Neurophysiological study on possible protective and therapeutic effects of Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol

Abstract

In this study, anti-convulsant effect of Sidr leaf extract was examined by using pentylenetetrazol (PTZ) model on male albino rat by evaluating the changes in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) contents in different brain regions (cerebellum, brainstem, striatum, cerebral cortex, hypothalamus and hippocampus). The administration of subconvulsive dose of PTZ (40 mg/kg i.p.) every other day for 9 days caused a significant decrease in monoamine content in different brain areas, this is may be due to the increase in nitric oxide levels, although antagonized the GABAA receptors which led to neurotransmitter release so the content is decreased. Administration of PTZ after treatment with Sidr (50 mg/kg i.p.) leaf extract for 3 weeks as a protective group and administration of Sidr leaf extract for 3 weeks after treatment of PTZ as a therapeutic group caused significant increase in NE, DA, and 5-HT contents in all tested brain regions at most of the time intervals studied. This may be due to the presence of peptide and cyclopeptide alkaloids in the extract which inhibit neurotransmitter activity which led to the inhibition of neurotransmitter release. From these results, we can say that the Sidr leaf extract has neuroprotective and therapeutic roles against pentylenetetrazol convulsant effect.

Full text

REVIEW
Neurophysiological study on possible protective
and therapeutic effects of Sidr (Zizyphus spina-christi L.)
leaf extract in male albino rats treated with
pentylenetetrazol
Abeer M. Waggas
a,
*
, Reem H. Al-Hasani
b
a
Department of Biology (Zoology), Faculty of Girls Education, Scientific Department,
King Abdulaziz University, Jeddah, Saudi Arabia
b
Department of Biology (Zoology), Faculty of Girls Education, Scientific Department,
Umm Al-qura University, Makah, Saudi Arabia
Received 12 April 2010; revised 25 April 2010; accepted 16 May 2010
KEYWORDS
Zizyphus;
Pentylenetetrazol;
NE;
DA;
5-HT;
Seizure
Abstract this study, anti-convulsant effect of Sidr leaf extract was examined by using pentylene-
tetrazol (PTZ) model on male albino rat by evaluating the changes in norepinephrine (NE), dopa-
mine (DA) and serotonin (5-HT) contents in different brain regions (cerebellum, brainstem,
striatum, cerebral cortex, hypothalamus and hippocampus). The administration of subconvulsive
dose of PTZ (40 mg/kg i.p.) every other day for 9 days caused a significant decrease in monoamine
content in different brain areas, this is may be due to the increase in nitric oxide levels, although
antagonized the GABAA receptors which led to neurotransmitter release so the content is
decreased. Administration of PTZ after treatment with Sidr (50 mg/kg i.p.) leaf extract for 3 weeks
as a protective group and administration of Sidr leaf extract for 3 weeks after treatment of PTZ as
a therapeutic group caused significant increase in NE, DA, and 5-HT contents in all tested brain
regions at most of the time intervals studied. This may be due to the presence of peptide and cyclo-
peptide alkaloids in the extract which inhibit neurotransmitter activity which led to the inhibition of
*
Corresponding author. Tel.: +966 505659845; fax: +966 6918920.
E-mail address: ombaderm@yahoo.com (A.M. Waggas).
1319-562X ª 2010 King Saud University. All rights reserved. Peer-
review under responsibility of King Saud University.
doi:10.1016/j.sjbs.2010.05.003
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neurotransmitter release. From these results, we can say that the Sidr leaf extract has neuroprotec-
tive and therapeutic roles against pentylenetetrazol convulsant effect.
ª 2010 King Saud University. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Chemicals and plant extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Experimental work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction
Convulsion, sudden, violent, involuntary contraction of the
muscles of the body is often accompanied by loss of conscious-
ness. It is not known what causes the abnormal impulses from
the brain that result in convulsive seizures, since the distur-
bance may arise in normal brain tissue as well as in diseased
or injured tissue. Convulsion may occur in such conditions
as epilepsy, poisoning, high fever (especially in young chil-
dren), disturbances of calcium or phosphorus metabolism,
alkalosis, diabetes, oxygen insufficiency, and low blood-sugar
content, as well as in local irritation and injury of the brain
(Bornstein et al., 1998).
Chemical kindling is widely used as an experimental model
of convulsion. This phenomenon is characterized by progres-
sive intensification of seizure activity after repeated adminis-
tration of doses of different central nervous system (CNS)
stimulants, including pentylenetetrazol (PTZ) (Racine, 1972).
Pentylenetetrazol is commonly used as a convulsant drug, act-
ing as a GABA
A
antagonist. Studies have shown that the
mechanisms involved in PTZ kindling may include a decrease
in central GABAergic function (Berman et al., 2000). The en-
hanced seizure susceptibility induced by kindling is probably
attributable to plastic changes in the synaptic efficacy (Morim-
oto et al., 2004).
Zizphus spina-christi (L.), locally known as Sidr, is a multi-
purpose tree species belonging to the botanical family Rhamn-
aceae. It is an important cultivated tree and one of the few
truly native tree species of Arabia that is still growing along
with many newly introduced exotic plants (Mandavillae,
1990). Flowering and fruiting occur in this species during Sep-
tember–November. The flowers are important for the produc-
tion of wild bee honey (Ghazanfar, 1994). The winter honey
(i.e. nabk honey) collected during November from the flowers
of the Sidr is in high demand among the citizens for its medic-
inal qualities in addition to its excellent taste and fragrant
smell. Sidr is one of the important fruit crops in the dry parts
of tropical Asia and Africa. Its fruit is highly nutritious and
rich in vitamin C. From the different species of genus Zizy-
phus, peptide and cyclopeptide alkaloids, flavonoids, tannins,
betulinic acid and triterpenoidal saponin glycosides have been
isolated and chemically identified (Ikram and Tomlinson,
1976; Duke, 1985; Morishita et al., 1987; Cheng et al., 2000;
Shahat et al., 2001).
Zizyphus has been used in folk medicine as a demulcent,
depurative, anodyne, emollient, stomachic, for tooth aches,
astringents and as a mouth wash (Nazif, 2002). Since Zizy-
phus is a wild tree commonly available in Saudi Arabia and
its leaves are used in folk medicine for treatment, it is there-
fore deemed interesting to examine the neuroprotective and
therapeutic effects of Z. spina-christi (L.) leaf extract as a
anti-convulsant effect in the pentylenetetrazol (PTZ)-induced
seizure in male albino rat by evaluating the changes in norepi-
nephrine (NE), dopamine (DA) and serotonin (5-TH) con-
tents in different brain regions (cerebellum, brainstem,
striatum, cerebral cortex, hypothalamus and hippocampus)
of male albino rats.
2. Materials and methods
2.1. Chemicals and plant extract
Pentylenetetrazol (PTZ) of highest purity supplied from Sigma
chemical company was used in the present work. The applied
dose of 40 mg/kg BW was dissolved in 0.2 ml saline solution.
The applied dose was selected according to Akula et al.
(2007), Dhir et al. (2007).
Fresh Z. spina-christi (L.) leaf were collected from trees
growing in Taif (Saudi Arabia) between September and
November of 2008. Zizyphus leaf were extracted according
to the method described by Adzu et al. (2001). The plant
was dried under shade at 25 C and the dried leaves of the
plant were grounded with a blender. The powder part was kept
in nylon bags in deep freezer until the time of use. It was
weighted (100 g) and cold distilled water was poured into it
to give a final volume of 200 ml as reported previously
(Abdel-Wahhab et al., 2007).
2.2. Experimental work
Thirty-six adult male albino rats (Rattus norvegicus) weighing
approximately 150 g BW were used for experimentation. Free
access of standard diet and water was allowed ad-libitum. They
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Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi Journal of Biological Sciences (2010),
doi:10.1016/j.sjbs.2010.05.003

were kept under good ventilation with 12 h light and dark
cycles. The rats were arranged into six groups, six per group.
1. The 1st group (n = 6), the animals were injected daily with
saline vehicle and served as control.
2. The 2nd group (n = 6), the rats were injected in a subcon-
vulsive dose (40 mg/kg, i.p.) every other day for 9 days
(Akula et al., 2007 and Dhir et al., 2007).
3. The 3rd group ( n = 6), the rats were treated with PTZ
(40 mg/kg) every other day for 9 days after Zizyphus
(50 mg/kg) leaf extract treated for 3 weeks.
4. The 4th, 5th and 6th groups were injected in a subconvul-
sive dose (40 mg/kg, i.p.) every other day for 9 days then
injected with single dose of Zizyphus (50 mg/kg i.p.) for
three weeks, respectively.
At the end of the treatment, the rats of both control and exper-
imental groups were sacrificed, and the brain was rapidly dis-
sected and separated into two equal halves. Each half was then
separated into the following regions according to the method
of Glowinski and Iversen (1966): cerebellum, brainstem, stria-
tum, cerebral cortex, hypothalamus and hippocampus. The
brain tissues were wiped dry, weighed and wrapped into
quickly plastic frozen in dry ice pending analysis. NE, DA
and serotonin were extracted and estimated according to the
method of Chang (1964) and modified by Ciarlone (1978).
The fluorescence was measured in Jenway 6200 fluorometer.
2.3. Statistical analysis
The results are reported as the mean ± SEM of values ob-
tained from multiple observations. Differences between means
were considered statistically significant at P < 0.01 variance
(Woolson, 1987). All statistical analyses were computed by
SPSS version 10.
3. Result
Figs. 1–3 illustrate protective and therapeutic effects of Zizy-
phus on norepinephrine (NE), dopamine (DA) and serotonin
(5-TH) contents (lg/g fresh tissue) in brain regions (cerebel-
lum, brainstem, striatum, cerebral cortex, hypothalamus and
hippocampus) of albino rats treated with pentylenetetrazol
(PTZ), respectively.
Fig. 1 shows that the injection of PTZ (40 mg/kg i.p.) every
other day for 9 days (G2) caused a significant decrease in NE
content in all investigated brain regions compared to control
group (G1). There was a significant increase in NE content
in protective group G3 (rats were treated with PTZ for 9 days
after Zizyphus leaf extract treated for 3 weeks) in all tested
brain regions compared to G1 (control rats were treated with
saline) and G2 (control rats were treated with PTZ). A signif-
icant increase in NE content was also found in therapeutic
groups G4, G5 and G6 (rats were treated with Zizyphus leaf
extract after PTZ injection) in all brain regions compared to
G1 and G2 except in cerebral cortex and hypothalamus in
G4 which significantly increased compared to G2 only.
A result from Fig. 2 revealed that PTZ treatment led to the
inhibition of dopamine content in the investigated brain re-
gions compared to control group (G1). Whereas there was a
significant increase in DA content in protective group (G3)
in all tested brain regions compared to G1 and G2. A signifi-
cant increase was also found in therapeutic groups (G4, G5
and G6) in investigated brain regions compared to G1 and G2.
As shown in Fig. 3, treatment with 40 mg/kg of PTZ caused
depletion of 5-HT content in investigated brain regions com-
pared to control group (G1). However, protective group signif-
icantly increases 5-HT content in all brain regions compared to
G1 and G2. A significant increase was also found in therapeu-
tic groups (G4, G5 and G6) compared to G1 and G2 except in
Figure 1 Protective and therapeutic effects of Zizphus spina-christi (L.) leaves on norepinephrine (NE) content in different brain regions
of male albino rats treated with pentylenetetrazol (PTZ). (a) Statically significant (P < 0.01) compared to group 1. (b) Statically significant
(P < 0.01) compared to group 2. G1, control rats were treated with saline; G2, control rats were treated with PTZ; G3, rats were treated
with PTZ for 9 days after Zizyphus leave extract treated for 3 weeks; G4, rats were treated with Zizyphus leave extract treated for 1 weeks
after PTZ injection for 9 days; G5, rats were treated with Zizyphus leave extract treated for 2 weeks after PTZ injection for 9 days; G6, rats
were treated with Zizyphus leave extract treated for 3 weeks after PTZ injection for 9 days.
Neurophysiological study on possible protective and therapeutic effects 3
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Please cite this article in press as: Waggas, A.M., Al-Hasani, R.H. Neurophysiological study on possible protective and therapeutic effects of
Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi Journal of Biological Sciences (2010),
doi:10.1016/j.sjbs.2010.05.003

cerebral cortex, hypothalamus and hippocampus in G4 which
significantly increases compared to G2 only.
4. Discussion
Pentylenetetrazol (PTZ) is commonly used as a convulsant
drug. The enhanced seizure susceptibility induced by kindling
is probably attributable to plastic changes in the synaptic effi-
cacy (Oses et al., 2007). Dazzi et al. (1997) indicated that PTZ
kindling enhances the basal activity and sensitivity of dopa-
mine neurons in rat brain and suggested that mesocortical,
mesoaccumbens and nigrostiatal dopaminergic neurons con-
tribute to the central alterations associated with experimental
epilepsy.
Figure 3 Protective and therapeutic effects of Zizphus spina-christi (L.) leaves on serotonin (5-HT) content in different brain regions of
male albino rats treated with pentylenetetrazol (PTZ). (a) Statically significant (P < 0.01) compared to group 1. (b) Statically significant
(P < 0.01) compared to group 2. G1, control rats were treated with saline; G2, control rats were treated with PTZ; G3, rats were treated
with PTZ for 9 days after Zizyphus leave extract treated for 3 weeks; G4, rats were treated with Zizyphus leave extract treated for 1 weeks
after PTZ injection for 9 days; G5, rats were treated with Zizyphus leave extract treated for 2 weeks after PTZ injection for 9 days; G6, rats
were treated with Zizyphus leave extract treated for 3 weeks after PTZ injection for 9 days.
Figure 2 Protective and therapeutic effects of Zizphus spina-christi (L.) leaves on dopamine (DA) content in different brain regions of
male albino rats treated with pentylenetetrazol (PTZ). (a) Statically significant (P < 0.01) compared to group 1. (b) Statically significant
(P < 0.01) compared to group 2. G1, control rats were treated with saline. G2, control rats were treated with PTZ; G3, rats were treated
with PTZ for 9 days after Zizyphus leave extract treated for 3 weeks; G4, rats were treated with Zizyphus leave extract treated for 1 weeks
after PTZ injection for 9 days; G5, rats were treated with Zizyphus leave extract treated for 2 weeks after PTZ injection for 9 days; G6, rats
were treated with Zizyphus leave extract treated for 3 weeks after PTZ injection for 9 days.
4 A.M. Waggas, R.H. Al-Hasani
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Please cite this article in press as: Waggas, A.M., Al-Hasani, R.H. Neurophysiological study on possible protective and therapeutic effects of
Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi Journal of Biological Sciences (2010),
doi:10.1016/j.sjbs.2010.05.003

