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Casimiroa edulis seed extracts show anticonvulsive properties in rats
Journal of Ethnopharmacology 68 (1999) 275 – 282 www.elsevier.com/locate/jethpharm
Casimiroa edulis seed extracts show anticonvulsive properties in rats P. Garzo´n-De la Mora a,c, P.M. Garcı´a-Lo´pez b, J. Garcı´a-Estrada a, A. Navarro-Ruı´z a,*, T. Villanueva-Michel c, L.Ma. Villarreal-de Puga b, J. Casillas-Ochoa c a
b
C.I.BO., I.M.S.S, Guadalajara, PO Box 1 -3838, Jalisco, Mexico Laboratorio de Biotecnologı´a, Instituto de Bota´nica y Zoologı´a, C.U.C.B.A., Uni6ersidad de Guadalajara, Guadalajara, Mexico c Laboratorio de Bioquı´mica, Depto. de Fisiologia, Centro Uni6ersitario de Ciencias de la Salud, Uni6ersidad de Guadalajara, Guadalajara, Mexico Received 13 July 1998; received in revised form 22 June 1999; accepted 6 July 1999
Abstract A single dose of 5, 10 and 100 mg/kg of Casimiroa edulis aqueous extract (AQ); 10, 100 and 1000 mg/kg of C. edulis ethanolic extract (E-OH); in addition, 10, 30 and 12 mg/kg of propyleneglycol (Pg), phenytoin (Phen) and phenobarbital (Phb) was orally given to adult male Wistar rat groups. Thereafter, all groups were assayed for protection against maximal electroshock (MES) and pentylenetetrazole (METsc) seizure inducing tests at hourly intervals throughout 8 h. For MES, a maximal protection of 70% at the 2nd and 4th h with 10 mg/kg AQ and 100 mg/kg E-OH doses, occurred. That of Phen, Phb and Pg was 80, 90 and 10% at the 8th, 6th and 2nd h, respectively. The averaged values of the MES unprotected rats under 10 and 100 mg/kg of AQ and E-OH extracts, showed that a shortened reflex duration as well as a delayed latency and uprising times occurred. On the other hand, just an enlarged latency and no protection against METsc device in AQ and EOH was observed. Phen and Phb maximal protection was 80 and 100% at the 4th and 6th hour against METsc. Thus, AQ is tenfold more potent anticonvulsive extract than E-OH against MES. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Casimiroa edulis; Antiepileptic drugs; Epilepsy treatment; Anticonvulsive activity
1. Introduction Casimiroa edulis, Llave et Lex. (La Llave and Lexarza, 1825) is a leafy and fruitful tree that * Corresponding author. E-mail address: [email protected] (A. NavarroRuı´z)
belongs to the Rutaceae family. In Me´xico, C. edulis has been found able to grow abundantly in the west, center, and southeast states of the country (Martı´nez, 1944, 1951). In most states, this tree is known to be medicinal because its leaves and seeds have been used by people in folkloric medicine to prepare teas in order to relief patients from anxiety, insomnia and hypertensive disease
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 2 5 - 7
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(Sahagu´n, 1571; Xime´nez, 1615; Robin and Coyon, 1909; De Lille, 1937; Estrada, 1989; Magos and Vidrio, 1991).Thus, following an ethnomedicinal criteria, scientists have performed their experimental work using leaves and seed hydrolates as well as specific organic solvent fractions of C. edulis to imbibe smooth muscle fibers taken from arteries ‘in vitro’ to demonstrate that after an electrical stimulus, muscle contractile response is less intense than the control. These experiments have been developed to the aim of explaining the assured antihypertensive effects of C. edulis extracts (Lozoya et al., 1977, 1978; Garcı´a-Gonza´lez et al., 1994; Magos et al., 1995). Also, under the same criteria, C. edulis leaves hydrolates were orally given to rats in order to prove that leaf components as a whole exert protective effects against pentylenetetrazole. Under these conditions, the percentage of protection found against METsc induced seizures in rats receiving 100 mg/kg of C. edulis. leaves hydrolates was nearly as efficacious as phenobarbital (12 mg/kg) to inhibit pentylenetetrazole induced seizures in rats that are equivalent to the non-convulsive ones of epileptic patients (Browning and Nelson, 1985; Garzo´n-De la Mora, 1986; Fisher, 1989; Navarro-Ruı´z et al., 1995). On the other hand, these experimental models of epileptiform seizures (Swinyard, 1972; Reinhard and Reinhard, 1977; Fisher, 1989; Swinyard et al., 1989), have been used to validate the anticonvulsive properties of leaves and seed extracts of about 100 plants from 33 families (Chauhan et al., 1988; Garzo´nDe la Mora et al., 1990). However, pure compounds have still not been isolated from them except for C. edulis. Since there are no references pertaining to biological assays using any of the about 25 seed isolated compounds from C. edulis, neither these are available anymore, we thought it important to study C. edulis aqueous (AQ) and ethanolic (E-OH) seed extracts capability to protect rats against the development of maximal electroshock (MES) and pentylenetetrazole (METsc) elicited seizures (Toman et al., 1946; Swinyard et al., 1952; Swinyard, 1972). Therefore, we thought of the possibility of applying a systematic approach to screen C. edulis seed AQ and E-OH extracts anticonvulsive properties using both MES
and METsc experimental models of epileptiform seizures (EMES) as have been done to validate the anticonvulsive activity of all antiepileptic drugs currently in use (Merrit and Putman, 1938; Morselli et al., 1989; Schmidt, 1989).
