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Journal of Medicinal Plants Research Vol.6(19), pp. 3535-3544, 23 May, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1060
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
The protective effect of Chrysanthemum fantanesii
extract, vitamin E and C on sodium valproate-induced
embryotoxicity in pregnant mice
Amrani Amel1, Zama Djamila1*, Boubekri Nassima1, Benaissa Ouahiba1, Meraihi Zahia2
Benayache Fadila3, Benayache Samir1 and Bettuzzi Saverio4
1Laboratoire de Valorisation des Ressources Naturelles et Synthèses de Substances Biologiquement Actives, Faculté
des Science Exactes, Université Mentouri, Constantine, Algérie.
2Laboratoire de Génie Microbiologique et Applications, Département des Sciences de la Vie et de la Nature, Faculté des
Sciences Université Mentouri, 25000 Constantine, Algeria.
3Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Faculté des Science Exactes, Université
Mentouri, Constantine, Algérie.
4Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico,
Plesso Biotecnologico Integrato, Università degli Studi di Parma, via Volturno 39, 43100 Parma, Italy.
Accepted 30 November, 2011
Valproate (VPA) has been shown to induce neural tube defect in human and mice. In this study, we
investigated the ability of butanolic extract from leaves of Chrysanthemum fantanesii, vitamin E and C
to modulate VPA-induced toxicity and oxidation damage in maternal and fetal tissues in mice. Plant
extract, VPA and vitamins were administered orally to pregnant mice from the 6 to 17th day of
gestation. Animals received plant extract (200 mg/kg per day), vitamin E (100 mg/kg per day) and
vitamin C (8.3 mg/kg per day) with an oral administration of VPA (400 mg /kg per day) under the same
conditions. On day 18 of gestation pregnant mice were sacrificed, fetuses, placenta and maternal
tissues were removed, homogenized and used for the determination of lipid peroxidation rates (LPO)
using thiobarbituric acid reactive substance (TBARS) method. Embryotoxicity was assessed by
counting the number of implants, live and dead fetuses, and resumptions. The fetuses were observed
for malformation including neural tube defect (Exencephaly), open eyes and skeletal malformation. The
results show clearly that there is a positive correlation between the increase in LPO and congenital
malformation. Plant extract, vitamin E and C caused partial decrease of embryo toxicity and congenital
malformation induced by VPA in mice.
Key words: Valproic acid, congenital malformation, embryotoxicity, oxidative stress, lipid peroxidation,
Chrysanthemum fantanesii, vitamin E and C.
INTRODUCTION
Valproic acid (VPA) is a potent teratogen agent in mice
(Nau et al., 1986; Ehlers et al., 1992; Nau, 1994;
Padmanabhan and Hammed, 1994). Marked strain
differences occur in the teratogenic sensitivities to VPA
(Finnel et al., 1997; Bennett et al., 2000 2000). The most
prominent effects induced by VPA in the fetus of VPA-
*Corresponding author. E-mail: atrouzl@umc.edu.dz. Tel:
00213776342823. Fax: 00213818849.
sensitive strains mice are: neural tube defects
(Exencephaly) reduced fetal weight and death and
skeletal malformation (Elmazar et al., 1988; Ehlers et al.,
1992; Nau, 1994; Al Deeb et al., 2000). The mechanisms
of developmental toxicity of VPA are not well understood,
although several teratogenic mechanisms have been
proposed, including the suppression of the glutathion
(GSH) system in maternal blood within 1h after
application (Hishida and Nau, 1998). It has been also
shown that VPA induced activation of the peroxisome
proliferation (Werling et al., 2001). It is believed that VPA
3536 J. Med. Plants Res.
consumption constitute a problem for liver cells, in such a
way that VPA acts as substrate for cytochrome P450 and
its dependent monoxygenase and glutathione-transferase
(Rogier et al., 1995), leading to the generation of
chemically reactive and potentially toxic intermediates
capable of reacting with and depleting GSH stores, this
would result in oxidative stress (Tang and Abott, 1996).
However, embryonic tissues, during the critical period
of organogenesis, are remarkably deficient in the
synthesis of many enzymes, including most of those
providing cytoprotection against oxidative stress, such as
glutathione reductase, superoxyde dismutase and
catalase (Yoshioka et al., 1982).
