolated the prenylated pterocarpan (-)-edunol ([3] in Fig. 1) (Rail et al., 1971). Structurally (-)- edunol is closely related to (-)-cabenegrins A-I [ 1) and A-II [2] (Nakawa et al., 1982). In a dog these substances counteracted the cardiovascular effects of Borhrops atrox venom and increased the survi- val of envenomed mice. It is noteworthy that cabenegrins were isolated by a bioassay directed fractionation of the hydroalcoholic root extract of the amazonian plant named ‘cabeca de negra’ (its botanical identity is unknown) used by plantation workers as an oral antidote against snake and spider venoms (Nakawa et al., 1982). Considering these antecedents, we decide to investigate the

Structures of (-)-cabenegrins A-l [I]. A-II 121 and (-)-edunol [3]

R. Reyes-Chifpa et al. I J. ~t~~o~~ar~arol. 42 (1994) W-203 201

effect of edunol on mice pretreated with the venom of Bothrops atrox, a south American snake of the Crotalinae subfamily. Our preliminary results showed that edunol can reduce the mortality of the envenomed mice with the LDso. (-)-Edunol was also obtained from the roots of Brongniarria in- termedia.

2. Materials and methods

2. I. Plant material Brongn~artia poda~yriaides was collected at ‘El

Rodeo’, State of Morelos, and B. intermedia at ‘Cerro de1 Tetzcutzingo’, State of Mexico, Mexico. Vouchers were authenticated by Dr Mario Sousa and are deposited at the National Herbarium of Mexico (MEXU).

2.2. Isolation of edunol Drillings from the roots (556 g) were extracted

at room temperature with petrol and methylene chloride. Solvents were removed in vacua. The petrol extract (2.9 g) was subjected to column chromatography (cc) on Silica Gel 60 (60 g). Frac- tions obtained with petrol-methylene chloride (3:7) were combined and rechromatographed by cc and preparative thin layer chromatography. Edunol [3] was obtained as a yellowish oil which was used for pharmacological tests. Further crystalization was achieved.

Edunol. C21H200Sr colourless needles, m.p. 154-155’ (reported 146-147”; Rail et al., 1971).

h]D = -208” (c. 2.13 mglml, CHC&) UV h,,, (MeOH) nm(e): 290 (2633), 306 (3120). IR vrnax (CHC13): 3540, 3420, 1625, 1590, 1460,

1330, 1140, 1045, 1037, 930. EIMS 70 ev m/z (rel. int.): 352 [M]+ (100). 297

(58), 296 (21), 283 (7.5X 279 (7) 176 (lo), 175 (22.5), 162 (20),, 161 (lo), 115 (12.5), 77 (7.5) 69 (20).

‘HNMR (300 MHz, CDClj-T-MS): 7.22 (s, 1H) H-l, 6.72(s, 1H) H-7,6.44(s, IH) H-4,6.40(s, IH) H-10, 5.46 (d, lH, f = 7 Hz) H-l la, 4.20 (dd, lH, J = 9.2, 5 Hz) H&c, 3.62 (t. IH, J = 9.2 Hz) H- 6ax, 3.45 (ddd, IH, f = 9.2, 7, 5 Hz) H-fia, 5.88 (d, IH, J = 1.2 Hz). 5.91 (d. IH, f = 1.2 Hz) OCH:O, 1.78 (s, 6H) 2 CHr, 3.32 (d, ZH, J = 7 Hz) H-l ‘.

5.31 (t, IH, J = 7 Hz) H-2’, 5.35 fs, 40 ex- changeable, 1H) Ar-OH.

13CNMR (50 MHz, CDCI,): 131.7 (C-l), 112.1 (C-la), 121.1 (C-2), 155.7 (C-3), 103.9 (C-4), 154.2 (C-4a), 66.4 (C-6), 40.2 (C-6a), 118.0 (C-7a), 141.6 (C-8),*148.0 (C-9), 93.7 (C-lo), 154.9 (C-lOa), 78.6 (C-lla), 29.1 (C-l’), 121.8 (C-2’), 134.7 (C-3’), 25.7 and 17.8 (2 CH3), 101.2 (OCH20).

(-)-Edunol was also isolated as described above from the petrol extract of B. intermedia roots.

2.3. Determination of LDsO in mice Male mice (28-35 g) strain CD-1 from the

Biotery of the School of Medicine UNAM, were used in all the experiments. The LDso of the venom of Bofhrops atrox (Sigma) was determined i.p. with groups of ten mice per dose (3.1, 4.6, 6.8, 10 and 14 mg/kg). The venom was dissolved in saline 0.9%. Each mouse was injected first with the venom and immediately with a solution of 5% Tween 20 in saline. Afterwards the mice were returned to the cages and maintained with food (Purina chow) and water ad libitum at laboratory conditions. Mortality was recorded after 24 h and transformed to probits units for linear regression analysis. LD,, was determined as 6.85 mg/kg

2.4. Pharmacological tests Four edunol dosis following a 0.5 logarithmic

series (0.31, 1.0, 3.1 and 10 mglkg) were tested i.p. with ten mice per dose. Each mouse was first injected with the LD% or 2 x LDss of the venom and immediately with the edunol solution. Edunol was dissolved in 0.9% saline containing Tween 20 (Sigma) to a final concentration of 5%, and son- nicated for 5 min. The control group was treated as indicated above, but the second injection con- tained only 5% Tween 20 in saline. Mortality was recorded after 24 h. Edunol solutions were very stable, and the compound could be purified and recovered unaltered (as indicated by ‘HNMR) after 3 days from the refrigerated surplus sol- utions.