2. Materials and methods
2.1. Animals Adult male Wistar rats (200–300 g) were randomly grouped in polypropylene cages under animal facility conditions and freely fed with Chow-Purina and water. All experiments took place from 07:00 to 16:00 h.
2.2. Plant collection Ripe fruits from C. edulis were collected at Nextipac, Jalisco, Mexico, botanically identified and registered with the specimen number 95693 for further reference at the Herbarium of the Botany Institute of the University of Guadalajara. Fruit seeds were knife-chopped into small pieces, shade-dried and ground until a moderately coarse powder was obtained and, thereafter were stored in darkness in a dry place at room temperature until the preparation of aqueous extracts and further liophylization.
2.3. C. edulis aqueous seed extract (AQ) C. edulis seeds were shade dried, moderately coarse powdered and kept dried within a chamber containing CaCO3. Afterwards, AQ seed extracts were always prepared at a ratio of 80 g of powder in 1000 ml of boiling distilled water, heated for 5 min, allowed to cool to room temperature and filtered several times through gauze and Whatman paper No. 40 until no precipitate was observed. These extracts were vacuum dried hydrolates in a Model 75050 Labconco freeze dryer for storage until the performance of pharmacological assays. Each 80 g of dried seed powder yielded 2.47 g of lyophilized powder. A solution of 70 mg/ml was prepared using the propylenglycol –ethanol–water (40:10.6:49.4) mixture as vehicle. All experimental
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solutions were freshly prepared at the beginning of each anticonvulsant test.
2.4. Ethanolic extract (E-OH) Seed powder (50 g) was submitted to ethanol (99.99%) soxhlet extraction for 4 h. The solvent was then evaporated in a rotavapor and the ratio of the obtained material was 3.65 g/50 g of seed powder. A solution of 100 mg/ml was prepared using the propylenglycol – ethanol – water (40:10.6:49.4) mixture as vehicle. All experimental solutions were freshly prepared at the beginning of each pharmacological test.
2.5. Maximal electroshock seizure (MES) inducing test A 60 Hz alternating current of 120 V and 150 mA shock was applied to the animals through corneal electrodes for 0.2 s. Electroconductivity was enhanced with two drops of 0.9% NaCl on each eye. The main measurable component of this experimental model is the hind-limb tonic extensor reflex (Swinyard, 1972). The efficiency of a substance to inhibit this reaction suggests an anticonvulsant activity. On the other hand, in order to assure whether a partial protection occur or not, the elapsed time from stimulus application until reflex appearance (latency), length of reflex (reflex) and the elapsed time from reflex ending until the recovery of a normal orthostatic position (uprising) and the spontaneous walking (recovery) is measured. After extracts or drug pre-treatment, those animals that show no hind-limb reflex are considered protected against MES. Such protection is plotted from 0 to 100% of non-covulsing animals. Those convulsing ones might be either unprotected or partly protected whether they are 0% non-convulsing or fall between the range of 10 and 90% hind-limb reflex inhibition. On the other hand, when we plot the hourly means of each hind-limb reflex stage duration (latency, reflex duration, uprising and recovery) of their entire 8 h period of the partly protected animals, the all or none electrical pulse triggering from central nervous system is evaluated (Garzo´n-De la Mora et al., 1990).
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2.6. Metrazole induced seizure (METsc) test The equivalence to 99% convulsive dose (70 mg/kg) of metrazole was dissolved in physiological saline solution (3.5 mg/ml) and then 0.1 mg of metrazole solution was injected into a loose fold of the dorsal skin. Animals were placed in individual cages and their pharmacological response was analyzed throughout 60 min for appearance of at least a 5 s clonic episode (Swinyard et al., 1952). The lack of this crisis after the administration of any substance submitted to study reveals its anticonvulsant properties. Time of latency, which is the elapsed time from injection until spasm occurrence was measured in those animals that developed clonic convulsions. Animals displaying the convulsion within an observational period of 30 min were classified as ‘METsc (+ )’ and the ones that did not develop a crisis as ‘METsc (− )’. Drug protection against METsc is measured in % of non-convulsing animals that were previously classified as METsc (+ ).
2.7. Experimental assays MES or METsc tests were performed in all rats previously to any treatment, in order to assess individual seizure susceptibility and to define the control parameter values. Thereafter, these animals were allowed to recover for 7 days from MES or METsc in order to return to basal central nervous system responses as well as for metrazole clearance (Toman et al., 1946; Garzo´n-De la Mora et al., 1990). Only seizure-susceptible rats (+ ) were included in the experimental groups and were divided into 12 groups to perform MES and 12 for METsc seizure inducing tests.
2.8. Procedure Acute convulsive assays were performed in 12 groups of 160 MES ( +) and 160 METsc ( + ) rats using MES and METsc devices. Such groups were pre-treated as follows: 5, 10 and 100 mg/kg of AQ and 10, 100 and 1000 mg/kg of E-OH extracts as well as 10, 30 and 12 mg/kg of propyleneglycol (Pg), phenytoin (Phen) and phenobarbital (Phb). Seizure protective effects were
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measured in subgroups at hourly intervals throughout 8 h. AQ, E-OH extracts, Phen, Phb and Pg were orally given once in propylenglycol – ethyl alcohol–water (40:10.6:49.4), through an esophageal cannula. Eight subgroups of 20 rats per dose were tested in order to measure the exact time when the maximal inhibitory activity against induced convulsions appeared, along the 8 h period. Duration of hind-limb stages in the MES test and that of latency in METsc was measured in seconds in each partly protected rat of every hourly tested subgroup, at each dose of the C. edulis extracts and drugs throughout 8 h.
