Anticonvulsant activity of Casimiroa edulis in comparison to phenytoin and phenobarbital

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Journal of

ETHNO PHARMACOLOGY E LSEVI E R

Journal of Ethnopharmacology 45 (1995) 199-206

Anticonvulsant activity of Casimiroa edulis in comparison to phenytoin and phenobarbital A. Navarro Ruiz *a, B.E. Bastidas Ramirez b, J. Garcia Estrada a, P. Garcia L6pez b, P. Garz6n a aDivisi6n de Bioquimica-Farmacol6gica, Unidadde lnvestigaci6n Biom6dica de Occidente, C.M.0., lnstituto Mexicano de/Seguro Social, Mexico, Mexico bDepartamento de lnveMiqaci6n Cientifica de la Facultad de Medicina, Universidadde Guadalajara, Guadalajara, Jalisco, M~xico

R~eived 26 April 1993; revision received 23 May 1994; accepted 30 November 1994

Abstract

An aqueous extract of Casimiroa edulis leaves was tested in adult male Wistar rats for anticonvulsant activity utilizing two models of experimental epilepsy: maximal electroshock seizure (MES) and subcutaneously injected metrazole (METsc). Single dose of 100 mg/kg C edulis vacuum dried aqueous extracts (VDA) orally administered to experimental animals elicited 50% and 70% abolition of MES and METsc-induced seizures, respectively. Two firmly established antiepileptic drugs in human therapy, phenytoin (PHT) and phenobarbital (PB), abolished 90% of MES-induced seizures, whereas an 80% and 100% absence of clonic seizures was attained in METsc test, correspondingly. The seizure abolition observed in C. edulis VDA treated rats was comparable with the anticonvulsive pattern exhibited by PHT and PB. These results suggest that potencially antiepileptic compounds are present in C edulis extracts that deserve the study of their identity and mechanism of action. Keywords: Anticonvulsants; Casimiroa edulis; Medicinal plants; Phenobarbital; Phenytoin

1. Introduction Casimiroa edulis Llave et Lex. (Rutaceae), commonly known as 'zapote blanco', is a tree widely distributed throughout the central and southeastern States of Mexico (Marinez, 1951). Some of the drug aspects of the white 'sapotes' seeds have been compiled elsewhere (Morton,

* Corresponding author, P.O. Box 1-3838, Guadalajara, Jaliseo, M6xico.

1962). Aqueous extracts of 'zapote blanco' leaves have been popularly used as a sedative and to reduce high blood pressure (Martinez, 1944; Lozoya and Enriquez, 1981; Magos and Vidrio, 1991). The sedative activity of C. edulis leaves extracts described in humans (Estrada, 1989) as well as the anti-strychnine activity reported in dogs (Villacis, 1978), led us to think that C. edulis would possess anticonvulsive properties of clinical usefulness comparable with those shown by phenytoin (PHT) and phenobarbital (PB) in the treatment of

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A. Navarro Ruiz et al./Journal of Ethnopharmacology 45 (1995) 199-206

human epilepsies (Merrit and Putnam, 1938; Gal et al., 1982). Therefore, infusions of leaves were investigated for anticonvulsant profile in rats employing two routine laboratory tests firmly established in the study of candidate anticonvulsant agents: maximal electroshock seizure (MES) and subcutaneously injected metrazole (METsc). In this respect, MES and METsc-induced seizures resemble convulsive and non-convulsive patterns of human epilepsies, respectively (Woodbury, 1972). Although these experimental seizureevoking procedures have predominantly been used to study the seizure-inhibiting properties of pure substances (Swinyard et al., 1989), they can be used in the evaluation of plant extracts as well (Chauhan et al., 1988; Garz6n et al., 1990). Thus, an attempt was made in this study to investigate the anticonvulsant effects of extracts from C. edulis leaves in comparison with PHT and PB, as reference antiepileptic drugs. 2. Material and methods 2.1. Preparation of extracts Leaves of C. edulis trees were collected in Nex-

