Anticonvulsant activity of aqueous extract of Leonotis leonurus.

Summary

Water extract of Leonotis leonurus was tested for anticonvulsant activity against seizures produced in mice by pentylenetetrazole, picrotoxin, bicuculline and N-methyl-DL-aspartic acid (intraperitoneal injections). L. leonurus extract in the doses of 200 and 400 mg/kg respectively protected 37.5% and 50% of animals used and significantly (p <0.05; Student's t-test) delayed pentylenetetrazole (90 mg/kg)-induced tonic seizures. Similarly, the same doses of L. leonurus extract significantly (p < 0.05; Student's t-test) delayed the onset of tonic seizures produced by picrotoxin (8 mg/kg) and N-methyl-DL-aspartic acid (400 mg/kg). However, all the doses of aqueous extract of L leonurus used did not alter the seizures induced by bicuculline (20 mg/kg) to any significant extent. The data suggest that the extract of L. leonurus has anticonvulsant activity and may probably be acting through non-specific mechanisms, since it affects both gahaergic and glutaminergic systems. High performance liquid chromatography (HPLC) and phytochemical tests carried out respectively show a spectrum profile, characteristic of L. leonurus and the presence of alkaloids, saponins and tannins in the extract.

Key words: Leonotis leonurus, aqueous extract, anticonvulsant activity, seizures, gabaergic and glutaminergic mechanisms, mice

Introduction

The present study has been focused on the crude water extract of Leonotis leonurus (L.) R. BR. of the family, Lamiaceae, which has a wide natural distribution over large parts of South Africa including the Western Cape Province (Hutchings et al., 1996; Van Wyk et al., 2000). It is known in Afrikaans as "wilde dagga", in Zulu as "umunyane", in Sotho as "lebake", in Xhosa as "umfincafincane" and in Shona as "umhlahlampetu" (Hutchings et al., 1996; Van Wyk et al., 2000). The leaves of L. leonurus have traditionally been smoked for the relief of epilepsy. An infusion and a decoction of the leaf and stem have been used internally for coughs, colds, influenza, bronchitis, high blood pressure and headaches. A tincture of the flower has also been used for the same purpose. Externally, decoctions have been applied to treat boils, eczema, skin disease, itching and muscular cramps (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996, Van Wyk et al., 2000). This study, therefore, investigated the effect of the water extract of L. leonurus on chemically induced seizures by pentylenetetrazole, picrotoxin, bicuculline and N-methyl D-L-aspartic acid in mice. The study also established the chromatographic profile using high performance liquid chromatography (HPLC) method, and identified the groups of chemical compounds present in the water extract using standard phytochemical tests.

Material and methods

Collection and identification of plant material

The plant material was collected from Kirstenbosch Botanic Garden, Cape Town, South Africa and authenticated by Dr Gillian Scott, taxonomist of the South African Traditional Medicine Research Group, School of Pharmacy, University of the Western Cape. A voucher specimen (number TRAD 10) was also deposited in the Herbarium of the Department of Botany, University of the Western Cape.

Preparation of aqueous extract of Leonotis leonurus

The leaves of plant species collected were washed with distilled water, dried in a ventilated oven for 72 hours at 35 [degrees]C and afterwards ground into fine powder (850 [micro]m) using the Waring Commercial laboratory blender. A known quantity of the powder (20 g) was refluxed for 5 hours in boiled water (11), allowed to cool for 24 hours and filtered. The resultant filtrate was freeze-dried (LSL Secfroid SR, Model 3021, Switzerland), for 72 hours to obtain the dried plant extract which was kept in a dessicator. The Leonotis leonurus solution used in the test against the chemically-induced seizures was freshly prepared on each day of the experiment by dissolving a given quantity of the dried extract in an appropriate volume of physiological saline.

Animals

Male albino mice bought from the University of Cape Town, Cape Town, Republic of South Africa, weighing 15--25 g each, were used in groups of eight per dose of drug or plant extract. Each mouse was housed singly in a transparent perspex cage for 30 minutes before the commencement of the experiment, in order to habituate them to their new environment. They all had access to food and water. Each mouse was used for one experiment only.

