Journal of Ethnopharmacology 155 (2014) 736–743

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

The protective effect of Phoenix dactylifera L. seeds

against CCl4-induced hepatotoxicity in rats
Dalia H.A. Abdelaziz n, Sahar A. Ali 1
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Cairo, Egypt

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: In traditional Egyptian medicine, Phoenix dactylifera L. (date palm) seeds
Received 24 January 2014 are listed in folk remedies for the management of diabetes, liver diseases and gastrointestinal disorders.
Received in revised form The present study was conducted to investigate the protective effect of Phoenix dactylifera L. seeds
18 April 2014
aqueous suspension against the chemically-induced hepatic injury in rats.
Accepted 6 June 2014
Available online 16 June 2014
Methods: Liver injury was achieved by exposing Wistar rats to CCl4 (10% in olive oil; 0.5 mL/rat; IP) twice
a week for 4 weeks. Along with CCl4, aqueous suspensions of raw or roasted Phoenix dactylifera seeds
Keywords: (1.0 g/kg) were administered orally in a daily manner.
Phoenix dactylifera seeds Results: Our results demonstrated that Phoenix dactylifera seeds significantly improved the CCl4-induced
Carbon tetrachloride
alterations in liver function parameters (AST, ALT, ALP and albumin). Moreover, the CCl4-induced
Hepatoprotective
oxidative stress, represented by elevated thiobarbituric acid reactive substance (TBARS), nitric oxide and
Antioxidants
Oxidative stress oxidative DNA damage, was ameliorated by Phoenix dactylifera seeds treatment. In addition, Phoenix
dactylifera seeds restored the activities of hepatic antioxidant enzymes (superoxide dismutase and
glutathione S-transferase) that were declined after CCl4 treatment. Examination of liver histopathology
revealed that Phoenix dactylifera seeds attenuate the incidence of liver lesions (including vacuolization
and fibroblast proliferation) triggered by CCl4 intoxication.
Conclusion: The Phoenix dactylifera seeds could be a promising candidate for protection against the CCl4-
induced liver intoxication, and this hepatoprotective effect might be attributed to the antioxidant and
free radical scavenging activities.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction species and free radicals generated during its metabolism

For several years, a remarkable attention has been grabbed to
oxidative stress; a status of an excessive production of reactive
oxygen species in the organism. It has been demonstrated that
reactive oxygen species (ROS) are key players in the hepatic
pathophysiologic changes caused by chemicals (Tsukamoto et al.,
1990), Hepatitis B virus (Lee et al., 2004), Hepatitis C virus (Okuda
et al., 2002) and steatosis (Oliveira et al., 2002). The pathophysiologic
changes which lead to liver cirrhosis and finally hepatocellular
carcinoma (Tanikawa and Torimura, 2006; Tsukiyama-Kohara,
2012). Accordingly, this concept represents a rationale for using
antioxidant supplements in treatment of liver disorders.
Carbon tetrachloride (CCl4) has been widely used for induction
of liver damage in experimental animals (Tsukamoto et al., 1990).
The liver injury caused by CCl4 is attributed to the reactive oxygen
n
Corresponding author. Tel.: þ 20 1149790788; fax: þ 20 25541601.
E-mail addresses: dalia_abdelaziz@pharm.helwan.edu.eg (D.H.A. Abdelaziz),
ganah_nour@yahoo.com (S.A. Ali).
1
Tel.: þ20 1005288921; fax: þ20 25541601.
(Rechnagel and Glende, 1973).
The hepatotoxic effect of CCl4 is partially circumvented by
antioxidant compounds including α-tocopherol (Parola et al.,
1992; Halim et al., 1997), ascorbic acid (Ozturk et al., 2009) and
silymarin (Mourelle et al., 1989). Induction of liver injury by CCl4
has been used vastly as a model for investigation of hepatopro-
tective agents (Naziroğlu et al., 1999; Kanter et al., 2005; Ozturk
et al., 2009).
Phoenix dactylifera L. (date palm) seeds have been reported to
be a rich source of antioxidants (Al-Farsi and Lee, 2008; Habib and
Ibrahim, 2011; Juhaimi et al., 2012). The antioxidant activity of
Phoenix dactylifera seeds is attributed mainly to the presence of
high content of phenolics, flavonoids and vitamin C (Habib and
Ibrahim, 2011; Juhaimi et al., 2012). Substantial amount of the
polyphenols could be isolated from Phoenix dactylifera seeds
ranging from 31–44 g gallic acid equivalent kg
1 depending on
the variety (Al-Farsi et al., 2007). Based on a recent study by Habib
et al. (2014), Phoenix dactylifera seeds constitute one of the highest
sources of total polyphenols, excelling tea, grapes, flaxseed, nut
seeds and even date flesh. Furthermore, a recent phytochemical