3. Results and discussion

(-)-Edunol was isolated from the roots of Bron~~iar~ia pada~yrioides and B. intermedia, and

202 R. Reyes-Chilpu et al. /J. E~hn~~~ffrrnuc51. 42 f 1994) 199-203

was identified by its spectral and physical data (Rail et al., 1970; Chalmers et al., 1977). Edunol has been previously isolated from the roots of Neorautanenia edufis C.A. Smith (Rall et al., 1970) and N. amboensis Schinz (Breytenbach et al., 1983). Ethnobotany of this genus resembles that of Brongniartia. Some species are used in Central and South Africa against lice, fleas, acarus and as fish poisons (Van Puyvelde et al., 1987); nevertheless, it is not known if they are used for snakebite treatments. The pharmacological assays (Table 2) showed that administration of 3.1 mglkg of edunol decreased the mortality of mice pretreated with the LD,, of B. utrox venom. This dose was still effec- tive when tested with 2 x LDse: mortality drop- ped from 100% to 70%. A lower dose, 1 mg/kg was only effective against the LDSo, reducing mortality from 40% to 20%. The highest dose, 10 mg/kg, had a poor survival value when tested with the LDw and failed completely with 2 x LDse. The above results can be reconciled if edunol is toxic at this concentration. The available evidence indicates that some pterocapans can inhibit mit~hondrial respiration. A mixture of gliceollins I, II, and III blocked electron transport in a similar way to rotenone (Boydston et al., 1983).

Considering the protective dose of 3.1 mg/kg, edunol seems to be less potent than cabenegrins A- l and A-11. In mice pretreated with 2.5 x LDso these pterocarpans showed a minimum effective dose of 2.8 and 2.0 mgJkg i-p., respectively. Therefore, within this group of compounds the dif- ference in the protective effect might be related

Table 2 Effect of edunol on mice pretreated with Burhrops atrox venom

Edunol -

(mgikg)

Mortality

Venom

with dimethylallyl side chain modi~cations. Apart from the cabenegrins A-I and A-II, the molecular structure and biological properties of edunol are also related to those reported for wedelolactone. This coumestan was isolated from Eclipta pro- strata L. (Asteraceae) and neutralized the lethal and myotoxic activities of Crotalus durissus ier- rificus venom (Mors et al, 1989). Structurally, edunol shares with cabenegrins A-I and A-II and wedelolactone some features - an isoflavone skel- eton, acidic nature, a dioxygenated functionality -which have been suggested to be relevant to the biological activity of several of the currently known antivenom plant metabolites (Mors, 1991). Antivenom compounds so far isolated from plants include protocatechuic acid, a catechin-gallo- catechin tannin, the alkaloid aristolochic acid, and the triterpenoids sitosterol, stigmasterol and gym- nagenin acid (Mors, 1991), as well as the alkaloids allantoin (Tsai et al., 1980), and shumaniolioside (Akunyili and Akubue, 1987). It has been sug- gested that these substances could possibly act by interaction with the enzymes and toxins of the snake venoms (Mors, 1991). A similar phenome- non could be involved in the protective activity of edunol, but this could not be established since a general survival test was used. Further specific pharmacological assays for evaluating anticar- diotoxic, antimyotoxic activity, etc. (Mors, 1991) are necessary. Finally, since prenylated pterocar- pans, or ring substitutes derived from the former, have been found only in some tribes of the Papilionoideae - Desmodieae, Dalbergieae, Phaseoleae, Sophoreae, and now Brongniartieae - (Ingham, 1981; Reyes Chilpa et al., 1992) it is possible to suggest that the alexiteric plant ‘cabeca de negra’, the source of cabenegrins (Nakawa et al., 1982), could possibly belong to to one of these taxa. Further ethnobotanical studies could test this suggestion.

Wo 2 J-&o (6.85 mg/kg) ( 13.7 mg/kg)

Acknowledgments

0.0 4/lO IO/IO 0.31 4110 IO/IO I.0 2110 IO/IO 3.1 O/IO 7110 10.0 3110 IO/IO

We are grateful to Dr Mario Sousa, Dr Robert Bye, and Biol. Jose C. Soto for their botanical ad- vice, to Dr Iioracio Vidrio and Dr Cristina Lemini for their facilities during pha~acological experi- ments, and to Dr Jaco C. Breytenbach who kindly

R. Reyes-Chilpa et al. /J. Ethnophurmacol. 42 (1994) 199-203 203

provided some pterocarpan samples. This project was finnanced partially by PADEP-UNAM and CONACYT funds granted to R.R.C.

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