2.9. Data analysis Our experimental design was set up to seek the events which occur at a confidence level of P B 0.05 in order to analyze whether a uniform response of each stage of the hind-limb reflex in partly protected rats occurs or not. The anticonvulsant effect of rats with extracts, drugs and vehicle against MES or METsc convulsing devices was expressed as percentage of non-convulsed
animals (abolition of seizure). Hind-limb reflex duration, latency, recovery and uprising times as well as METsc latency were reported as the mean and standard error of the mean (in seconds) and ANOVA treated. All results were Student t-test validated at PB 0.05.
3. Results
3.1. Anticon6ulsi6e acti6ity A single oral administration of 10 mg/kg of AQ extract was able to inhibit the MES induced hindlimb reflex in 30, 70, 20, 55, 50, 50, 40 and 40% of the assayed population at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th h, respectively. The subgroup at the 2nd h displayed the maximal protection value of 70% against MES. Those rats under 5 mg/kg displayed a maximum of 30% protection against MES at the 2nd, 3rd and 6th h and those under 100 mg/kg showed a maximum value of 40% protection at the 5th and 7th h, respectively (Fig. 1). A single oral administration of 100 mg/kg of E-OH extract was able to inhibit the MES induced hind-limb reflex in 10, 30, 60, 70, 50, 35, 50 and 30% of the assayed population at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th h, respectively. As above, the subgroup of the 4th h showed the maximal value of (70%) protection against MES elicited seizures. The lowest dose of 10 mg/kg and the highest of 1000 mg/kg of E-OH had a lower protection pattern than 100 mg/kg showing maximal inhibition values of 30 and 10%, respectively (Fig. 2).
3.2. Latency
Fig. 1. Anticonvulsive pattern of 5, 10 and 100 mg/kg of C. edulis AQ against MES induced seizures. Hind-limb extensor reflex was MES elicited at hourly intervals throughout an 8 h consecutive period in individual subgroups of 20 rats per hour. Values are expressed in % of abolition of the reflex.
The MES latency duration of hind-limb reflex hourly means in partly protected rats were pooled and for the three dose levels of 5, 10 and 100 mg/kg of AQ. The obtained values were 2.6 9 0.01 (P B0.05), 3.09 0.01 (NS) and 3.699 0.01 (PB 0.05), respectively, in comparison to the control that corresponded to 2.96 9 0.01. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a latency of
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values were 125.389 2.11 (PB 0.05), 201.759 18.13 (PB0.05) and 202.88 9 19.14 (PB 0.05), in comparison to the control that corresponded to 151.359 1.51. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a latency of 227.38912.86 (PB 0.05), 194.25 9 13.59 (PB 0.05) and 162.5 9 7.86 (PB 0.05).
3.5. Reco6ery
Fig. 2. Anticonvulsive pattern displayed by 10, 100 and 1000 mg/kg of C. edulis E-OH against MES induced seizures. Convulsive seizures were elicited at hourly intervals during an 8 h consecutive period in individual subgroups of 20 rats per hour. Values are expressed in % of abolition of the reflex.
2.999 0.01 (NS), 3.05 90.03 (P B0.05) and 2.289 0.05 (PB 0.05).
3.3. Reflex duration Hind-limb reflex duration in partly protected groups under 10 and 100 mg/kg AQ or 10 and 100 mg/kg E-OH extracts had a significantly lower duration than the control (9.9390.05). The lowest duration of the hind-limb reflex of 6.9 90. 02 (PB 0.05) and 7.890.04 (P B 0.05) was seen in those subgroups of rats receiving 10 and 100 mg/kg AQ. Also, statistically significant were those values of 8.49 0.05 (P B 0.05) and 7.79 0.036 (P B 0.05) for 10 and 100 mg/kg of E-OH except in groups under 5 mg/kg AQ and 1000 mg/kg E-OH whose values were 10.059 0.05 (NS) and 9.6890.02 (NS), respectively.
3.4. Uprising The MES uprising duration after the hind-limb reflex values in partly protected rats for 5, 10 and 100 mg/kg of AQ were plotted. The obtained
The MES recovery duration after hind-limb reflex in partly protected rats was plotted for dose leels of 5,10 and 100 mg/kg of AQ whose values were 138.98 9 11.16 (PB 0.05), 256.5 9 24.1 (PB 0.05) and 466.59 39.53 (PB0.05), in comparison to the control that corresponded to 342.639 31.24. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a recovery time of 176.259 13.01 (PB 0.05), 158.5 9 28.33 (PB0.05) and 169.82 9 16.89 (PB 0.05).
3.6. Latency (METsc) In METsc tested groups, latency was always longer in experimental animals than the control and Pg, correspondingly. The averaged latency values of METsc tested groups were 11.69 0.1, 11.09 0.2, 16.79 0.14, 16.690.14 and 15.79 0.2 min for the control, Pg, 5, 10 and 100 mg/kg of AQ and 16.490.15, 16.19 0.2 and 11.69 0.7 min. for 10, 100 and 1000 mg/kg of E-OH, all respectively. A statistically significant difference was observed in the latency of those animals treated with 5, 10 and 100 mg/kg AQ (PB 0.05) or 10 and 100 mg/kg of E-OH (PB0.05).