http://dx.doi.org/10.1016/j.jep.2014.06.026
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743
report has revealed that the antioxidant flavonoid contents of
roasted Phoenix dactylifera seeds were superior to that of raw ones
(Paranthaman et al., 2012).
In Middle East, people believe that eating dates (Phoenix
dactylifera) on an empty stomach will reverse the effect of any
toxin for the whole day. Interestingly, roasted Phoenix dactylifera
seeds are traditionally used in Arab countries in making caffeine-
free beverage with common conception that it is effective against
gastric upsets and indigestion (Ali-Mohamed and Khamis, 2004).
Phoenix dactylifera seed is listed in remedies of Egyptian folk
medicine for the management of various infectious diseases, liver,
diabetes and cancer (Duke, 1992). A recent study by Habib and
Ibrahim (2011) has demonstrated that diet containing Phoenix
dactylifera seeds reduces the basal level of lipid peroxidation in
liver of normal rats while does not affect the antioxidant enzyme
capacity of the normal tissues. Furthermore, it has been shown
that the aqueous and ethanolic extracts of Phoenix dactylifera seeds
were effective in ameliorating gastric ulceration in rats (Al-Qarawi
et al., 2005).
To our knowledge, the data concerning the hepatoprotective
effect of Phoenix dactylifera seeds are scanty. A study by Al-Qarawi
et al. (2004) has demonstrated that Phoenix dactylifera seeds have
hepatoprotective effect on CCl4 treated rats . However, in this
study; the Phoenix dactylifera seeds extract was added to the
drinking water with no definite dose/rat, only the raw (unroasted)
Phoenix dactylifera seeds have been used. In addition, they
assessed only the serum markers of liver function. However, the
antioxidant status and the histopathology of liver tissues have not
been investigated in their study.
The objective of the present study was to investigate the
hepatoprotective effect of both raw and roasted Phoenix dactylifera
seeds on CCl4-treated rats. In our study we investigated the
hepatoprotective effect on different levels, serum parameters,
tissue oxidative stress, including oxidative DNA breaks, and
histopathological changes of liver. In addition, silymarin, a poly-
phenolic flavonoid isolated from milk thistle with clinically proven
hepatoprotective effect (Lin et al., 2012), was used as a reference
hepatoprotective agent in our study.
2. Materials and methods
2.1. Chemicals
All chemicals required for all biochemical assays were of
analytical grade and were obtained from Sigma-Aldrich Chemicals
Co., St. Louis, USA.
2.2. Preparation of Phoenix dactylifera seeds suspension
Seeds of Phoenix dactylifera L. variety Hayani (family Arecaceae)
were obtained from Elsharkia date factory (El-Sharkia Governor-
ate, Egypt). A voucher specimen number ph-d.1 was kept in the
herbarium of the Department of Pharmacognosy, Faculty of
Pharmacy, Helwan University, Cairo, Egypt. A part of the seeds
was sun dried and the other part was roasted in the oven (140 1C
for 3 h) then ground to a fine powder by hammer-mil. The Phoenix
dactylifera seeds (raw or roasted) suspension was freshly prepared
by mixing Phoenix dactylifera seeds powder (1 g) with one drop of
tween 80 then 10 ml water was added gradually.
2.3. Preliminary phytochemical analysis
The powder of Phoenix dactylifera seeds was subjected to
phytochemical tests to determine the amounts of total phenolics
using a Folin–Ciocalteu reagent method (Singleton and Rossi,
737
1965) and the results were expressed as mg gallic acid equivalent
g
1. In addition, total flavonoids were assessed according to
Zhishen et al. (1999) and the results were expressed as mg rutin
equivalent g
1 of Phoenix dactylifera seeds
2.4. Animals
This study was conducted on adult male Wistar rats (Rattus
Norvegicus, strain Wistar) weighing 180–200 g provided by Insti-
tutional Breeding House, Egypt. Animals were kept in controlled
environment of humidity and temperature with alternating 12 h
light/dark cycle for one week for acclimatization, with free access
to standard rat chow and drinking water ad libitum. The protocol
of the study was approved by the Animal Ethics Committee of the
Faculty of Pharmacy, Helwan University on 01/11/2012. The study
was conducted in accordance with EC Directive 86/609/EEC for
animal experiments.
2.5. Experimental groups
Thirty five rats were randomly divided into five groups (seven
rats per group). Group I served as normal control and was given
olive oil (0.5 ml/rat) intraperitoneally twice a week. To induce
hepatic injury (in vivo), the animals in Groups II–V received 0.5 ml
of CCl4 (10% CCl4 in olive oil) intraperitoneally twice a week (Lin
et al., 2008). Group II (CCl4 group) received CCl4 only. Group III
(reference group) was given silymarin (50 mg/kg) orally on daily
basis (Lin et al., 2012). Silymarin was obtained from crushed
Legalons tablets (Madaus, Egypt). Group IV received oral aqueous
suspension of raw Phoenix dactylifera seeds (1 g/kg) daily. Group V
received oral aqueous suspension of roasted Phoenix dactylifera
seeds (1 g/kg) daily. The suspension of Phoenix dactylifera seeds
was vigorously shaken before administration to ensure equivalent
dose for each rat. All groups were treated for four weeks.
2.6. Preparation of serum and tissue homogenate
At the end of the experiment, blood samples were collected
from the retro-orbital puncture after anesthesia. Serum was
separated and stored at
80 1C until analysis. Then animals were
sacrificed by cervical dislocation and the liver was rapidly removed
on ice, rinsed using ice-cold saline. Liver tissue samples were cut
into two pieces. One small piece was fixed in formalin for
histopathological examination. The other piece was utilized for
the biochemical analyses. Liver homogenates (20% w/v) were
prepared in normal saline (pH 7.0) and stored at
80 1C for
analysis.
2.7. Determination of liver function
Alanine aminotransferase (ALT), aspartate aminotransferase
(AST) activities were assayed in serum samples obtained from all
groups of rats by a colorimetric method (Reitman and Frankel,
1957) using commercial diagnostics kits (Diamond Diagnostics,
Egypt). Whereas alkaline phosphatase (ALP) activity was assayed