Drugs and chemicals

Pentylenetetrazole (PTZ; Sigma), picrotoxin (Sigma), N-methyl-DL-aspartic acid (NMDLA; Sigma) and phenobarbitone (Gardenal, Rhone-poulenc Rorer, South Africa) were all dissolved in physiological saline. Bicuculline (Sigma) and diazepam (Valium[R], Roche, South Africa) were suspended in a minimum amount of Tween 80 and Polyethylene glycol 400 (Flucka AG, Buchs) respectively, and adjusted to an appropriate volume with physiological saline. Preliminary studies were carried out to establish the doses of drugs and plant extract used. All drugs and L. leonurus solutions were injected intraperitoneally (i.p.). Control animals received equal volume injections of the appropriate vehicles which included Tween 80 or polyethylene glycol 400, all dissolved in physiological saline. Fresh drug solutions were prepared on the days of the experiment.

Anticonvulsant properties assessment

The investigation of anticonvulsant properties of aqueous extracts of L. leonurus was carried out using the method of Vellucci and Webster (1984), and modified by Amabeoku and Chikuni (1993). Seizures were induced in animals with convulsant drugs such as pentylenetetrazole, picrotoxin, bicuculline and NMDLA. Mice were observed for 30 minutes for tonic convulsion episode. Seizures were manifested as tonic hind-limb extension. The ability of the plant extract to prevent this feature or prolong the latency or onset of the tonic hind-limb extension was taken as an indication of anticonvulsant activity (Navarro-Ruiz et al., 1995, 1996; Williamson et al., 1996; Amabeoku et al., 1998). The onset of tonic convulsions and the proportion of mice presenting convulsions were recorded. Animals that did not convulse during the period of observation were considered not having convulsed. Experiments were repeated with mice pretreated for 15 minutes with L. leonurus extract, the standard antiepileptic drug, phenobarbitone or diazepam, or the control vehicle, before the administration of the convulsant drug. Control experiments were also carried out concurrently with the test experiments.

HPLC

The chromatographic system includes Beckman HPLC system consisting of double pump Programmable Solvent Module model 126; Diode Array detector Module model 168; Samsung computer 386 with management System Gold (GoldV601) software supplied by Beckman; Column, C18 Bondapak 5 [micro]m and dimensions (250 x 4.6 mm). The chromatographic conditions include Mobile phase: solvent A: 1% acetic acid; solvent B: methanol; Mode: gradient; flow rate, 1 ml/min; injection volume, 10 [micro]l; detector, UV at 360 nm; reference standard, Rutin (2.5 g dissolved in 100 ml of methanol). The HPLC operating conditions were programmed to give the following: at 0 min, solvent B: 20%; 5 min, solvent B: 40%; 15 min, solvent B: 60%; 20 min, solvent B: 80% and at 27 min, solvent B: 20%. The run rate was 30 min.

Phytochemical analysis

Using standard chemical tests, the water extract of L. leonurus was tested for compounds reported to be bioactive (Sofowora, 1982; Harborne, 1984; Moffat et al., 1986). The compounds and tests used are presented in Table 1.

Statistical analysis

The results on the onset of seizures were analyzed using the paired Student's t-test while the proportion of animals that exhibited tonic seizures was analyzed using the chi-squared test (Tallarida et al., 1981).

Results

Anticonvulsant properties assessment

Effect of aqueous extract of L. leonurus on pentylenetetrazole-induced seizures

Pentylenetetrazole (90 mg/kg) produced tonic seizures in all the animals used. A dose of 100 mg/kg of L. leonurus extract protected 25% of animals against pentylenetetrazole-induced seizures and did not affect the onset of the seizures to any significant extent. L. leonurus (200 mg/kg) protected 37.5% of mice against pentylenetetrazole-induced seizures, and significantly delayed the latency of the seizures. Similarly, 400 mg/kg of L. leonurus significantly prolonged the latency of the seizures produced by pentylenetetrazole, and protected 50% of animals against the seizures. The standard antiepileptic drugs, phenobarbitone (10 mg/kg) and diazepam (0.50 mg/kg) profoundly antagonized the seizures produced by pentylenetetrazole (Table 2). The control vehicle did not affect the gross behavior of mice or alter pentylenetetrazole-induced seizures to any significant extent (results not shown).