3.7. Maximal protection of extracts and antiepileptic drugs The percentage of maximal protection (%) as well as its occurrence time (h) within the 8 h experimental period for either extracts or drugs against induced seizures using MES and METsc devices, were compared. The maximum protection found against MES for AQ, E-OH, Phen, Phb and Pg was 70, 70, 80, 90 and 10% at the 2nd,
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4th, 8th, 6th and 2nd h, respectively. On the other hand, the maximal protection found for AQ, EOH, Phen, Phb, and Pg groups against METsc was 10, 0, 80, 100 and 0, at the 2nd, 1st to 8th, 4th, 6th h, and 1st to 8th h, respectively.
4. Discussion The strategy of investigating the most commonly used popular hypnogenic remedies in our country has allowed us to test 12 medicinal plants for their anticonvulsive properties in our laboratory. Although all of the 12 plants were said to exert hypnogenic and sedative effects in people, only C. edulis leaf hydrolates showed the most promising anticonvulsive activity in Wistar rats when we assayed it against EMES (Garzo´n-De la Mora, 1986); however, it was not until 1995 when we finished a series of experiments that were performed using MES and METsc among EMES that we were able to assert that C. edulis leaf extracts are effective at inhibiting pentylenetetrazole elicited seizures in rats and, that this knowledge is in accordance with the conventionally accepted screening processes that have been used to evaluate new anticonvulsive compounds (Toman et al., 1946; Swinyard et al., 1952; Swinyard, 1972). Therefore, we were able to ascertain leaf hydrolates efficacy to prevent METsc elicitable seizures to appear and, by this means would potentially block the appearance of non-convulsive seizures in epileptic patients (Garzo´n-De la Mora, 1986; Navarro-Ruı´z et al., 1995). On the other hand, although we know that about 25 substances of known chemical structure from C. edulis leaves and seeds were identified by investigators from 1910 to 1970 (Power and Callan, 1911; Morton, 1962; Lozoya, 1977; Lozoya and Enrı´quez, 1981), they still have not been biologically assayed. Because such isolated compounds from C. edulis are not available anymore nor are there some in enough quantity to screen their anticonvulsive properties, nobody knows what substance would be better to synthesize in order to perform biological assays. Therefore, we decided to apply the nowadays accepted EMES to ascertain that C. edulis seed AQ and E-OH are
efficacious in preventing 70% of pre-treated animals from the development of hind-limb reflex after MES. On these grounds, AQ was able to suppress the hind-limb extensor reflex appearance in rats under 10 mg/kg at the 2nd h and was 10-fold more effective than E-OH in attaining the same effects at the 4th h. On the other hand, an average behavior of the hind-limb reflex stages such as long latency, shortened reflex, recovery and uprising duration in both AQ and E-OH treated groups was observed. Also, it is noticeable that the average of the eight subgroups mean values allowed us to assert that: (1) the hind-limb reflex is an all or none physiological response in control rats as well as in experimental animals. (2) Each hind-limb extensor reflex stage is similarly affected by AQ and E-OH during the 8 h experimental period as described in the results. It means that the physiological response ‘hind-limb extensor reflex’ after the electrical stimulus at any hour of the 8 h experimental period is proportionally time-duration modified by AQ, E-OH and drugs when compared to the control; however, neither the elapsed time after the single oral intake of AQ or E-OH nor their doses affect the quality of the physiological response. (3) AQ and E-OH only delayed the response to the MES and METsc devices. The AQ and E-OH maximal protection of 70% against MES that was registered to occur at the 2nd and 4th h, respectively, is close to the maximal protection of 80 and 90% recorded for phenytoin and phenobarbital at the 2nd and 6th h, respectively. Phen response in these experiments performed un rats was attained with a dose that is four times higher than the ussual one of 10 mg/kg to prevent epileptic patients from the development of tonic-clonic epileptic seizures. AQ and E-OH were useless in protecting against the METsc device when compared to the maximal protection found for Phen and Phb of 70 and 100% at the 4th and 6th h. Vehicle (Pg) did not protect animals from the development of MES and METsc elicited seizures. Although AQ and E-OH were unable to protect against METsc, the averaged latency values in min of METsc tested groups were 16.21 AQ, 16.06 E-OH, 38 Phen, \ 60 Phb in comparison to
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11.2 control and 11.7 Pg. This means that almost no protection occurred in animals that received either AQ or E-OH when tested against METsc and, that seed extracts are useless to control nonconvulsive seizures in epileptic patients; however, both AQ and E-OH would be efficacious to control convulsive epileptic seizures in patients. Lastly, it can be assured from these experiments that enough data pertaining to toxicological assays have been achieved for AQ and E-OH in the herein described results, because the administered doses of 1000 mg/kg of E-OH yielded no casualties during the 8 h assay period in so many subgroups of 20 rats each. On the other hand, since we know that a plant extract can be considered non-toxic at 1000 mg/kg, also, because we have found that the more efficacious dose of 10 mg/kg and 100 mg/kg of AQ and E-OH were able to inhibit seizures maximally and, these doses are above the ED50, then the TD50 of C. edulis extracts is superior to 1000 mg/kg and might allow us to begin a stage 1 study in epileptic patients who suffer difficult to control epileptic seizures despite of receiving three to four antiepileptic drugs simultaneously.
Acknowledgements The authors of this work gratefully acknowledge the technical assistance of A.A. Gonza´lezBallesteros. We also gratefully acknowledge L. Miguel Aguilar-De la Mora, Humberto BasultoVelasco and Rau´l Orozco-Martı´nez for their constant and excellent work at the Bioterium.
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Casimiroa edulis seed extracts show anticonvulsive properties in rats P. Garzo´n-De la Mora a,c, P.M. Garcı´a-Lo´pez b, J. Garcı´a-Estrada a, A. Navarro-Ruı´z a,*, T. Villanueva-Michel c, L.Ma. Villarreal-de Puga b, J. Casillas-Ochoa c a
b
C.I.BO., I.M.S.S, Guadalajara, PO Box 1 -3838, Jalisco, Mexico Laboratorio de Biotecnologı´a, Instituto de Bota´nica y Zoologı´a, C.U.C.B.A., Uni6ersidad de Guadalajara, Guadalajara, Mexico c Laboratorio de Bioquı´mica, Depto. de Fisiologia, Centro Uni6ersitario de Ciencias de la Salud, Uni6ersidad de Guadalajara, Guadalajara, Mexico Received 13 July 1998; received in revised form 22 June 1999; accepted 6 July 1999
Abstract A single dose of 5, 10 and 100 mg/kg of Casimiroa edulis aqueous extract (AQ); 10, 100 and 1000 mg/kg of C. edulis ethanolic extract (E-OH); in addition, 10, 30 and 12 mg/kg of propyleneglycol (Pg), phenytoin (Phen) and phenobarbital (Phb) was orally given to adult male Wistar rat groups. Thereafter, all groups were assayed for protection against maximal electroshock (MES) and pentylenetetrazole (METsc) seizure inducing tests at hourly intervals throughout 8 h. For MES, a maximal protection of 70% at the 2nd and 4th h with 10 mg/kg AQ and 100 mg/kg E-OH doses, occurred. That of Phen, Phb and Pg was 80, 90 and 10% at the 8th, 6th and 2nd h, respectively. The averaged values of the MES unprotected rats under 10 and 100 mg/kg of AQ and E-OH extracts, showed that a shortened reflex duration as well as a delayed latency and uprising times occurred. On the other hand, just an enlarged latency and no protection against METsc device in AQ and EOH was observed. Phen and Phb maximal protection was 80 and 100% at the 4th and 6th hour against METsc. Thus, AQ is tenfold more potent anticonvulsive extract than E-OH against MES. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Casimiroa edulis; Antiepileptic drugs; Epilepsy treatment; Anticonvulsive activity
1. Introduction Casimiroa edulis, Llave et Lex. (La Llave and Lexarza, 1825) is a leafy and fruitful tree that * Corresponding author. E-mail address: [email protected] (A. NavarroRuı´z)
belongs to the Rutaceae family. In Me´xico, C. edulis has been found able to grow abundantly in the west, center, and southeast states of the country (Martı´nez, 1944, 1951). In most states, this tree is known to be medicinal because its leaves and seeds have been used by people in folkloric medicine to prepare teas in order to relief patients from anxiety, insomnia and hypertensive disease
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 2 5 - 7
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(Sahagu´n, 1571; Xime´nez, 1615; Robin and Coyon, 1909; De Lille, 1937; Estrada, 1989; Magos and Vidrio, 1991).Thus, following an ethnomedicinal criteria, scientists have performed their experimental work using leaves and seed hydrolates as well as specific organic solvent fractions of C. edulis to imbibe smooth muscle fibers taken from arteries ‘in vitro’ to demonstrate that after an electrical stimulus, muscle contractile response is less intense than the control. These experiments have been developed to the aim of explaining the assured antihypertensive effects of C. edulis extracts (Lozoya et al., 1977, 1978; Garcı´a-Gonza´lez et al., 1994; Magos et al., 1995). Also, under the same criteria, C. edulis leaves hydrolates were orally given to rats in order to prove that leaf components as a whole exert protective effects against pentylenetetrazole. Under these conditions, the percentage of protection found against METsc induced seizures in rats receiving 100 mg/kg of C. edulis. leaves hydrolates was nearly as efficacious as phenobarbital (12 mg/kg) to inhibit pentylenetetrazole induced seizures in rats that are equivalent to the non-convulsive ones of epileptic patients (Browning and Nelson, 1985; Garzo´n-De la Mora, 1986; Fisher, 1989; Navarro-Ruı´z et al., 1995). On the other hand, these experimental models of epileptiform seizures (Swinyard, 1972; Reinhard and Reinhard, 1977; Fisher, 1989; Swinyard et al., 1989), have been used to validate the anticonvulsive properties of leaves and seed extracts of about 100 plants from 33 families (Chauhan et al., 1988; Garzo´nDe la Mora et al., 1990). However, pure compounds have still not been isolated from them except for C. edulis. Since there are no references pertaining to biological assays using any of the about 25 seed isolated compounds from C. edulis, neither these are available anymore, we thought it important to study C. edulis aqueous (AQ) and ethanolic (E-OH) seed extracts capability to protect rats against the development of maximal electroshock (MES) and pentylenetetrazole (METsc) elicited seizures (Toman et al., 1946; Swinyard et al., 1952; Swinyard, 1972). Therefore, we thought of the possibility of applying a systematic approach to screen C. edulis seed AQ and E-OH extracts anticonvulsive properties using both MES
and METsc experimental models of epileptiform seizures (EMES) as have been done to validate the anticonvulsive activity of all antiepileptic drugs currently in use (Merrit and Putman, 1938; Morselli et al., 1989; Schmidt, 1989).
2. Materials and methods
2.1. Animals Adult male Wistar rats (200–300 g) were randomly grouped in polypropylene cages under animal facility conditions and freely fed with Chow-Purina and water. All experiments took place from 07:00 to 16:00 h.
2.2. Plant collection Ripe fruits from C. edulis were collected at Nextipac, Jalisco, Mexico, botanically identified and registered with the specimen number 95693 for further reference at the Herbarium of the Botany Institute of the University of Guadalajara. Fruit seeds were knife-chopped into small pieces, shade-dried and ground until a moderately coarse powder was obtained and, thereafter were stored in darkness in a dry place at room temperature until the preparation of aqueous extracts and further liophylization.
2.3. C. edulis aqueous seed extract (AQ) C. edulis seeds were shade dried, moderately coarse powdered and kept dried within a chamber containing CaCO3. Afterwards, AQ seed extracts were always prepared at a ratio of 80 g of powder in 1000 ml of boiling distilled water, heated for 5 min, allowed to cool to room temperature and filtered several times through gauze and Whatman paper No. 40 until no precipitate was observed. These extracts were vacuum dried hydrolates in a Model 75050 Labconco freeze dryer for storage until the performance of pharmacological assays. Each 80 g of dried seed powder yielded 2.47 g of lyophilized powder. A solution of 70 mg/ml was prepared using the propylenglycol –ethanol–water (40:10.6:49.4) mixture as vehicle. All experimental
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solutions were freshly prepared at the beginning of each anticonvulsant test.
2.4. Ethanolic extract (E-OH) Seed powder (50 g) was submitted to ethanol (99.99%) soxhlet extraction for 4 h. The solvent was then evaporated in a rotavapor and the ratio of the obtained material was 3.65 g/50 g of seed powder. A solution of 100 mg/ml was prepared using the propylenglycol – ethanol – water (40:10.6:49.4) mixture as vehicle. All experimental solutions were freshly prepared at the beginning of each pharmacological test.
2.5. Maximal electroshock seizure (MES) inducing test A 60 Hz alternating current of 120 V and 150 mA shock was applied to the animals through corneal electrodes for 0.2 s. Electroconductivity was enhanced with two drops of 0.9% NaCl on each eye. The main measurable component of this experimental model is the hind-limb tonic extensor reflex (Swinyard, 1972). The efficiency of a substance to inhibit this reaction suggests an anticonvulsant activity. On the other hand, in order to assure whether a partial protection occur or not, the elapsed time from stimulus application until reflex appearance (latency), length of reflex (reflex) and the elapsed time from reflex ending until the recovery of a normal orthostatic position (uprising) and the spontaneous walking (recovery) is measured. After extracts or drug pre-treatment, those animals that show no hind-limb reflex are considered protected against MES. Such protection is plotted from 0 to 100% of non-covulsing animals. Those convulsing ones might be either unprotected or partly protected whether they are 0% non-convulsing or fall between the range of 10 and 90% hind-limb reflex inhibition. On the other hand, when we plot the hourly means of each hind-limb reflex stage duration (latency, reflex duration, uprising and recovery) of their entire 8 h period of the partly protected animals, the all or none electrical pulse triggering from central nervous system is evaluated (Garzo´n-De la Mora et al., 1990).
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2.6. Metrazole induced seizure (METsc) test The equivalence to 99% convulsive dose (70 mg/kg) of metrazole was dissolved in physiological saline solution (3.5 mg/ml) and then 0.1 mg of metrazole solution was injected into a loose fold of the dorsal skin. Animals were placed in individual cages and their pharmacological response was analyzed throughout 60 min for appearance of at least a 5 s clonic episode (Swinyard et al., 1952). The lack of this crisis after the administration of any substance submitted to study reveals its anticonvulsant properties. Time of latency, which is the elapsed time from injection until spasm occurrence was measured in those animals that developed clonic convulsions. Animals displaying the convulsion within an observational period of 30 min were classified as ‘METsc (+ )’ and the ones that did not develop a crisis as ‘METsc (− )’. Drug protection against METsc is measured in % of non-convulsing animals that were previously classified as METsc (+ ).
2.7. Experimental assays MES or METsc tests were performed in all rats previously to any treatment, in order to assess individual seizure susceptibility and to define the control parameter values. Thereafter, these animals were allowed to recover for 7 days from MES or METsc in order to return to basal central nervous system responses as well as for metrazole clearance (Toman et al., 1946; Garzo´n-De la Mora et al., 1990). Only seizure-susceptible rats (+ ) were included in the experimental groups and were divided into 12 groups to perform MES and 12 for METsc seizure inducing tests.
2.8. Procedure Acute convulsive assays were performed in 12 groups of 160 MES ( +) and 160 METsc ( + ) rats using MES and METsc devices. Such groups were pre-treated as follows: 5, 10 and 100 mg/kg of AQ and 10, 100 and 1000 mg/kg of E-OH extracts as well as 10, 30 and 12 mg/kg of propyleneglycol (Pg), phenytoin (Phen) and phenobarbital (Phb). Seizure protective effects were
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measured in subgroups at hourly intervals throughout 8 h. AQ, E-OH extracts, Phen, Phb and Pg were orally given once in propylenglycol – ethyl alcohol–water (40:10.6:49.4), through an esophageal cannula. Eight subgroups of 20 rats per dose were tested in order to measure the exact time when the maximal inhibitory activity against induced convulsions appeared, along the 8 h period. Duration of hind-limb stages in the MES test and that of latency in METsc was measured in seconds in each partly protected rat of every hourly tested subgroup, at each dose of the C. edulis extracts and drugs throughout 8 h.