tipac, Jalisco (Mexico), by the Unidad de Investigaci6n Biom6dica de Occidente of the Instituto Mexicano del Seguro Social (IMSS). The botanical identity of the plant material was verified and vouchered specimens were deposited at the herbarium of the Institute of Botany University of Guadalajara for reference. Authenticated leaves were washed in tap water, shade-dried, ground to a moderately coarse powder and stored in a chamber containing CaCo3. Thereafter, leaf aqueous extract (LA) was prepared in a ratio of 20 g of leaf powder to 500 ml of boiling distilled water. Once LA reached room temperature, it was filtered several times through a gauze and Whatman paper No. 40, until no precipitate was observed. Vacuum dried aqueous extract (VDA) was prepared from LA by using a Labconco freeze-dryer model 75050 (Labconco Corporation, 8811 Prospect, Kansas City, Mo. 64132). 2.2. M E S test

This assay was performed by applying corneal

electrodes to rats using an alternating current of 60 Hz at 150 mA for 0.2 s preceded by an application of one drop of 0.9% saline solution (SS) to both eyes. Elapsed time from electrical stimulus until hind-limb extensor reflex appearance (latency) was measured. Also, duration of the reflex was registered from the time of full hind-limb extension until the legs became perpendicular to the body (Swinyard, 1972). Animals presenting the extensor reflex were MES(+), whereas MES(-) classification was given if the animal was unresponding. 2.3. METsc test

Metrazole (Sigma Chemical Co.), was subcutaneously injected to rats at a dose of 70 mg/kg (CD97) in a volume of approximately 0.1 ml SS. The animals were placed in isolated cages and observed during the next 60 min for the presence or absence of a clonic spasm episode persisting for at least 5 s (Swinyard et al., 1952). Elapsed time from metrazole injection until occurrence of clonic spasm was measured (latency). Those animals presenting convulsions were classified as METsc(+), whereas non-convulsed animals were classified as METsc(-). 2.4. Animals

Adult male Wistar rats (200-300 g) were housed in groups of 10 per polypropylene cage and maintained at 22°C room temperature with day and night cycles of 12 h each. Free access to standard rodent diet (Purina-Chow) and tap water was allowed, unless stated otherwise. All experiments were carried out between 07:00 and 16:00 h. The MES or METsc test was performed in all rats prior to any treatment in order both to assess individual seizure susceptibility and to implement the control parameter values. Only seizure-susceptible rats constituted experimental groups. Animals were divided into 37 MES(+) and 37 METsc(+) groups of 20 rats each, then distributed in the following order. LA chronic assay: Two groups, one MES(+) and one METsc(+), were under ad libitum fresh LA ingestion during 7 days; no water intake was allowed within this period. On the 7th day the MES or METsc test was performed.

A. Navarro Ruiz et al,/ Journal o f Ethnopharmacology 45 (1995) 199-206

P H T and PB chronic assay: One MES(+) and one METsc(+) group were utilized to test each drug. Two oral daily doses of 30 mg/kg PHT or 12 mg/kg PB were administered through an esophageal canulae during 7 days. Subsequently, the MES or METsc test was performed on the seventh day. PHT was dissolved in a mixture of propyleneglycol, ethyl-alcohol and water in a 40:10.6:49.4 ratio. PB vehicle was 0.11 M phosphate buffer, pH 7.9. VDA acute assay: A single oral dose of VDA was administered through an esophageal canulae and three different closes were studied: 10 100 and 1000 mg/kg (VDA was dissolved in distilled water). Six MES(+) and six METsc(+) groups were assigned to test each dose. The anticonvulsant effect in MES or METsc-induced seizures was assayed at hourly intervals following VDA administration throughout 6 consecutive hours, utilizing one different group for each hour. P H T and PB acute assay: These experiments were similarly performed as described above for VDA acute assay. Eight MES(+) and eight METsc(+) groups were necessary to test each drug. A single intake of 30 mg/kg PHT or 12 mg/kg PB was given to rats. Animal groups were individually tested hourly after drug administration for 8 consecutive hours.