An earlier report revealed that VPA exposure caused
significant depletion of GSH levels in blood (Gupta et al.,
2004), liver (Baran et al., 2004), brain (Vidya and
Subramanian, 2006) and kidneys as well as significant
increased in LPO formation in this tissues, The resulting
oxidative stress could then contribute to tissue injury
(Raza et al., 1997). Thus, it is reasonable to assume that
peroxidative damage may play a major role in
developmental toxicity induced by VPA. Free-radical
scavenging systems, such as antioxidant enzymes, lipid
and water soluble antioxidants, can protect the cell from
tissue damage caused by free radicals. Small molecule
antioxidants, such as flavonoids, vitamin E and C are
able to interact with oxygen radicals directly. Vitamin E
and flavonoids terminates the chain reaction of LPO
(Halliwell et al., 1992), whereas Vitamin C scavenges
aqueous-phase reactive oxygen species by very rapid
electron transfer, thus inhibiting LPO as well as reducing
the oxidized antioxidants (Carr and Frei, 1999). In this
study, we have examined the effect of Chrysanthemum
fontanesii extract, vitamin E and C on VPA induced
developmental toxicity and oxidative damage in maternal,
fetal and placental tissues in mice.
MATERIALS AND METHODS
Plant material
C. fontanesii (Boiss. et Reut.) Q. et S., synonyms : Leucanthemum
fontanesii (Boiss. et Reut.), Plagius virgatus (B. et T.) non DC, is a
plant of 100 to 150 cm height (Figure 1). It grows in the
undergrowths and forests and is endemic to North Africa. C.
fontanesii was collected during the flowering stage in April 2003
from the area of Bejaia, north east of Algeria and authenticated by
Pr. M. Kaabeche (University of Sétif, Algeria).
Extraction procedure
Air-dried leaves: (1500 g) were powdered and macerated at room
temp with EtOH–H2O (8:2 v/v) for 48 h three times. After filtration,
the filtrates were combined, concentrated under vacuum (at 35°C),
diluted with 600 ml H2O, filtered to remove chlorophyll and
successively extracted with (3x400 ml), chloroform, ethyl acetate
and n-butanol. The organic layers were dried with Na2SO4.
Removal of solvents under reduced pressure, CHCl3 (5.0 g), EtOAc
(28.0 g), n-butanol (42.0 g) resulted in final extracts.
Animals and treatment
Female Albino Swiss mice aging from 6 to 8 weeks were purchased
from Pasteur Institute Algiers. The animals were kept in 12 h light/
dark cycles and maintained in an air-conditioned room at 22to
25°C, with free access to food and water ad libitum. The general
guidelines for the use and care of living animals in scientific
investigations were followed (Council of European Communities,
1986). Animals were caged over night with breeder males. In the
morning, when a vaginal plug was observed, it was considered as
the first day of gestation. Pregnant mice were divided in to eight
groups. Treatments were given from the 6 to 17 day of gestation.
Group1 served as control, Group 2 received an oral administration
of VPA (400 mg/kg per day). Group 3 received plant extract (200
mg/kg by gavages). Group 4 received plant extract (200 mg/kg) and
VPA at the same dose mentioned earlier, Group 5 received vitamin
E (100 mg/kg by gavages). Group 6 received vitamin E (100 mg/kg)
and VPA at the same dose mentioned earlier, Group 7 received an
oral administration of vitamin C (8.3 mg/kg) and Group 8 received
vitamin C (8.3 mg/kg) and VPA at the same dose mentioned earlier.
Embryotoxicity estimation
Pregnant mice were sacrificed on the 18 day of gestation. Fetuses
were eviserded (the uterus was opened to eviscerate fetuses), from
the uterus and living fetuses were examined grossly for external
malformation under a dissecting microscope. Some fetuses were
fixed in 95% ethanol to stain the skeletal system with Alizarin red S
(Dawson, 1926) or by Alcian blue and Alizarin red S (Leod, 1980).
Embryotoxicity was assessed by counting the number of implants,
live and dead fetuses, resorptions and fetal body weight.
Malondialdehyde (MDA) measurement
LPO was determined by measuring the formation of TBARS using
the colorimetric method of Uchiyama and Mihara (1978). Liver,
spleen, kidneys, fetuses and placenta were removed and
homogenized in cold KCl 1, 15% to make a10% homogenate. 3 ml
of 1% phosphoric acid and 1ml of 0, 67% thiobarbituric acid (TBA).
aqueous solution were added to 0.5 ml of 10% homogenate
pipetted into 10 ml centrifuge tube. The mixture was heated for 45
min in a boiling water bath. The mixture was cooled to room
temperature, and then 4 ml of n- butanol was added and mixed
vigorously. The butanol phase was separated by centrifugation and
absorbance was measured at 532 nm. MDA was employed as the
standard.
Statistical analysis
Data are expressed as the mean ± SD. Differences between means
were evaluated by one-way analysis of variance (ANOVA).
Statistical interferences were based on student's t-test for mean
values comparing control and treated animals.
RESULTS
Effect of VPA, plant extract, vitamin E and C on
fetuses toxicity
VPA produced a significant decrease in fetal and
placental weight (Figures 2 and 3) as well as increase in
embryo-lethality (Figure 6), exencephaly formation
Amel et al. 3537
Figure 1. C. fontanesii.
Control VPA Ext200 VPA+Ext200 Vitamin E VPA+ vitamin E Vitamin C VPA+ vitamin
Weight (g)
Weight (g)
Control VPA Ext200 VPA+Ext200 Vitamin E VPA+ vitamin E Vitamin C VPA+ vitamin
Weight (g)
Figure 2. Effect of VPA, plant extract, vitamin E and C on fetuses weight. Control; VPA, treated with 400 mg/kg valproic acid;
VPA + Ext200, treated with 400 mg/kg valproic acid and plant extract 200 mg/kg ; VPA+Vitamin E, treated with 400 mg/kg
valproic acid and 100 mg/kg vitamin E ; VPA+Vitamin C, treated with 400 mg/kg valproic acid and 8.6 mg/kg Vitamin C. All
substances were given by gavage from the 6 to 17 day of gestation. The fetal weight was significantly decreased by 2.32-fold
from that control (p<0.001) but it increased significantly in the VPA+Vitamin E (p<0.01), VPA+Vitamin C (p<0.05) and
VPA+Ext200 (p<0.001) groups. Placental weight was significantly decreased by 1.2– fold from that control (p<0.05) but No
significant differences in the placenta weight found among other groups. Data are mean±SD. a: Compared with control; b:
Compared with animals given VPA alone. *: Significant p<0.05 **: Highly significant p<0.01 ***: Very highly significant p<0.001.
(Figure 5) open eyes (Figure 7) and skeletal malformation
(Figure 9 and 11) relative to control values (Figure 4, 8
and 10). Plant extract, vitamin E or C given alone did not
show any teratogenic effect relative to controls (Table 1).
However, plant extract and Vitamin E caused significant
decreases in VPA-induced embryo-lethality and fetal
growth retardation, while vitamin C was only slightly
effective. These data show that plant extract, vitamin E
3538 J. Med. Plants Res.
Control VPA Ext200 VPA+Ext200 Vitamin E VPA+ vitamin E Vitamin C VPA+ vitamin
Weight (g)
Figure 3. Effect of VPA, plant extract, vitamin E and C on placenta weight. Control; VPA, treated with 400 mg/kg valproic acid;
VPA + Ext200, treated with 400 mg/kg valproic acid and plant extract 200 mg/kg ; VPA+Vitamin E, treated with 400 mg/kg
valproic acid and 100 mg/kg vitamin E ; VPA+Vitamin C, treated with 400 mg/kg valproic acid and 8.6 mg/kg Vitamin C. All
substances were given by gavage from the 6 to 17 day of gestation. The fetal weight was significantly decreased by 2.32-fold
from that control (p<0.001) but it increased significantly in the VPA+Vitamin E (p<0.01), VPA+Vitamin C (p<0.05) and
VPA+Ext200 (p<0.001) groups. Placental weight was significantly decreased by 1.2– fold from that control (p<0.05) but No
significant differences in the placenta weight found among other groups. Data are mean±SD. a: Compared with control; b:
Compared with animals given VPA alone. *: Significant p<0.05 **: Highly significant p<0.01 ***: Very highly significant p<0.001.