2.9. Data analysis Our experimental design was set up to seek the events which occur at a confidence level of P B 0.05 in order to analyze whether a uniform response of each stage of the hind-limb reflex in partly protected rats occurs or not. The anticonvulsant effect of rats with extracts, drugs and vehicle against MES or METsc convulsing devices was expressed as percentage of non-convulsed
animals (abolition of seizure). Hind-limb reflex duration, latency, recovery and uprising times as well as METsc latency were reported as the mean and standard error of the mean (in seconds) and ANOVA treated. All results were Student t-test validated at PB 0.05.
3. Results
3.1. Anticon6ulsi6e acti6ity A single oral administration of 10 mg/kg of AQ extract was able to inhibit the MES induced hindlimb reflex in 30, 70, 20, 55, 50, 50, 40 and 40% of the assayed population at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th h, respectively. The subgroup at the 2nd h displayed the maximal protection value of 70% against MES. Those rats under 5 mg/kg displayed a maximum of 30% protection against MES at the 2nd, 3rd and 6th h and those under 100 mg/kg showed a maximum value of 40% protection at the 5th and 7th h, respectively (Fig. 1). A single oral administration of 100 mg/kg of E-OH extract was able to inhibit the MES induced hind-limb reflex in 10, 30, 60, 70, 50, 35, 50 and 30% of the assayed population at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th h, respectively. As above, the subgroup of the 4th h showed the maximal value of (70%) protection against MES elicited seizures. The lowest dose of 10 mg/kg and the highest of 1000 mg/kg of E-OH had a lower protection pattern than 100 mg/kg showing maximal inhibition values of 30 and 10%, respectively (Fig. 2).
3.2. Latency
Fig. 1. Anticonvulsive pattern of 5, 10 and 100 mg/kg of C. edulis AQ against MES induced seizures. Hind-limb extensor reflex was MES elicited at hourly intervals throughout an 8 h consecutive period in individual subgroups of 20 rats per hour. Values are expressed in % of abolition of the reflex.
The MES latency duration of hind-limb reflex hourly means in partly protected rats were pooled and for the three dose levels of 5, 10 and 100 mg/kg of AQ. The obtained values were 2.6 9 0.01 (P B0.05), 3.09 0.01 (NS) and 3.699 0.01 (PB 0.05), respectively, in comparison to the control that corresponded to 2.96 9 0.01. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a latency of
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values were 125.389 2.11 (PB 0.05), 201.759 18.13 (PB0.05) and 202.88 9 19.14 (PB 0.05), in comparison to the control that corresponded to 151.359 1.51. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a latency of 227.38912.86 (PB 0.05), 194.25 9 13.59 (PB 0.05) and 162.5 9 7.86 (PB 0.05).
3.5. Reco6ery
Fig. 2. Anticonvulsive pattern displayed by 10, 100 and 1000 mg/kg of C. edulis E-OH against MES induced seizures. Convulsive seizures were elicited at hourly intervals during an 8 h consecutive period in individual subgroups of 20 rats per hour. Values are expressed in % of abolition of the reflex.
2.999 0.01 (NS), 3.05 90.03 (P B0.05) and 2.289 0.05 (PB 0.05).
3.3. Reflex duration Hind-limb reflex duration in partly protected groups under 10 and 100 mg/kg AQ or 10 and 100 mg/kg E-OH extracts had a significantly lower duration than the control (9.9390.05). The lowest duration of the hind-limb reflex of 6.9 90. 02 (PB 0.05) and 7.890.04 (P B 0.05) was seen in those subgroups of rats receiving 10 and 100 mg/kg AQ. Also, statistically significant were those values of 8.49 0.05 (P B 0.05) and 7.79 0.036 (P B 0.05) for 10 and 100 mg/kg of E-OH except in groups under 5 mg/kg AQ and 1000 mg/kg E-OH whose values were 10.059 0.05 (NS) and 9.6890.02 (NS), respectively.
3.4. Uprising The MES uprising duration after the hind-limb reflex values in partly protected rats for 5, 10 and 100 mg/kg of AQ were plotted. The obtained
The MES recovery duration after hind-limb reflex in partly protected rats was plotted for dose leels of 5,10 and 100 mg/kg of AQ whose values were 138.98 9 11.16 (PB 0.05), 256.5 9 24.1 (PB 0.05) and 466.59 39.53 (PB0.05), in comparison to the control that corresponded to 342.639 31.24. Those subgroups of partly protected rats receiving 10, 100 and 1000 mg/kg of E-OH showed a recovery time of 176.259 13.01 (PB 0.05), 158.5 9 28.33 (PB0.05) and 169.82 9 16.89 (PB 0.05).
3.6. Latency (METsc) In METsc tested groups, latency was always longer in experimental animals than the control and Pg, correspondingly. The averaged latency values of METsc tested groups were 11.69 0.1, 11.09 0.2, 16.79 0.14, 16.690.14 and 15.79 0.2 min for the control, Pg, 5, 10 and 100 mg/kg of AQ and 16.490.15, 16.19 0.2 and 11.69 0.7 min. for 10, 100 and 1000 mg/kg of E-OH, all respectively. A statistically significant difference was observed in the latency of those animals treated with 5, 10 and 100 mg/kg AQ (PB 0.05) or 10 and 100 mg/kg of E-OH (PB0.05).