201

Following plant extract or drug treatment, either complete abolition of hind-limb extensor reflex (MES test) or inhibition of clonic spasm appearance (METsc test) was conclusive of anticonvulsant activity. 2.5. Statistics

Protection against MES or METsc-induced seizures was expressed as percentage of abolition of seizure. Latency and hind-limb extensor reflex duration of experimental groups were compared with control values and analysed by Student's ttest; P values were verified through the MannWhitney test. The percentage of mortality both in the control and the experimental groups were studied employing the X2 test. 3. Results

PHT, PB and LA treated animals during 7 days, showed inhibitory responses of 85%, 90% and 70% to METsc test and 70%, 100% and 6.4% to MES seizure-inducing test on the 7th day, respectively (Fig. 1). A maximum hind-limb extensor reflex inhibition of 10%, 50% and 30% was attained when 10 100 or 1000 mg/kg VDA was orally administered to rats in a single dose, respectively (Fig. 2). Although in-

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PHT

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~

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Fig. I. Protection to METsc and MES-induced seizures after 7 days of oral treatment with PHT (30 mg/kg, twice a day), PB (12 mg/kg, twice a day) or C. edulis LA (ad libitum) in Wistar male rats.

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A. Navarro Ruiz et al. /Journal of Ethnopharmacology 45 (1995) 199-206

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Fig. 2. MES test after a single oral dose of 10, 100 or 1000 mg/kg C. edulis VDA. Assay was performed hourly following VDA administration employing an individual group each hour interval (n = 20/h).

hibition of reflex did not occur in 100% of the animals, prolonged latencies and shortened reflex duration appeared in 16 out of 18 groups, when compared with the controls. Statistically significant longer latency periods were found in the following groups: first and fifth hour of 100 mg/kg (P < 0.01, P < 0.05); and third, fourth and fifth hour of 1000 mg/kg groups (P < 0.01, P < 0.01 and P < 0.05) (Table 1). A maximum inhibition of 70% and 55% of METsc-elicited seizures was observed in animals receiving a single oral dose of 100 and 1000 mg/kg VDA correspondingly (Fig. 3). Latency was found significantly prolonged in 7 out of 12 groups when compared with controls (Table 2). No protection was attained when 10 mg/kg VDA was administered. An increasing protection of 30% at the first hour and 90% 6 h later to MES elicited seizures was exhibited in PB acute assayed groups. PHT receiving animals showed hind-limb reflex abolition of 6% from the second hour and it reached a maximum of 80% in the group tested in the eighth hour (Fig. 4). PB acute assayed groups showed a 60% protection to METsc-induced seizures in the first and

second hours, followed by a decrease to 40% in the third and fourth hours and an increase to 95% and 100% on the fifth and sixth hour groups, correspondingly. Unexpected diminution of protection to 40% and 30% on the seventh and eighth hour groups was observed. Similarly, PHT protected a maximum of 80% of the experimental animals to METsc-elicited seizures in the fourth hour and 60% on the fifth hour. A decrease to 20% at the sixth and eighth hours was recorded. No protection was observed at first, second, third and seventh hours (Fig. 5). No dead animals were found after the METsc test in C. edulis treated groups in comparison with a recorded average of 10% deaths observed in control group and this difference was statistically significant (P < 0.01). 4. Discussion and conclusions

Experimental models of epileptiform seizures (EMES) have constituted the nearest physiological approach to the development of new anticonvulsant drugs for seizure control in epileptic patients (Putnam and Merrit, 1937; Fisher, 1989). Among EMES, MEG and METsc are the primary assays

A. Navarro Ruiz et al./ Journal of Ethnopharmacology 45 (1995) 199-206

203

Table 1 Hind-limb extensor reflex partial abolition (MES test) in convulsed C. edulis VDA treated animals Dose (mg/kg)

Elapsed time (h)

Convulsed rats (%)

Latency (s)

Hind-limb extensor reflex (s)

