anticonvulsant effects of aqueous and ethanolic extracts of crocus sativus l. stigmas in mice

Hossein Hosseinzadeh PhD, Vahid Khosravan

Faculty of Pharmacy, Mashad University of Medical Sciences, Mashad, Iran

Correspondence: H. Hosseinzadeh PhD, Faculty of Pharmacy, Mashad University of Medical Sciences, Mashhad, Iran. P.O.Box: 91775-1365, Fax: +98-511-8437075, E-mail: hosseinzadehh@yahoo.com.


Abstract

Background-Crocus sativus L. stigma (CSS) has sedative properties and is used in traditional medicine for its anticonvulsant property.

Objective-We studied the anticonvulsant activity of the aqueous and ethanolic extracts of CSS in mice in order to evaluate the traditional use of this plant.

Methods-The pentylenetetrazole (PTZ) and the maximal electroshock seizure (MES) tests were used for assessing the anticonvulsive effects of this plant.

Results-In the PTZ test, CSS delayed the onset of tonic convulsions, but failed to produce complete protection against mortality. In the MES test, both extracts decreased the duration of tonic seizures.

Conclusion-The results of this study indicate that the extracts of CSS may be beneficial in both absence and tonic clonic seizures.


Keywords Crocus sativus L. stigmas anticonvulsant activity herbal medicine

Introduction

The stigma of the plant Crocus sativus L., commonly known as saffron is used in traditional medicine as an aphrodisiac, antispasmodic and expectorant.1 Recent pharmacological studies have demonstrated that saffron extract has antitumor2-4 and hypolipidemic effects5 as well as radical scavengering and learning or memory-improving properties.5-6

Chemical studies have shown that C. sativus contains constituents such as crocin, crocetin safranal and picrocrocin.7-9 Among the constituents of saffron extract, crocetin is mainly responsible for the above pharmacological activities.5

In traditional medicine, the stigmas of this plant have been used as an anticonvulsant remedy.1 The aim of this study was to evaluate the anticonvulsant effect of C. sativus stigma by the maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) tests.

Materials and Methods

Animals

Male and female albino mice, weighing 25 to30 g, were obtained from a randomly-bred colony and maintained on a special diet (Khorassan Javaneh Co, Mashad, Iran). They were kept in the animal house of Mashad University of Medical Sciences, in colony rooms with 12/12 h light/dark cycle at 21±2˚ C. The animals had free access to food and water. The mice were divided into 4 groups as follows: 144 mice in the PTZ seizure test group, 144 mice in the MES group, 96 mice in the toxicity and 20 mice in the best time of treatment group.

Plant material

Stigmas were collected from the suburbs of Ghaen City (North Iran) in October 1998, dried in the shadow, and subsequently grounded. The C. sativus L. plants were identified by Ferdowsi University (Ms Molaei) and voucher samples were preserved for reference in the herbarium of the Faculty of Pharmacy of Mashhad Universityof Medical Sciences. The specimen number of the plant is 134-0319-1.

Table 1. Effect of aqueous extract of Crocus sativus stigmas on the onset of seizure of pentylenetetrazole-induced convulsion and death in mice.

Treatment

Dose

Onset of seizure (sec)

Mortality protection (%)

Normal saline

20 mL/kg

43.9±0.9

0

Phenobarbital

10 mg/kg 20 mg/kg 30 mg/kg 40 mg/kg

90.0±1.7* 172.3±3.3* 296.5±3.9* 372.9±2.4*

0 37.5 62.5 100

Aqueous extract

0.08 g/kg 0.32 g/kg 0.56 g/kg 0.80 g/kg

86.5±4.2* 135.32±1.6* 300.3±0.8* 267.3±3.8*

0 0 25 25

The extracts and phenobarbital were injected intraperitoneally 20 and 45 min before the administration of pentylenetetrazole (IP, 90 mg/kg), respectively. Values are presented as mean±SEM for the duration of tonic seizure for 8 mice; *p<0.001, compared to normal saline using Tukey-Kramer test.

Preparation of extracts

In the maceration method, 3g of stigma was macerated in 400 mL ethanol (80%, v/v) (yield: 62.43) or distilled water (yield: 60.67) for three days. The mixture was subsequently filtered and concentrated under reduced pressure at 35˚ C.

Anticonvulsant activity

Pentylenetetrazole (PTZ) seizure test10

A total of 144 mice were divided into 8 groups. The aqueous and ethanolic extracts as well as phenobarbital (Merck, Germany) were injected intraperitoneally 20 and 45 min before administration of 90mg/kg pentylenetetrazole (Aldrich, Germany), respectively. Time before onset of clonic convulsions and the percentage of mortality were recorded.

Maximal electroshock seizure (MES) test10

A total of 144 mice were divided into 8 groups. An alternating current of 50 Hz and 150 mA was delivered to experimental animals through bicorneal electrodes for 0.2 sec. A drop of 0.9% saline solution was poured into both eyes prior to placing the electrodes. The aqueous and ethanolic extracts as well as phenobarbital were injected intraperitoneally 45 min before induction of MES. Duration of tonic convulsions (a hindlimb tonic extension) and the percentage of seizure protection and mortality were recorded.

The maximum non-fatal dose and acute toxicity

A total of 96 mice were divided into 4 groups and different doses of the extracts were injected to them. After 48 h, the maximum dose that had not induced mortality, was considered as the maximum non-fatal dose. LD50 values and the corresponding confidence intervals were determined by the Litchfield and Wilcoxon methods (PHARM/PCS Version 4, USA).

Data were expressed as mean values±SEM and tested with ANOVA and Tukey-Kramer tests.

Results

The maximum non-fatal doses of the aqueous and ethanolic extracts were 0.8 and 2 g/kg, respectively. LD50 values of aqueous and ethanolic extracts were 1.6 g/kg (95% CI: 1.2-2.2) and 3.4 g/kg (95% CI: 2.5-4.5), respectively.

Table 2. Effect of ethanolic extract of Crocus sativus stigma on the onset of seizure of pentylenetetrazole-induced convulsion and mortality in mice.

Treatment

Dose

Onset of seizure (sec)

Mortality protection (%)

Normal saline

20 mL/kg

58.5±3.7

0

Phenobarbital

10 mg/kg 20 mg/kg 30 mg/kg 40 mg/kg

87.8±2.7* 178.3±2.9* 308.5±3.8* 379.1±3.7*

0 25

75 100

Ethanolic extract

0.2 g/kg 0.8 g/kg 1.4 g/kg 2.0 g/kg

95.9±3.0* 118.1±3.4* 147.6±3.7* 244.4±5.3*

0 0 12.5 25

The extracts and phenobarbital were injected intraperitoneally 20 and 45 min before the administration of pentylenetetrazole (i.p., 90 mg/kg), respectively. Values presented as the mean±SEM duration of tonic seizure for 8 mice; *p<0.001, compared to normal saline using Tukey-Kramer test.

Table 3. Effect of aqueous extract of Crocus sativus stigma on the duration and inhibition of seizure and mortality in maximal electroshock-induced seizure in mice.

Treatment

Dose

Duration of tonic seizure (sec)

Seizure protection(%)

Mortality protection(%)

Normal saline

20 mL/kg

27.8±1.0

0

87.5

Phenobarbital

10 mg/kg 20 mg/kg 30 mg/kg 40 mg/kg

18.0±0.6* 14.4±0.4* 9.6±0.5* 5.8±0.5*

25 37.5 62.5 75

87.5 100 100 100

Aqueous extract

0.08 g/kg 0.32 g/kg 0.56 g/kg 0.80 g/kg

17.1±0.7* 17.0±1.4* 12.3±0.3* 10.5±0.5*

0 0 0 0

75 87.5 100 100

The extracts and phenobarbital were injected intraperitoneally 20 and 45 min before induction of maximal electroshock seizures, respectively. Values presented as the mean±SEM duration of tonic seizure for 8 mice; *p<0.001, compared to the normal saline using Tukey-Kramer test.

Both ethanolic and aqueous extracts increased the latency of convulsions induced by PTZ dose-dependently, but failed to produce complete protection against mortality (Tables 1 and 2). At the dose of 80 mg/kg, the aqueous extract had an efficacy similar to 10 mg/kg of phenobarbital used in the PTZ test.

In the MES test, the aqueous (0.8 g/kg) and ethanolic (2 g/kg) extracts as well as phenobarbital (40 mg/kg) decreased the duration of tonic seizures by 62%, 49% and 79%, respectively (Tables 3 and 4). Neither extracts had seizure-protective effects in the MES test.

Discussion

The present study indicates that the aqueous and ethanolic extracts of C. sativus have anticonvulsant activity in PTZ and MES-induced seizures.

Compared with a toxicity classification11, the extracts are relatively toxic. The ethanolic extract was found to be more toxic than the aqueous extract.

Agents affecting the PTZ test can inhibit absence seizures.10 Thus C. sativus may have some beneficial effect on this kind of seizure in clinical trials. The extracts showed activity against maximal electroshock seizures. This implies that the extracts can improve tonic clonic seizures.10 The mechanism (s) of anticonvulsant activity of the extracts is not clear. Saffron has been reported to have some behavioral effects on the central nervous system. In one study an alcoholic extract of C. sativus decreased the motor activity and prolonged the sleeping time induced by hexobarbital.6 This study suggests that the ethanolic extract possesses a sedative effect, which is probably responsible for the anticonvulsant effect of the extracts. In another study the plant was found to increase learning and memory performance in experimental animals.5

Table 4. The effect of ethanolic extracts of C. sativus stigmas on the duration, inhibition of seizure and mortality protection against seizure induced by maximal electroshock in mice.

Treatment

Dose

Duration of tonic seizure (sec)

Seizure protection percent

Mortality protection percent

Normal saline

20 mL/kg

28.1±0.30

0

87.5

Phenobarbital

10 mg/kg 20 mg/kg 30 mg/kg 40 mg/kg

16.3±0.3* 12.5±0.5* 9.6±0.5* 5.9±0.3*

25 25 75 75

87.5 100

100 100

Ethanolic extract

0.2 g/kg 0.8 g/kg 1.4 g/kg 2.0 g/kg

22.8±0.5* 19.0±0.3* 17.5±1.1* 14.4±0.5*

0 0 0 0

75 75 87.5 100

The extracts and phenobarbital were injected intraperitoneally 20 and 45 min before induction of maximal electroshock seizures, respectively. Values presented as the mean±SEM duration of tonic seizure for 8 mice; *p<0.001, compared to the normal saline using Tukey-Kramer test.

We conclude that the aqueous and ethanolic extracts of CSS have anticonvulsant activity in PTZ and MES tests that may help control petit mal and grand mal seizures.

References

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  8. Escribano J, Alonso GL, Coca-Prados M, Fernandez JA. Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro. Cancer Lett. 1996; 100: 23-30.
  9. Lozano P, Delgado D, Gomez D, Rubio M, Iborra JL. A non-destructive method to determine the safranal content of saffron (Crocus sativus L.) by supercritical carbon dioxide extraction combined with high-performance liquid chromatography and gas chromatography. J Biochem Biophys Method. 2000; 43: 367-8.
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