3.7. Maximal protection of extracts and antiepileptic drugs The percentage of maximal protection (%) as well as its occurrence time (h) within the 8 h experimental period for either extracts or drugs against induced seizures using MES and METsc devices, were compared. The maximum protection found against MES for AQ, E-OH, Phen, Phb and Pg was 70, 70, 80, 90 and 10% at the 2nd,
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4th, 8th, 6th and 2nd h, respectively. On the other hand, the maximal protection found for AQ, EOH, Phen, Phb, and Pg groups against METsc was 10, 0, 80, 100 and 0, at the 2nd, 1st to 8th, 4th, 6th h, and 1st to 8th h, respectively.
4. Discussion The strategy of investigating the most commonly used popular hypnogenic remedies in our country has allowed us to test 12 medicinal plants for their anticonvulsive properties in our laboratory. Although all of the 12 plants were said to exert hypnogenic and sedative effects in people, only C. edulis leaf hydrolates showed the most promising anticonvulsive activity in Wistar rats when we assayed it against EMES (Garzo´n-De la Mora, 1986); however, it was not until 1995 when we finished a series of experiments that were performed using MES and METsc among EMES that we were able to assert that C. edulis leaf extracts are effective at inhibiting pentylenetetrazole elicited seizures in rats and, that this knowledge is in accordance with the conventionally accepted screening processes that have been used to evaluate new anticonvulsive compounds (Toman et al., 1946; Swinyard et al., 1952; Swinyard, 1972). Therefore, we were able to ascertain leaf hydrolates efficacy to prevent METsc elicitable seizures to appear and, by this means would potentially block the appearance of non-convulsive seizures in epileptic patients (Garzo´n-De la Mora, 1986; Navarro-Ruı´z et al., 1995). On the other hand, although we know that about 25 substances of known chemical structure from C. edulis leaves and seeds were identified by investigators from 1910 to 1970 (Power and Callan, 1911; Morton, 1962; Lozoya, 1977; Lozoya and Enrı´quez, 1981), they still have not been biologically assayed. Because such isolated compounds from C. edulis are not available anymore nor are there some in enough quantity to screen their anticonvulsive properties, nobody knows what substance would be better to synthesize in order to perform biological assays. Therefore, we decided to apply the nowadays accepted EMES to ascertain that C. edulis seed AQ and E-OH are
efficacious in preventing 70% of pre-treated animals from the development of hind-limb reflex after MES. On these grounds, AQ was able to suppress the hind-limb extensor reflex appearance in rats under 10 mg/kg at the 2nd h and was 10-fold more effective than E-OH in attaining the same effects at the 4th h. On the other hand, an average behavior of the hind-limb reflex stages such as long latency, shortened reflex, recovery and uprising duration in both AQ and E-OH treated groups was observed. Also, it is noticeable that the average of the eight subgroups mean values allowed us to assert that: (1) the hind-limb reflex is an all or none physiological response in control rats as well as in experimental animals. (2) Each hind-limb extensor reflex stage is similarly affected by AQ and E-OH during the 8 h experimental period as described in the results. It means that the physiological response ‘hind-limb extensor reflex’ after the electrical stimulus at any hour of the 8 h experimental period is proportionally time-duration modified by AQ, E-OH and drugs when compared to the control; however, neither the elapsed time after the single oral intake of AQ or E-OH nor their doses affect the quality of the physiological response. (3) AQ and E-OH only delayed the response to the MES and METsc devices. The AQ and E-OH maximal protection of 70% against MES that was registered to occur at the 2nd and 4th h, respectively, is close to the maximal protection of 80 and 90% recorded for phenytoin and phenobarbital at the 2nd and 6th h, respectively. Phen response in these experiments performed un rats was attained with a dose that is four times higher than the ussual one of 10 mg/kg to prevent epileptic patients from the development of tonic-clonic epileptic seizures. AQ and E-OH were useless in protecting against the METsc device when compared to the maximal protection found for Phen and Phb of 70 and 100% at the 4th and 6th h. Vehicle (Pg) did not protect animals from the development of MES and METsc elicited seizures. Although AQ and E-OH were unable to protect against METsc, the averaged latency values in min of METsc tested groups were 16.21 AQ, 16.06 E-OH, 38 Phen, \ 60 Phb in comparison to
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11.2 control and 11.7 Pg. This means that almost no protection occurred in animals that received either AQ or E-OH when tested against METsc and, that seed extracts are useless to control nonconvulsive seizures in epileptic patients; however, both AQ and E-OH would be efficacious to control convulsive epileptic seizures in patients. Lastly, it can be assured from these experiments that enough data pertaining to toxicological assays have been achieved for AQ and E-OH in the herein described results, because the administered doses of 1000 mg/kg of E-OH yielded no casualties during the 8 h assay period in so many subgroups of 20 rats each. On the other hand, since we know that a plant extract can be considered non-toxic at 1000 mg/kg, also, because we have found that the more efficacious dose of 10 mg/kg and 100 mg/kg of AQ and E-OH were able to inhibit seizures maximally and, these doses are above the ED50, then the TD50 of C. edulis extracts is superior to 1000 mg/kg and might allow us to begin a stage 1 study in epileptic patients who suffer difficult to control epileptic seizures despite of receiving three to four antiepileptic drugs simultaneously.
Acknowledgements The authors of this work gratefully acknowledge the technical assistance of A.A. Gonza´lezBallesteros. We also gratefully acknowledge L. Miguel Aguilar-De la Mora, Humberto BasultoVelasco and Rau´l Orozco-Martı´nez for their constant and excellent work at the Bioterium.
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