Effect of aqueous extract of L. leonurus on picrotoxin-induced seizures

Picrotoxin (8 mg/kg) produced tonic seizures in all the animals used. L. leonurus (100 mg/kg) did not affect the incidence nor the latency of picrotoxin-induced seizures. L. leonurus (200 mg/kg) significantly delayed the latency, but did not alter the incidence of seizures produced by picrotoxin to any significantly extent. A dose of 400 mg/kg of L. leonurus did not affect the incidence, but significantly prolonged the latency of picrotoxin-induced seizures. Similarly, the standard antiepileptic drug, phenobarbitone (10 mg/kg) did not alter the incidence, but profoundly delayed the onset of picrotoxin-induced seizures. Diazepam (0.50 mg/kg) significantly reduced the number of animals convulsing and significantly prolonged the latency of seizures produced by picrotoxin (Table 3). The control vehicle did not alter the seizures or affect the gross behavior of mice (results not shown).

Effect of aqueous extract of L. leonurus on bicuculline-induced seizures

Bicuculline (20 mg/kg) elicited tonic seizures in all the animals used. However, all the doses of L leonurus used (100, 200 and 400 mg/kg) did not alter the incidence of bicuculline-elicited seizures significantly. Besides, 400 mg/kg of L leonurus significantly shortened the latency of bicuculline-induced seizures. Both of the standard antiepileptic drugs, phenobarbitone (10 mg/kg) and diazepam (0.50 mg/kg) profoundly antagonized seizures produced by bicuculline (Table 4). The control vehicle did not affect the gross behavior of mice or the seizures produced by bicuculline significantly (results not shown).

Effect of aqueous extract of L. leonurus on NMDLA-induced seizures

N-methyl-D-L-aspartic acid (NMDLA; 400 mg/kg) elicited tonic seizures in all the animals used. L. leonurus (100 mg/kg) did not affect NMDLA-induced seizures significantly. L. leonurus (200-400 mg/kg) significantly prolonged the latency of the seizures. A dose of 200 mg/kg of L. leonurus did not affect the incidence of the seizures while 400 mg/kg protected 12.5% of the mice convulsing. However, the standard antiepileptic drugs, phenobarbitone (10 mg/kg) and diazepam (0.50 mg/kg) did not affect the seizures produced by NMDLA to any significant effect (Table 5). The control vehicle did not affect the gross behavior of the mice or alter the seizures significantly (results not shown).

HPLC analysis

According to the HPLC spectrum of water extract of L. leonurus obtained, the major peaks presented the following retention times (mm): 17.69, 19.22 and 20.76 (Figure 1). The peak for the reference standard, Rutin, appeared at the retention time (mm) of 21.47.

Phytochemical analysis

The standard chemical tests used showed the positive reactions for alkaloids, saponins (of both steroid and/or triterpenoid groups) and tannins, and the negative reactions for the rest of compounds tested. In addition, the literature reports the presence of volatile oil, several unusual diterpenoids (labdane type lactones) in Leonotis species. Marrubiin has been isolated as the main diterpenoid lactone from L. leonurus, but its precursor, premarrubiin, was not found. Two labdane terpenoids, compounds X and Y occur also in L. leonurus (Van Wyk et al.; 2000).

Discussion and conclusion

In the present study, both the antiepileptic activity of L. leonurus and the probable involvement of GABA (gamma amino butyric acid) and glutamic acid systems in this activity were investigated. GABA is the major inhibitory neurotransmitter in the brain whereas glutamic acid is an excitatory neurotransmitter in the brain. The inhibition of GABA neurotransmission and the enhancement of the action of glutamic acid have been shown to be the underlying factors in epilepsy (Westmoreland et al., 1994; Rang et al., 1999). Our study shows that the water extract of L. leonurus protected some of the animals against seizures induced by pentylenetetrazole (90 mg/kg), picrotoxin (8 mg/kg) and NMDLA (400 mg/kg), and also delayed the latency of the seizures. This is in conformity with the claims by traditional medicine practitioners that the plant is effective in the treatment of convulsion.

In this study, pentylenetetrazole was shown to induce seizures in all the mice used. The mechanism of pentylenetetrazole-induced seizures is uncertain (Rang et al., 1999). However, according to De Sarro et al. (1999), pentylenetetrazole may be producing seizures by inhibiting gabaergic mechanisms. Standard antiepileptic drugs, phenobarbitone and diazepam are thought to produce their effects by enhancing GABA-mediated inhibition in the brain (Rang et al., 1999). It is therefore possible that the anticonvulsant effects shown in this study by the said drugs against seizures produced by pentylenetetrazole might be due to the activation of GABA neurotransmission. Since L. leonurus extract similarly antagonized seizures elicited by pentylenetetrazole in mice, it is probable therefore that it may also be exerting its anticonvulsant effects by affecting gabaergic mechanisms.