Control 10

0 I 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

100 100 100 90 90 90 100 90 80 70 80 50 85 90 70 85 70 70 90

2.90 2.82 3.60 3.40 3.88 3.70 3.20 4.44 3.90 3.90 3.50 4.20 3.20 2.20 3.50 4.20 4.30 4.20 3.80

10.10 9.80 9.20 9.20 8.80 8.40 10.80 8.70 8.10 8.40 9.60 9.90 9.30 9.20 8.50 7.76 8.46 7.93 8.70

100

1000

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.20 0.20 0.34 0.30 0.26 0.36 0.35 0.27** 0.67 0.45 0.30 0.30* 0.23 0.35 0.61 0.38** 0.34** 0.24* 0.69

± 0.60 ± 0.50 ± 0.38 ± 0.64 ± 0.48 ± 0.64 ± 0.44 ± 0.42 ± 0.84 ± 1.01 ~- 0.47 ± 0.64 ± 0.31 ± 0.88 ± 0.79 ± 0.54** 4- 0.52* 4- 0.36* ± 0.85

Latency and hind-limb extensor reflex values are expressed as the average -~- the standard error. 'Convulsed rats' refers to the pereentag¢ of rats responding to MES test. *P < 0.05, in comparison with control group (Student's t-test). **P < 0.01, in comparison with control group (Student's t-test).

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Fig. 3. Anticonvulsant activity of C. edulis VDA in METsc-induced seizures. Assay was performed hourly following a single intake of 100 mg/kg or 1000 m ~ k g VDA (n = 20/h).

204

A. Navarro Ruiz et al. /Journal of Ethnopharmacology 45 (1995) 199-206

to be developed in the conventionally accepted anticonvulsant screening procedure (Swinyard and Kupferberg, 1985). Formerly, this procedure was restricted only to test compounds of known chemical structure; however, now it has been also applied to validate anticonvulsant activity of medicinal plant extracts as well (Chauhan et al., 1988). It has been reported that PB, carbamazepine, primidone and ethosuximide inhibit METscinduced seizures in experimental animals as well as generalized non-convulsive seizures in epileptic patients. Also, the MEG protection by PHT described herein is comparable with the efficacious control of generalized convulsive disorders reported in humans (Swinyard et al., 1989). The maximal inhibition of 70% METsc and 50% MES-induced seizures in VDA C. edulis treated animals, heretofore described, in comparison with the values observed in those animals receiving PHT or PB, suggests that this plant contains compounds potentially efficaceous to inhibit mainly non-convulsive seizures in epileptic patients. Convulsed VDA treated animals showed

Table 2 Partial protection to METsc-induced seizures in convulsed C. edulis VDA treated Wistar rats Dose (mg/kg)

Elapsed time (h)

Convulsed rats (%)

Latency (s)

Control 100

0

100 60 30 75 80 50 70 80 80 90 70 60 55

16.00 14.10 36.00 23.30 23.00 17.20 22.40 19.40 21.40 25.70 25.80 27.50 8.30

1000

1

2 3 4 5 6 1 2 3 4 5 6

± ± ± ± ± ± ± ± ± ± ± ± ±

0.90 1.59 7.50*** 2.78** 1.90'* 2.90 2.60** 2.20 2.10 3.50** 3.80** 5.10"* 2.30

Latency values are expressed as the average ± the standard error. 'Convulsed rats' refers to the percentage of rats presenting convulsions. *P < 0.050 in comparison to control group (Student's t-test). **P < 0.010 in comparison to control group (Student's t-test). ***P < 0.001 in comparison to control group (Student's t-test).

I00

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2

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I L

6

5 TiME

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7

8

( hrs )

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Fig. 4. MES test hourly performed throughout 8 consecutive hours in individual Wistar male rat groups following a single orally administered dose of PHT or PB (30 mg/kg and 12 mg/kg, respectively).