by kinetic method by measuring the rate of hydrolysis of
p-nitrophenyl phosphate in accordance to the protocol of the
commercial diagnostic kits (Spectrum Diagnostics, Egypt). The
activities of ALT, AST and ALP were expressed as U/L. Serum
albumin level was assessed colorimetrically using commercial
diagnostic kits obtained from Diamond Diagnostics, Egypt. All
serum parameters were measured using a Spectrophotometer
1200 UNICO Instruments Inc. USA.
738 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743
2.8. Determination of lipid peroxide level
Lipid peroxidation level in the liver homogenate was determined
as thiobarbituric acid reactive substances (TBARS) level spectropho-
tometrically in liver homogenates (Mihara and Uchiyama, 1978).
Values were expressed as nmoles of TBARS/mg protein.
2.9. Determination of glutathione S-transferase (GST) activity
GST activity was determined spectrophotometrically in liver
homogenate by measuring the increase in absorbance of the
reaction mixture at 340 nm (Habig et al., 1974). The GST activity,
was defined as the amount of enzyme producing 1 μmol of
1-chloro-2,4-dinitrobenzene (CDNB)-Glutathione conjugate/min
under the conditions of the assay. The GST activity was calculated
using an extinction coefficient of 9.6 mM
1 cm
1. The results
were expressed in U/min/mg of protein.
2.10. Determination of nitric oxide level
Nitrite level was assayed in liver homogenate supernatant by
reacting with Griess reagent (1:1 solution of 1% sulfanilamide in
5% phosphoric acid and 0.1% naphthyl-ethylenediamine dihydro-
chloric acid in water) and measuring the absorbance at 543 nm.
Nitrite level was calculated using a standard curve for sodium
nitrite and its level was expressed as mmol/mg protein (Green
et al., 1982).
2.11. Superoxide dismutase (SOD) activity
Superoxide dismutase (SOD) activity in liver homogenate was
assayed spectrophotometrically by measuring the % inhibition of
the auto-oxidation of pyrogallol in the presence of SOD enzyme
(Roth and Gilbert, 1984). One unit of SOD represents the amount of
enzymes required to inhibit the rate of pyrogallol oxidation by 50%
at 25 1C. The activity was expressed as units/mg protein.
2.12. Determination of tissue protein content
Protein content in liver homogenate was determined according
to Lowry's method using bovine serum albumin (BSA) as a
standard (Lowry et al., 1951).
2.13. Alkaline comet assay
The Comet assay was performed essentially as described by
Singh et al., (1988). Liver tissue (0.5 g) was crushed and transferred
to 1 ml ice-cold PBS (phosphate buffer saline). This suspension was
stirred for 5 min and filtered. Cell suspension (100 ml) was mixed
with 600 ml of low melting agarose (0.8% in PBS). The mixture
(100 ml) was spread on pre-coated slides. The coated slides were
immersed in lyses buffer for 15 min. The slides were placed in
electrophoresis chamber containing TBE buffer. The electrophor-
esis conditions were 2 V/cm for 2 min and 100 mA. Staining was
Table 1
Effect of Phoenix dactylifera seeds (PDS) and silymarin on the growth parameters in CCl4-intoxicated rats.
Parameter Control
Weight gain (g) 90.7 79.6
Relative liver weight (g/100 g BW) 4.34 70.11
PDS: Phoenix dactylifera seeds (1 g/kg suspension), silymarin (50 mg/kg). Data are presented as mean 7 SE, n¼ 7.
#
Po 0.05 compared with the control group.
##
P o0.01 compared with the control group.
###
Po 0.001 compared with the control group.
n
Po 0.05 compared with CCl4-treated group.
conducted using ethidium bromide 20 mg/ml at 4 1C. The DNA
fragment migration patterns were evaluated with a fluorescence
microscope (excitation filter 420–490 nm). The comet tail lengths
were measured from the middle of the nucleus to the end of the
tail. We used Komet 5 image analysis software developed by
Kinitic Imaging, Ltd. (Liverpool, UK) linked to a CCD camera to
assess the quantitative and qualitative extent of DNA damage.
Generally, 50 to 100 randomly selected cells were analyzed per
sample.
2.14. Histopathological examination
Immediately following sacrifice of the animals, liver tissues were
surgically excised, individually weighed, and liver slices were cut and
fixed in 10% neutral buffered formalin and embedded in paraffin.
Tissue sections (5 μm thick) were prepared, stained with hematoxylin-
eosin (H&E), and then examined under light microscope at 200 and
400 magnifications for determination of pathological changes.
The sections were analyzed blindly by a certified pathologist
and three different sections were examined in each sample of liver.
The severity of histopathological changes (fibrosis and apoptosis)
was scored according to an arbitrary scale (between
to þ þ þ).
2.15. Statistics
All data were expressed as mean7SEM for seven rats in each
group. Statistical analysis was performed by one-way analysis
of variance (ANOVA) followed by Tukey–Kramer test for multiple
comparisons using GraphPad Instat (Graph software Inc., V 3.05, Ralf
Stahlman, Purdue Univ.). Po0.05 was considered statistically signifi-
cant. Appropriate graphs were plotted using Microsoft Excel 2007.
3. Results
3.1. Phytochemical constituents of Phoenix dactylifera seeds
The preliminary phytochemical analysis revealed that Phoenix
dactylifera seeds contained significant amounts of total phenolics
(38.8 mg gallic acid equivalent g
1) and total flavonoids (87.86 mg
rutin equivalent g
1).
3.2. Effect of Phoenix dactylifera seeds on body weight gain and
relative liver weight
The growth performance of the studied groups was assessed
using the increase in body weight at the end of the experiment.
The body weight gain and the relative liver weights of all studied
groups are shown in Table 1. The administration of CCl4 for
4 weeks attenuated the weight gain significantly (22.8 710.3 vs.
90.77 9.6, P o0.001) compared to the control group. The group
treated with silymarin gained more weight than CCl4 group but
still significantly less than the control group (Po 0.01). Treatment
with raw or roasted Phoenix dactylifera seeds markedly improved
CCl4 Silymarin Raw PDS Roasted PDS
22.8 7 10.3### 41.6 7 6.16## 55.4 7 11.18 607 7.08n
4.8 7 0.14# 4.6 7 0.12 4.4 7 0.063 4.3 7 0.068n
 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743 739

the growth performance. Yet, only the group treated with roasted
Phoenix dactylifera seeds showed significant weight increment
compared to CCl4 group (P o0.05). Furthermore, CCl4 intoxication
increased the relative liver weights significantly compared to the
control group (P o0.05). Although the other groups showed
decrease in the relative liver weights, only the group treated with
roasted Phoenix dactylifera seeds showed significantly lower rela-
tive liver weight compared to the CCl4 group (P o0.05).
3.3. Effect of Phoenix dactylifera seeds on serum hepatic markers
NO (P o0.001 and Po0.01, respectively) compared to CCl4 group.
Furthermore, treatment with Phoenix dactylifera seeds, either raw
or roasted, caused efficient attenuation of TBARS and NO levels
compared to CCl4 group (Po 0.001 and P o0.01, respectively).
Despite the marked decrease in NO levels in both raw and roasted
Phoenix dactylifera seeds-treated groups, they did not restore the
liver NO values to the control values (P o0.05).
3.5. Effect of Phoenix dactylifera seeds on antioxidant enzymes in
liver tissue

The liver function parameters analyzed in this study are shown
in Fig. 1. Treatment with CCl4 for 4 weeks induced abnormal liver
function parameters as shown by elevation of serum levels of
hepatic enzymes AST, ALT and ALP (P o0.001) whereas, serum
albumin level significantly decreased (P o0.01) compared to con-
trol group. Administration of Phoenix dactylifera seeds (raw or
roasted) restored normal levels of ALT, AST and ALP (P o0.001) and
caused significant increase in serum albumin level (P o0.05)
compared to CCl4 group. Furthermore, the improvement of liver
function parameters achieved by Phoenix dactylifera seeds treat-
ment was equivalent to that of silymarin-treated group (Fig. 1).
3.4. Potency of Phoenix dactylifera seeds as a free radical scavenger
After four weeks of CCl4 exposure, levels of TBARS (marker of
lipid peroxidation) and NO in liver were significantly elevated
compared to the control group (P o0.001) (Fig. 2). Silymarin-
treated group showed significant decline in the level of TBARS and
To investigate the ability of Phoenix dactylifera seeds to enhance
the antioxidant capacity of liver, we assessed superoxide dismu-
tase (SOD) and glutathione S-transferase (GST) in liver tissue
homogenate. We found that 4 weeks of CCl4 treatment induced
significant decline in SOD and GST enzymes activities in the liver
homogenates compared to the control group (Po 0.05 and
Po 0.01, respectively). The liver homogenates of groups treated
with silymarin, raw and roasted Phoenix dactylifera seeds showed
higher levels of both SOD and GST. Yet, only the group treated
with roasted Phoenix dactylifera seeds demonstrated statistically
significant augmentation in the levels of both enzymes (P o0.05
for both) compared to the CCl4 group (Fig. 2).
3.6. Effect of Phoenix dactylifera seeds on oxidative DNA damage
in liver tissue
The comet assay (Single-cell gel electrophoresis) is a sensitive
method for the detection of DNA damage (Singh et al., 1988;