Figure 4. Control (18 day of gestation) × 5.
and C provided the same protective effect against VPA–
induced exencephaly, open eyes and skeletal
malformation (Table 1).
Effect of VPA, plant extract, vitamin E and C on LPO
(TBARs content) in fetal and maternal tissues
Significant increases in LPO were observed in fetal,
placental and maternal tissues 24 h after the
administration of VPA to pregnant mice from the 6 to the
17 day of gestation. When administrated together with
VPA, plant extract, vitamin E and C resulted in a
significant decrease in VPA-induced LPO in maternal,
fetal and placental tissues (Figure 12 and 13). Plant
extract provided more protection than vitamin E and C
against VPA-induced LPO in placenta, while vitamin E
and C provided more protection than plant extract in
maternal liver and kidneys. The data shown in Figure 13
demonstrated that plant extract, vitamin E and C had the
same protective effect against VPA-induced LPO in
fetuses and in the liver of fetuses.
DISCUSSION
The ability of VPA to produce teratogenic effects as well
as LPO in mice has been previously demonstrated (Nau
Amel et al. 3539
Figure 5. Exencephaly and cleft palate showing in treated fetus with
VPA × 5. Effect of VPA administration on the developmental degree
and the general morphology of Albino Swiss mouse fetuses. The
drugs (400 mg/kg) were administered by gavage from the 6 to 17day
of gestation. Uterine contents were evaluated on 18 day of gestation.
Figure 6. Fetolethality showing in treated fetus with VPA × 5. Effect of VPA
administration on the developmental degree and the general morphology of
Albino Swiss mouse fetuses. The drugs (400 mg/kg) were administered by
gavage from the 6 to 17day of gestation. Uterine contents were evaluated on 18
day of gestation.
Figure 7. Open eyes and fetal growth retardation showing in treated fetus
with VPA × 5. Effect of VPA administration on the developmental degree
and the general morphology of Albino Swiss mouse fetuses. The drugs
(400 mg/kg) were administered by gavage from the 6 to 17day of
gestation. Uterine contents were evaluated on 18 day of gestation.
3540 J. Med. Plants Res.
Figure 8. Control (Alizarin red S) × 5. Effect of VPA administration on skeletal development of Albino Swiss mouse fetuses. The drugs
(400 mg/kg) were administered by gavage from the 6 to 17 day of gestation. Uterine contents were evaluated on 18 day of gestation.
Fetuses displaying skeletal variations relative to controls (like ciphosis, scoliosis, inhibition of ossification and malformed ribs (curved)).
Photographs were taken on a gelatin base in a solution of 100% glycerine. Specimens are stained with alizarin red S (bone) according
to the method proposed by (Dawson, 1921) or with alcian blue and alizarin red S according to (Leod, 1980). Cartilage stain blue.
Fig.9 . Ciphosis (1); Scoliosis (2), inhibition of ossification (3) and
malformed ribs (curved) (4) showing in treated fetus with VPA(400mg/kg
per day) from the 6 th to 17th day of gestation (Alizarin red S) x 5
(1)
(2)
(4)
(1)
(4)
(3)
(3)
(3)
Figure 9. Ciphosis (1); Scoliosis (2), inhibition of ossification (3) and malformed ribs (curved) (4) showing in treated fetus with VPA
(400 mg/kg per day) from the 6 to 17 day of gestation (Alizarin red S) × 5. Effect of VPA administration on skeletal development of
Albino Swiss mouse fetuses. The drugs (400 mg/kg) were administered by gavage from the 6 to 17 day of gestation. Uterine contents
were evaluated on 18 day of gestation. Fetuses displaying skeletal variations relative to controls (like ciphosis, scoliosis, inhibition of
ossification and malformed ribs (curved)). Photographs were taken on a gelatin base in a solution of 100% glycerine. Specimens are
stained with alizarin red S (bone) according to the method proposed by (Dawson, 1921) or with alcian blue and alizarin red S according
to (Leod, 1980). Cartilage stain blue.
et al., 1991; Elmazar et al., 1992; Nau, 1994; Raza et al.,
1997; Ubedamartin, 1998). The current investigations
confirm previous observations. In this study, we showed
that LPO was involved in the teratogenic effects of VPA.
Moreover, C. fantanesii extract, vitamin E and C could
modulate the effects of VPA. In this investigation, VPA
Amel et al. 3541
Figure 10. Control (Alizarin red S and Alcian blue) × 5. Effect of VPA administration on skeletal development of Albino Swiss mouse
fetuses. The drugs (400 mg/kg) were administered by gavage from the 6 to 17 day of gestation. Uterine contents were evaluated on 18
day of gestation. Fetuses displaying skeletal variations relative to controls (like ciphosis, scoliosis, inhibition of ossification and
malformed ribs (curved)). Photographs were taken on a gelatin base in a solution of 100% glycerine. Specimens are stained with alizarin
red S (bone) according to the method proposed by (Dawson, 1921) or with alcian blue and alizarin red S according to (Leod, 1980).
Cartilage stain blue.
Figure 11. Ciphosis, delays of ossification showing in treated fetus with VPA (400 mg/kg per day) from the 6 to 17 day of gestation
(Alizarin red S and Alcian blue) × 5. Effect of VPA administration on skeletal development of Albino Swiss mouse fetuses. The drugs (400
mg/kg) were administered by gavage from the 6 to 17 day of gestation. Uterine contents were evaluated on 18 day of gestation. Fetuses
displaying skeletal variations relative to controls (like ciphosis, scoliosis, inhibition of ossification and malformed ribs (curved)).
Photographs were taken on a gelatin base in a solution of 100% glycerine. Specimens are stained with alizarin red S (bone) according to
the method proposed by (Dawson, 1921) or with alcian blue and alizarin red S according to (Leod, 1980). Cartilage stain blue.
exposure in vivo was associated to LPO generation in
maternal, fetal and placental tissues of pregnant albino Swiss mice. A possible second mechanism by which VPA
affected liver and kidney may involve an increase in LPO
3542 J. Med. Plants Res.
Table 1. Teratological effects of VPA, plant extract, vitamin E and C in pregnant mice.
Treatment
Percent of fetuses
dead or resorbed
Percent of fetuses
having exencephaly
Percent of fetuses
having open eyes
Percent of fetuses having
skeletal malformation
Control
6.77±4.28
0
0
0
VPA
38.05±23.47a*
37.33±16.00a**
63.10±23.14a***
79.59±24.18a***
Plant extract (200 mg/kg)
6.39±3.77
0
0
0
VPA+ plant extract
13.07±5.15b*
14.67±6.47b*
24.4±4.85b**
27.61±12.63b**
Vitamin E (100 mg/kg)
6.68±3.77
0
0
0
VPA+ vitamin E
14.33±7.13b*
18.04±6.13b*
25.53±4.47b**
34.48±6.88b**
Vitamin C (8.33 mg/kg)
5.43±3.17
0
0
0
VPA+ vitamin C
17.87±8.33
19.28±4.85b*
28.17±10.20b**
35.33±14.40b**
VPA (400 mg/kg) was administered by gavage from the 6to 17 day of gestation, after gavages of plant extract (200 mg/kg), vitamin E (100
mg/kg) and vitamin C (8.3 mg/kg). Animals were sacrificed on day 18 of gestation.
Control VPA Ext200 VPA+Ext200 Vitamin E VPA+ vitamin E Vitamin C VPA+ vitamin
MDA (nmol / g)
Figure 12. Effect of VPA, plant extract, vitamin E and vitamin C on LPO (TBARs content) in maternal liver, kidneys and spleen.
LPO was estimated by the measurement of MDA. data are mean±SD The level of MDA in group given VPA alone(400 mg/ kg)
was significantly increased by 1.54-, 1.31-, 1.59-, 1.37- and 1.41-fold, respectively, in maternal liver, kidney (p<0.01), fetus, liver
of fetus and placenta over that in control group (p<0.001), but it decreased significantly in groups received vitamin E (100 mg/kg)+
VPA, vitamin C (8.6 mg/kg)+ VPA, and plant extract (200 mg/kg) + VPA. No significant differences in the level of MDA of maternal
spleen found among groups. a : Compared with control; b : Compared with animals given VPA alone *: Significant p<0.05 **:
Highly significant p<0.01 ***: Very highly significant p<0.001.
and a decrease in antioxidant enzymes, such as
superoxyde dismutase and catalase (Vidya and
Subramanian, 2006), and GSH content (Raza et al.,
1997; Tong et al., 2005). Schulpis et al. (2006) showed
that VPA impairs liver function causing in free radicals
production. This seems to produce deoxyribonucleic acid
(DNA) oxidative damage in liver, as evidenced by a
remarkable increase of 8-OHdG levels in serum.
Indeed, supplementation with vitamin E and C should
protect against this kind of tissue damage. Our data are
in agreement with those showed by Jurina-Romet et al.
(1996) who found that antioxidants, vitamins E and C
were found to be cyto-protective agents against VPA-
induced injury in GSH-depleted hepatocyte. Also, it was
demonstrated that vitamin E protects the brain against
ethanol induced oxidative stress and apoptosis (Shirpoor
et al., 2009). One possible explanation for lack of
increase in LPO in fetal organs treated with VPA is that
these tissues posses low activities of antioxidant enzyme
and extremely low concentration of vitamin E (Yoshioka
et al., 1982). An additional important issue could be the
activation of peroxisome proliferation receptor by VPA
and teratogenic derivatives (Werling et al., 2001). Thus, it
is reasonable to assume that antioxidantt play a role in
Amel et al. 3543
Control VPA Ext200 VPA+Ext200 Vitamin E VPA+ vitamin E Vitamin C VPA+ vitamin
Figure 13. Effect of VPA, plant extract, vitamin E and vitamin C on LPO (TBARs content) in fetus, liver of fetus and placenta.
LPO was estimated by the measurement of MDA. data are mean±SD The level of MDA in group given VPA alone(400 mg/
kg) was significantly increased by 1.54-, 1.31-, 1.59-, 1.37- and 1.41-fold, respectively, in maternal liver, kidney (p<0.01),
fetus, liver of fetus and placenta over that in control group (p<0.001), but it decreased significantly in groups received vitamin
E (100 mg/kg)+ VPA, vitamin C (8.6 mg/kg)+ VPA, and plant extract (200 mg/kg) + VPA. No significant differences in the level
of MDA of maternal spleen found among groups. a : Compared with control; b : Compared with animals given VPA alone *:
Significant p<0.05 **: Highly significant p<0.01 ***: Very highly significant p<0.001.
protecting against VPA toxicity. Achieving effective
protection in vivo requires adequate nutrition. Flavonoids
act as scavengers of oxygen radicals, thereby preventing
LPO of polyunsaturated fatty acids (Cook and Samman,
1996).
However, supplemental butanolic extract of the leaves
from C. fontanesii given to mice have been shown in our
experiments to ameliorate the cytotoxic effect of LPO
induced by VPA. This finding is in agreement with results
provided by others, who demonstrated that thymoquinone
(a major constituent of volatile oil of Nigella species) is an
antioxidant and protects liver against VPA induced toxic
damage (Raza et al., 2006). In the same study, Apium
graraveolens extract (apigenin is the major constituent of
active fraction) was shown to modulate VPA induced
reproductive toxicity in rats (Alaaeldin and Amr Amin,
2007). Our results clearly demonstrated that oral
administration of vitamin E, and C and plant extract
resulted in partial protection against VPA-induced
developmental toxicity. These results are consistent with
those previously presented by others (Aldeeb et al.,
2000; Baran et al., 2004; Baran et al., 2006) in that
vitamin E administrated orally protected against the
developmental toxicity induced by VPA. Furthermore,
vitamin E was found to be effective on oxidative stress
and teratogenic effects of diabetes (Viana et al., 1996).
Conclusion
VPA exposure increased LPO and induced develop-
mental toxicity. Supplementation with vitamin E, and C
and the butanolic extract of C. fontanesii inhibited VPA-
related toxicity. Butanolic extract of the leaves of C.
fontanesii have antioxidant properties. These findings
suggest that developmental toxicity produced by VPA
may be due to increased LPO in fetal, placental and
maternal organs, thus antioxidants show protective
activity. Further studies are needed to determine the
mechanisms of LPO generation in VPA-exposed animals.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Prof.
M. Kaabeche from the University of Setif, Algeria, for the
identification of the plant material. They are also very
grateful to Prof. S KHENNOUF (Setif University) for his
help in correction and critical review of the manuscript.
This work was supported by Algerian National Agency for
Development of Health Research (ANDRS).
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