205

A. Navarro Ruiz et al./ Journal of Ethnopharmacology 45 (1995) 199-206

100

bi

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4 ELAPSED 30 mg/kg

5 TIME

6

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8

(hrs)

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Fig. 5. Anticonvulsant activity of PHT and PB in hourly performed METsc test following a single oral dose of 30 mg/kg or 12 mg/kg, respectively.

delayed MES and METsc latency values. Seizure duration was found to be lower than that shown by the control group (Tables 1 and 2). These observations may lead one to think that although VDA was not able to completely inhibit the seizure appearance in all the experimental animals, a partial protection could have been exerted in convulsed rats against stimuli diffusing from forebrain to brainstem, instead of brainstem to spinal cord (Browning and Nelson, 1985). It is important to mention that mortality was zero in all C. edulis treated groups when subjected to METsc test, and the time of normal ambulatory activity recovery was found tO be lowered in all convulsed rats when compared with controls (data not shown). Since C. edulis exhibited anti-seizure activity, it might be clinically useful in the control of human epilepsies. Thus, we conclude that successive studies are mandatory to establish the precise nature of C. edulis active constituents as well as their mechanism of action. Acknowledgements. The statistical advisory of Rogelio Troyo Sanromfin, the technical assistance of Ang61ica

Gonz~ilez Ballesteros, the photography work done by Armando Alcaraz and care of experimental animals by Raul Orozco Martinez and Luis Miguel Aguilar de la Mora is gratefully acknowledged. References Browning, R.A. and Nelson, D.K. (1985) Variation in threshold and pattern of electroshock induced-seizures in rats depending on site of stimulation. Life Sciences 37, 2205-2211. Chauhan, A.K., Dobhal, M.P. and Joshi, B.C. (1988) A review of medicinal plants showing anticonvulsant activity. Journal of Ethnopharmacology 22, 11-23. Estrada. L.E. (1989) Plantas medicinales de M~xico, lntroducci6n a su Estudio. Universidad Aut6noma de Chapingo, Departamento de Fitotecnia, M~xico. Fisher, R.S. (1989) Animal models of the epilepsies. Brain Research Reviews 14, 245-278. Gal, P., Tobock, J., Boer, H., Erkan, N. and Wells, T. (1982) Efficacy of phenobarbital monotherapy in treatment of neonatal seizure-relationship to blood levels. Neurology 32, 1401-1404. Garz6n, P., Navarro Ruiz, A., Dominguez-Rodriguez, J.R., Garcia-Estrada, J., Gonz/dez-Hita, M., Bastidas-Ramlrez, B.E., Rom~.n-Maldonado, S. and Navarro-Ruiz, I. (1990) Modified anticonvulsant screening procedure. Phenytoin and phenobarbital reevaluation. Archivos de lnvestigaci6n M~dica (M~xico) 21, 57-63.

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Walter, R.D. (Eds.), Experimental Models of Epilepsy. Raven Press, New York, pp. 433-458. Swinyard, E.A. and Kupferbcrg, H.J. (1985) Antiepileptic drugs: detection, quantification and evaluation. Federation Proceedings 44, 2629-2633. Swinyard, E.A., Brown, W.C. and Goodman, L.S. (1952) Comparative assays of antiepileptic drugs in mice and rats. Journal of Pharmacology and Experimental Therapy 106, 319-330. Swinyard, E.A., Woodhead, J.H., White, H.S. and Franklin, M.R. (1989) General principles: experimental selection, quantification and evaluation of anticonvulsants. In: Levy, R., Mattson, R., Meldium, B., Penry, J.K. and Dreifuss, F. E. (Eds.), Antiepileptic Drugs. Raven Press, LTD New York, pp. 233-239. Villacis, R.L. (1978) Plantas medicinales de M~xico. Epoca, M~xico, pp. 134-135. Woodbury, D.M. (1972) Applications to drugs evaluations. In: Purpura, D.P., Penry, J.K., Tower, D.B., Woodbury, D.M., Walter, R.D. (Eds.), Experimental Models of Epilepsy. Raven Press, New York, pp. 557-584.