6 120
###
5 * * * 100

4 ## 80

3 60 **
*** ***
2 40

1 20

0 0

500 500
###
###
400 400

300 300
**
200 *** *** 200 ***

*** ***
100 100

0 0
CCl4 - + + + + - + + + +
Silymarin - - + - - - - + - -
Raw PDS - - - + - - - - + -
Roasted PDS - - - - + - - - - +
Fig. 1. Effects of Phoenix dactylifera seeds (PDS) and silymarin on CCl4-induced alterations in serum liver function parameters (ALT, AST, ALP and albumin). ALT: alanine
aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, CCl4 (0.5 ml of 10% CCl4 in olive oil), PDS: Phoenix dactylifera seeds (1 g/kg) for both raw and
roasted, silymarin (50 g/kg). Data are presented as mean 7SE, n¼ 7. ##Po 0.01, ###Po 0.001 as compared with the control group. nPo 0.05, nnPo 0.01, and nnnPo 0.001
compared with CCl4-treated group.
740 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743

TBARS 35 NO
0.3

30
0.25

(µmol/mg protein)
(nmol/mg protein) 0.2
25

20
0.15
15
0.1
10

0.05 5

0 0

120 SOD 16 GST

14
100

(U/min/mg protein)
12
(U/mg protein)

80
10

60 8

6
40
4
20
2

0 0

CCL4 - + + + + - + + + +
Silymarin - - + - - - - + - -
Raw PDS - - - + - - - - + -
Roasted PDS - - - - + - - - - +
Fig. 2. Effects of Phoenix dactylifera seeds (PDS) and silymarin on TBARS, NO, SOD and GST in CCl4-intoxicated rats. TBARS: thiobarbituric acid reactive substance, NO: nitric
oxide, SOD: superoxide dismutase, GST: glutathione S-transferase, PDS: Phoenix dactylifera seeds (1 g/kg suspension), silymarin (50 mg/kg). Data are presented as mean 7SE,
n¼ 7. #Po 0.05, ##Po 0.01, ###P o 0.001 compared with the control group. nP o0.05, nnPo 0.01, nnnPo 0.001 as compared with CCl4-treated.