From the present result it is clear that the administration of
pentylenetetrazol (40 mg/kg) caused a significant decrease in
NE, DA and 5-HT contents in all tested brain regions. The
earlier studies indicated that PTZ is a potent competitive
antagonist at inhibitory GABA
A
receptors that can induce sei-
zure activity by altering potassium permeability of the cell
membrane via a voltage-dependent mechanism, also, treat-
ment with PTZ significantly increased nitric oxide (NO) levels
in brain (Berman et al., 2000; Huang et al., 2001; Dhir et al.,
2005).
Sidr (Z. spina-christi) has been used as an anti-convulsant
and hypnotic in oriental countries due to its CNS inhibitory
activity (Yu-ching, 1983; Han et al., 1986; Zhang et al.,
2003; Park et al., 2004). Adzu et al. (2002) studied the effect
of Z. spina-christi aqueous extract (100–200 mg/kg, i.p.) on
the central nervous system in mice. It was observed that the
aqueous extract of Z. spina-christi root bark may have some
sedative activities, this is evident from the marked inhibition
of the exploratory behavior in mice spontaneous motor activ-
ity (SMA) and prolonged sleeping time. These results suggest
that the extract contained some constituents that depress the
central nervous system. These findings correlate with those ob-
served by Morishita et al. (1987) on the aqueous extract of Ziz-
yphus seeds.
The protective and therapeutic effects of Zizyphus leaf ex-
tract against Pentylenetetrazol-induced seizure were clear in
the present study. The extract improves the content of NE,
DA and 5-HT in all brain regions compared with animal group
that received pentylenetetrazol.
In 2006 Waggas reported that the daily i.p. injection of Sidr
(Z. spina-christi) leaf extract (50 mg/kg BW) for 15 days and
subsequent withdrawal caused a significant increase in epi-
nephrine (E), norepinephrine (NE), dopamine (DA), serotonin
(5-HT), 5-hydroxyindolacetic acid (5-HIAA) and gamma-ami-
nobutyric acid (GABA) contents in different brain areas. This
study suggested that the increase in neurotransmitter content
in CNS areas may be due to the inhibition of calcium-ATPase
and phosphodiesterase, at the same time inhibition of Ca2+/
calmodulin binding which plays an important role in the re-
lease of these neurotransmitters, which may be due to the pres-
ence of peptide and cyclopeptide alkaloids in the extract. These
agents share the ability to depress excitable tissue at all levels
of the CNS, leading to a decrease in the amount of transmitter
released by the nerve impulse, as well as to general depression
of postsynaptic responsiveness and ion movement (Levin and
Weiss, 1979; Bloom, 2001; Han et al., 2005).
In conclusion, the present study showed that the Pentylene-
tetrazol caused a decrease in NE, DA and 5-HT contents
which can be minimized by Sidr (Z. spina-christi ) which im-
proves the contents of these neurotransmitters in different
brain regions, this may be due to the presence of peptide and
cyclopeptide alkaloids in the Sidr leaf extract.
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Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi Journal of Biological Sciences (2010),
doi:10.1016/j.sjbs.2010.05.003

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6 A.M. Waggas, R.H. Al-Hasani
ARTICLE IN PRESS
Please cite this article in press as: Waggas, A.M., Al-Hasani, R.H. Neurophysiological study on possible protective and therapeutic effects of
Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi Journal of Biological Sciences (2010),
doi:10.1016/j.sjbs.2010.05.003