In the same study, picrotoxin also produced seizures in all the mice used. Picrotoxin has been shown to elicit seizures by antagonizing the effect of GABA by blocking the chloride channels linked to GABAA-receptors. This prevents the conductance of chloride ions into the brain, thus inhibiting GAB A-mediated inhibition and GABA neurotransmission (Westmoreland et al., 1994; Rang et al., 1999). In this study, phenobarbitone and diazepam were shown to antagonize the effect of picrotoxin and L. leonurus extract was also shown to delay the latency of picrotoxin-induced seizures. This suggests that L. leonurus may be affecting gabaergic mechanisms, probably by opening the chloride channels associated with GABAA-receptors.

Bicuculline is a selective antagonist of GABA at the GABAA-receptors (Rang et al., 1999). The fact that L. leonurus aqueous extract did not affect the seizures induced by bicuculline, suggests that its effects on gabaergic mechanisms do not affect directly GABAA-receptors.

NMDLA was also shown to elicit seizures in all the mice used. NMDLA, a specific agonist at the NMDA-receptors, mimics the action of glutamic acid and thus induces seizures by enhancing the glutaminergic system (Rang et al., 1999). It is not surprising that the standard antiepileptic drugs, phenobarbitone and diazepam did not alter NMDLA-induced seizures to any significant extent. In this study, L. leonurus extract was shown to delay the latency of seizures induced by NMDLA, it may probably, therefore, be exerting its anticonvulsant effect partly by affecting glutaminergic mechanisms.

The phytochemical and the HPLC analyses showed the presence of alkaloids, saponins (of steroid and/or triterpenoid groups) and tannins in the leaves of the plant, and distinct peaks which may characterize L. leonurus respectively. In the present state of knowledge of the chemical constituents of the extracts of this plant, it is not possible to attribute with certainty the anticonvulsant effect to one or several active principles among those detected in the aqueous extract of L. leonurus. This is supported by the literature, according to which some of the groups of compounds found in the aqueous extract of L. leonurus, such as alkaloids, did not show any anticonvulsant activity in the experimental animal model of epilepsy (Akah and Nwaiwu, 1988). Moreover, Van Wyk et al. (2000) also reported that the actual pharmacological effect of marrubiin, the main diterpenoid lactone of L. leonurus, is not known. However, Chauhan et al. (1988) reported that some natural plant triterpenoids evaluated for anticonvulsant ac tivities provided 10-40% protection against PTZ-induced convulsions in mice. This therefore suggests that saponin triterpenoids detected in the aqueous extract of the leaves of L. leonurus might be involved in the anticonvulsant effect shown by this plant.

In conclusion, the data obtained in this study indicate that L. leonurus may be said to exert its anticonvulsant effect via non-specific mechanisms, since it has been shown to delay the latency of seizures produced by agents affecting both gabaergic and glutaminergic systems. However more extensive pharmacological, toxicological and phytochemical studies of this plant are needed to fully elucidate the active constituents, mechanism of action and safety of the plant as medicinal remedy.

[FIGURE 1 OMITTED]

Table 1

Phytochemical screening of aqueous extract of L. leonurus.

Compounds           Tests/Reagents

Anthraquinones      Borntrager's test, BPC
Alkaloids           Dragendorif's reagent and
                    Mayer's reagent
Cardiac glycosides  -- Lieberman's test
                    -- Keller-Killiani test
Reducing sugars     Fehlings reagents
Saponins            Frothing test
Tannins             Ferric chloride reagent
Table 2

Effect of aqueous extract of L. leonurus (LST) on pentylenetetrazole
(PTZ)-induced seizures in mice.

Dose (mg/kg)                     No. convulsed/  Animals not
                                         No. used        convulsed (%)
PTZ       LST       Pheno-     Diazepam
                    barbitone

90        --        --         --        8/8               0
90        100       --         --        6/8              25
90        200       --         --        5/8              37.5
90        400       --         --        4/8              50
90        --        10         --        0/8 a           100
90        --        --         0.50      0/8 a           100

          Latency of tonic
          convulsion (min.)
PTZ       Mean [+ or -] S.E.M.