Fairbairn et al., 1995) and has been used extensively in order to
detect oxidative DNA breaks (Collins et al., 1995; Pfuhler and Wolf,
1996). In the present study, the comet assay results were repre-
sented as tail moment and tail DNA%. Tail moment was defined as
the distance between the center of the tail and the center of the
head, in microns, multiplied by the percentage of DNA in the tail
(Bowden et al., 2003). In the current study, dosing rats with CCl4
markedly damages DNA as shown by significant increase in tail
DNA% and tail moment (P o0.001 for both). However, treatment
with Phoenix dactylifera seeds, either raw or roasted, significantly
decreased the tail moment (Po 0.001 for both) and the tail DNA%
(P o0.05 and P o0.01, respectively). In addition, silymarin signifi-
cantly diminished the DNA breaks induced by CCl4 as shown
in Fig. 3.
3.7. Histopathology of liver
To confirm our findings on the sera and liver homogenates, we
investigated the pathologic changes in liver microscopically. The
examination of the liver histology of CCl4 group revealed sever
hepatic injury represented by cytoplasmic vacuolization of hepa-
tocytes, sever fibrosis in portal tract (Table 2) associated with
inflammatory cells infiltration, lipidosis of hepatocytes as well as
sever apoptosis of hepatocytes (Fig. 4; B and C). However, treat-
ment with Phoenix dactylifera seeds, either raw or roasted, ame-
liorated the histological manifestations of liver injury as confirmed
by less cytoplasmic vacuolization of hepatocytes, decreased fibro-
sis and apoptosis (Table 2) (Fig. 4; E–G). Furthermore, the
improvement of liver histology exerted by roasted Phoenix dacty-
lifera seeds treatment was significant ( mild to no fibrosis and
apoptosis) and this effect was superior to that achieved by
silymarin (Fig. 4; D) and (Table 2).
4. Discussion
The present study was undertaken to investigate the protective
effect of aqueous suspension of Phoenix dactylifera L. seeds against
the CCl4-induced hepatic injury in Wistar rats. To our knowledge,
the data published regarding the hepatoprotective effect of Phoe-
nix dactylifera seeds are scanty and limited to a study by Al-Qarawi
et al. (2004) that demonstrated that both Phoenix dactylifera seed
and fruit have hepatoprotective effect on CCl4 treated rats. More-
over, a recent study has reported that Phoenix dactylifera seeds
supplementation has in vivo antioxidant activity which repre-
sented by diminishing the lipid peroxidation product (malondial-
dahyde) in the serum and the liver of normal rats (Habib and
Ibrahim, 2011).
It is noteworthy that the powder of roasted Phoenix dactylifera
seeds is used in making caffeine-free drinks in Arab countries with
a common conception that it is effective against gastric upset and
indigestion. In addition, Phoenix dactylifera seeds have been used
in the Egyptian folk medicine for management of liver diseases for
many years without scientific evidences (Duke, 1992). Accordingly,
we used both raw (unroasted) and roasted Phoenix dactylifera
seeds in order to compare their effect with reference hepatopro-
tective drug. In addition, in the current study we used an aqueous
suspension of Phoenix dactylifera seeds powder to get the advan-
tages of all antioxidant contents.
There is a solid body of literature which has demonstrated that
CCl4 treatment induces hepatic damage in experimental animals
 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743 741

7 Kujawska et al. (2011)). In addition, lipid peroxidation is believed

to be implicated in the induction of liver fibrosis and cirrhosis
6
by upregulating collagen gene expression (Parola et al., 1993).
5 Our histopathological results are consistent with these data; we
Tail DNA %

detected fibrosis, apoptosis and vacuolization in liver tissue of

4
CCl4-treated group which were severe in the centrilobular area.
3 Several reports have demonstrated that the hepatic fibrosis can
2
be attenuated if treated at an early stage and then the irreversible
cirrhosis and hepatocellular carcinoma can be avoided (Dang et al.,
1 2007; Lin et al., 2008). Numerous studies have shown that
0 hepatotoxicity induced by CCl4 may be prevented by antioxidants
supplementation which represents a rationale for using them in
35 the treatment of liver disorders (Kamm et al., 1973; Naziroğlu
et al., 1999; Ozturk et al., 2009).
30
Our preliminary phytochemical analysis revealed that Phoenix
Tail Moment (U)

25 dactylifera seeds possessed substantial amounts of total phenolics

and flavonoids. This comes in accordance with several reports
20
which stated that, Phoenix dactylifera seeds have vast array of
15 antioxidants (phenolics and flavonoids) (Al-Farsi et al., 2007;
Al-Farsi and Lee, 2008; Juhaimi et al., 2012). A recent study has
10
revealed that Phoenix dactylifera seed constitutes one of the high-
5 est sources of the polyphenols, surpassing grapes, flaxseed, nut
seeds and even date fruit (Habib et al., 2014). Notably, the concept
0
of using natural products as antioxidants supplementation is
CCL4 - + + + + based on synergistic effect achieved by presence of different types
Silymarin - - + - - of antioxidant compounds in the same plant.
Raw PDS - - - + - In the current study, Phoenix dactylifera seeds (raw or roasted)
Roasted PDS - - - - + enhanced the antioxidant capacity of liver tissues as represented
by increased levels of SOD and GST. Interestingly, those enzymes
Fig. 3. Effect of Phoenix dactylifera seeds on DNA damage induced by CCl4. The
reach values equivalent to that of the control group only in roasted
oxidative DNA damage as evaluated by the comet assay was represented as Tail
DNA% and Tail moment (U). CCl4 (0.5 ml of 10% CCl4 in olive oil), PDS: Phoenix Phoenix dactylifera seeds-treated group. This indicates that roasted
dactylifera seeds (1 g/kg) for both raw and roasted, silymarin (50 mg/kg). Data are Phoenix dactylifera seeds are more effective than the raw ones in
presented as mean 7 SD, n¼ 7. ###Po 0.001 as compared with the control group. boosting the intracellular antioxidant capabilities. A recent phyto-
n
P o0.05, nnPo 0.01, nnnPo 0.001 compared with CCl4-treated. chemical report has revealed that the antioxidant flavonoid con-
tents of roasted Phoenix dactylifera seeds are superior to that of
Table 2 raw ones (Paranthaman et al., 2012).
Histopathological evaluations of effect of Phoenix dactylifera seeds (PDS) on the On the other hand, the marker of lipid peroxidation, TBARS,
liver lesions of CCl4-intoxicated rats.
decreased in the Phoenix dactylifera seeds treated group (both raw
Lesion Control CCl4 Silymarin Raw PDS Roasted PDS and roasted) to levels close to that of control. This finding is
consistent with a recent study by Habib and Ibrahim (2011) which
Fibrosis
þþþ þ /þ þ þ/ þ þ
/þ demonstrated that feeding normal rats with a diet containing
Apoptosis
þþþ þþ þ
/þ 70 mg/kg Phoenix dactylifera seeds powder decreased the TBARS
CCl4 (0.5 ml of 10% CCl4 in olive oil) was given intraperitoneal twice a week for
significantly in both serum and liver tissue. This denotes that
4 weeks, PDS: Phoenix dactylifera seeds (1 g/kg), silymarin (50 mg/kg). Data are Phoenix dactylifera seeds, with its antioxidant content, have the
presented as (
) Normal; ( þ ) mild; ( þ þ) moderate; and ( þ þ þ) sever. free radical scavenging activity that controls the CCl4-induced
oxidative stress in liver tissues. In addition, the extent of oxidative
(Rechnagel and Glende, 1973; Tsukamoto et al., 1990). In our study, DNA damage induced by CCl4 was mitigated when the animals
exposing Wistar rats to CCl4 for 4 weeks induced severe hepatic were treated with raw or roasted Phoenix dactylifera seeds. Inter-
injury represented by elevated serum levels of ALT, AST and ALP. In estingly, the effect of roasted has surpassed that of raw ones and
addition, elevated oxidative stress, which is represented by high was comparable to that of silymarin. Our results come in accor-
levels of TBARS and NO, was noticed in liver tissue of CCl4-treated dance with previous studies showing that the extent of DNA
rats compared to the control group. Furthermore, CCl4 treatment damage was alleviated when the animals were treated with
decreased the antioxidant capacity of liver tissue as shown by dietary antioxidants, flaxseed extract (Endoh et al., 2002), a
reduced activities of protective enzymes like superoxide dismu- carotenoid-producing algae (Vanitha et al., 2007) and cloudy apple
tase (SOD) and glutathione S-transferase (GST). juice (Kujawska et al., 2011).
It is well established that the disturbance of the pro-oxidant/ Our data revealed that treatment with Phoenix dactylifera seeds
antioxidant balance induced by CCl4 is attributed to the massive (either raw or roasted) significantly diminished the CCl4-induced
amount of reactive free radicals generated during its metabolism. elevations of liver function parameters (GOT, GPT and ALP)
These free radicals are thought to be responsible for lipid perox- in serum. The reduction in these parameters toward the normal
idation of plasma membrane with subsequent cellular necrosis control values is a sign of the stabilization of plasma membranes
(Comporti, 1985; Weber et al., 2003). Moreover, CCl4 induces DNA as well as repair of liver injury. Accordingly, the anti-lipid
damage, mainly due to the reactions of trichloromethyl and/or peroxidation achieved by Phoenix dactylifera seeds halted the
trichloromethyl peroxy radicals directly or indirectly via peroxida- damaging effects of free radicals generated by CCl4 leading to
tion products (Manibusan et al., 2007). Our comet results revealed healing of hepatic parenchyma and regeneration of hepatocytes.
massive DNA damage in CCl4 group which come in the same Moreover, the improvement of the hepatic integrity in the Phoenix
line with numerous reports (López-Diazguerrero et al., 2005; dactylifera seeds-treated groups was very obvious in our
742 D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743
Fig. 4. Effects of Phoenix dactylifera seeds (PDS) and silymarin on histopathological changes induced by CCl4 exposure in Wistar rats. (A) Control group, (B and C) animals
treated with CCl4 (0.5 ml of 10% CCl4 in olive oil), (D) animals treated with CCl4 and silymarin (50 mg/kg), (E and F) animals treated with CCl4 and raw Phoenix dactylifera
seeds (1 g/kg), and (G) animals treated with CCl4 and roasted Phoenix dactylifera seeds (1 g/kg). All sections were stained with Hematoxylin/eosin; 400  for all panels except
(C) and (F) which were 200  magnification.
histopathological results; in which decreased fibrosis, vacuoliza-
tion and apoptosis were observed compared to that of the
CCl4 group.
Interestingly, the protective effect of Phoenix dactylifera seeds
suspension against CCl4-induced liver injury was comparable to
that of silymarin. However, roasted Phoenix dactylifera seeds
suspension revealed superior effect in respect to the raw ones in
promoting the enzymatic antioxidant capability of liver cells.
5. Conclusions
Our results demonstrate that aqueous Phoenix dactylifera seeds
suspension (raw or roasted), at a dose of 1 g/kg, was able to
attenuate the pathological consequences of CCl4 treatment. This
was revealed by mitigation of DNA damage, decrease of lipid
peroxidation, less fibrotic changes in liver besides the normalization
of serum levels of hepatic markers (AST, ALT, ALP and albumin) in a
way comparable to that of silymarin. Moreover, roasted Phoenix
dactylifera seeds suspension showed superior effect on boosting the
antioxidant capacity of liver cells. The protective ability of Phoenix
dactylifera seeds can be explained, at least partially, by the high
content of antioxidants (polyphenols and flavonoids) which act as
free radical scavengers. Therefore, Phoenix dactylifera seeds (either
raw or roasted) may be a promising hepatoprotective agent against
chemically-induced liver damage in vivo. However, further studies
are required to investigate the active constituents responsible for
the effect of Phoenix dactylifera seeds and to explore the other
mechanisms implicated in this protective effect.
Acknowledgment
The authors would like to express their deep appreciation
to Prof. Kawkab Abdulaziz, Department of Pathology, Faculty of
Veterinary Medicine, Cairo University. We are grateful to Hassan
Ashry for his technical assistance. We would like to thank Radwa
Nour for her constructive criticism of the manuscript. This
research received no specific grant from any funding agency in
the public, commercial, or not-for-profit sectors
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