90         6.38    [+ or -] 0.88
90         9.67    [+ or -] 1.81
90        12.20 *  [+ or -] 2.73
90        16.00 ** [+ or -] 1.76
90        --
90        --

* p <0.50

** p < 0.025; vs pentylenetetrazole control (90 mg/kg; i.p.); Student's
t-test.

(a)p < 0.001 vs pentylenetetrazole control (90 mg/kg; i.p.); Chi-squared
test.
Table 3

Effect of aqueous extract of Leonotis leonurus (LST) on picrotoxin
(PIRC)-induced seizures in mice.

Dose (mg/kg)                                  No. convulsed/
                                              No. used
PICR      LST       Phenobarbitone  Diazepam

8         --        --              --        8/8
8         100       --              --        8/8
8         200       --              --        8/8
8         400       --              --        8/8
8         --        10              --        8/8
8         --        --              0.50      2/8 (a)

          Animals not       Latency of tonic
          convulsed (%)    convulsion (min.)
PICR                      Mean [+ or -] S.E.M.

8          0             14.13    [+ or -] 0.40
8          0             15.38    [+ or -] 0.56
8          0             17.00 *  [+ or -] 0.87
8          0             19.37 *  [+ or -] 1.00
8          0             24.38 ** [+ or -] 1.32
8         75             22.50 ** [+ or -] 1.75

* p < 0.05

** p < 0.025; vs picrotoxin control (8 mg/kg; i.p.), Student's t-test.

(a)p < 0.1 vs pocrotoxin control (8 mg/kg; i.p.); Chi-squared test.
Table 4

Effect of aqueous extract of Lenotis leonurus (LST) on
bicuculline(BIC)-induced seizures in mice.

Dose (mg/kg)                                  No. convulsed/
                                              No. used
BIC       LST       Phenobarbitone  Diazepam

20        --        --              --        8/8
20        100       --              --        8/8
20        200       --              --        8/8
20        400       --              --        8/8
20        --        10              --        0/8 (a)
20        --        --              0.50      0/8 (a)

          Animals not    Latency of tonic
          convulsed (%)  convulsion (min.)
BIC                      Mean [+ or -] S.E.M.

20          0            10.88   [+ or -] 0.48
20          0             9.88   [+ or -] 0.69
20          0            10.38   [+ or -] 0.75
20          0             6.63 * [+ or -] 0.48
20        100             --
20        100             --

* p < 0.025; vs bicuculline control (20 mg/kg; i.p.), Student's t-test.

(a)p < 0.001 vs bicuculline control (20 mg/kg; i.p.); Chi-squared test.
Table 5

Effect of aqueous extract of Leonotis leonurus (LST) on
N-methyl-DL-aspartic acid (NMDLA)-induced seizures in mice.

Dose (mg/kg)                                  No. convulsed/
                                              No. used
NMDLA     LST       Phenobarbitone  Diazepam

400       --        --              --        8/8
400       100       --              --        8/8
400       200       --              --        8/8
400       400       --              --        7/8
400       --        10              --        8/8
400       --        --              0.50      8/8

          Animals not      Latency of tonic
          convulsed (%)   convulsion (min.)
NMDLA                    Mean [+ or -] S.E.M.

400        0             3.50   [+ or -] 0.40
400        0             4.38   [+ or -] 0.26
400        0             5.75 * [+ or -] 0.31
400       12.5           8.71 * [+ or -] 1.42
400        0             2.25   [+ or -] 0.31
400        0             4.00   [+ or -] 0.42

* p < 0.05 vs NMDA control (400 mg/ kg; i.p.), Student's t-test. The
proportion of animals convulsing is compared using Chi-squared test.

Acknowledgements

The authors wish to thank Ms Celeste Farmer, Mr Brian Minnis and Mr Yussuf Alexander for their technical assistance, and Ms Renee Symonds for her help in scanning the spectra obtained from HPLC.

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E. Bienvenu (1), G.J. Amabeoku (1), P.K. Eagles (2), G. Scott (3) and E.P. Sringfield (3)

(1.) Department of Pharmacology

(2.) Department of Pharmaceutical Chemistry

(3.) South African Traditional Medicines Research Group; School of Pharmacy, University of the Western Cape, Bellville, South Africa

Address

E. Bienvenu, Department of Pharmacology, School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa