Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
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.
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) 736–743 737
report has revealed that the antioxidant flavonoid contents of 1965) and the results were expressed as mg gallic acid equivalent
roasted Phoenix dactylifera seeds were superior to that of raw ones g 1. In addition, total flavonoids were assessed according to
(Paranthaman et al., 2012). Zhishen et al. (1999) and the results were expressed as mg rutin
In Middle East, people believe that eating dates (Phoenix equivalent g 1 of Phoenix dactylifera seeds
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- 2.4. Animals
free beverage with common conception that it is effective against
gastric upsets and indigestion (Ali-Mohamed and Khamis, 2004). This study was conducted on adult male Wistar rats (Rattus
Phoenix dactylifera seed is listed in remedies of Egyptian folk Norvegicus, strain Wistar) weighing 180–200 g provided by Insti-
medicine for the management of various infectious diseases, liver, tutional Breeding House, Egypt. Animals were kept in controlled
diabetes and cancer (Duke, 1992). A recent study by Habib and environment of humidity and temperature with alternating 12 h
Ibrahim (2011) has demonstrated that diet containing Phoenix light/dark cycle for one week for acclimatization, with free access
dactylifera seeds reduces the basal level of lipid peroxidation in to standard rat chow and drinking water ad libitum. The protocol
liver of normal rats while does not affect the antioxidant enzyme of the study was approved by the Animal Ethics Committee of the
capacity of the normal tissues. Furthermore, it has been shown Faculty of Pharmacy, Helwan University on 01/11/2012. The study
that the aqueous and ethanolic extracts of Phoenix dactylifera seeds was conducted in accordance with EC Directive 86/609/EEC for
were effective in ameliorating gastric ulceration in rats (Al-Qarawi animal experiments.
et al., 2005).
To our knowledge, the data concerning the hepatoprotective
2.5. Experimental groups
effect of Phoenix dactylifera seeds are scanty. A study by Al-Qarawi
et al. (2004) has demonstrated that Phoenix dactylifera seeds have
Thirty five rats were randomly divided into five groups (seven
hepatoprotective effect on CCl4 treated rats . However, in this
rats per group). Group I served as normal control and was given
study; the Phoenix dactylifera seeds extract was added to the
olive oil (0.5 ml/rat) intraperitoneally twice a week. To induce
drinking water with no definite dose/rat, only the raw (unroasted)
hepatic injury (in vivo), the animals in Groups II–V received 0.5 ml
Phoenix dactylifera seeds have been used. In addition, they
of CCl4 (10% CCl4 in olive oil) intraperitoneally twice a week (Lin
assessed only the serum markers of liver function. However, the
et al., 2008). Group II (CCl4 group) received CCl4 only. Group III
antioxidant status and the histopathology of liver tissues have not
(reference group) was given silymarin (50 mg/kg) orally on daily
been investigated in their study.
basis (Lin et al., 2012). Silymarin was obtained from crushed
The objective of the present study was to investigate the
Legalons tablets (Madaus, Egypt). Group IV received oral aqueous
hepatoprotective effect of both raw and roasted Phoenix dactylifera
suspension of raw Phoenix dactylifera seeds (1 g/kg) daily. Group V
seeds on CCl4-treated rats. In our study we investigated the
received oral aqueous suspension of roasted Phoenix dactylifera
hepatoprotective effect on different levels, serum parameters,
seeds (1 g/kg) daily. The suspension of Phoenix dactylifera seeds
tissue oxidative stress, including oxidative DNA breaks, and
was vigorously shaken before administration to ensure equivalent
histopathological changes of liver. In addition, silymarin, a poly-
dose for each rat. All groups were treated for four weeks.
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. 2.6. Preparation of serum and tissue homogenate
2.8. Determination of lipid peroxide level conducted using ethidium bromide 20 mg/ml at 4 1C. The DNA
fragment migration patterns were evaluated with a fluorescence
Lipid peroxidation level in the liver homogenate was determined microscope (excitation filter 420–490 nm). The comet tail lengths
as thiobarbituric acid reactive substances (TBARS) level spectropho- were measured from the middle of the nucleus to the end of the
tometrically in liver homogenates (Mihara and Uchiyama, 1978). tail. We used Komet 5 image analysis software developed by
Values were expressed as nmoles of TBARS/mg protein. Kinitic Imaging, Ltd. (Liverpool, UK) linked to a CCD camera to
assess the quantitative and qualitative extent of DNA damage.
2.9. Determination of glutathione S-transferase (GST) activity Generally, 50 to 100 randomly selected cells were analyzed per
sample.
GST activity was determined spectrophotometrically in liver
homogenate by measuring the increase in absorbance of the 2.14. Histopathological examination
reaction mixture at 340 nm (Habig et al., 1974). The GST activity,
was defined as the amount of enzyme producing 1 μmol of Immediately following sacrifice of the animals, liver tissues were
1-chloro-2,4-dinitrobenzene (CDNB)-Glutathione conjugate/min surgically excised, individually weighed, and liver slices were cut and
under the conditions of the assay. The GST activity was calculated fixed in 10% neutral buffered formalin and embedded in paraffin.
using an extinction coefficient of 9.6 mM 1 cm 1. The results Tissue sections (5 μm thick) were prepared, stained with hematoxylin-
were expressed in U/min/mg of protein. eosin (H&E), and then examined under light microscope at 200 and
400 magnifications for determination of pathological changes.
2.10. Determination of nitric oxide level The sections were analyzed blindly by a certified pathologist
and three different sections were examined in each sample of liver.
Nitrite level was assayed in liver homogenate supernatant by The severity of histopathological changes (fibrosis and apoptosis)
reacting with Griess reagent (1:1 solution of 1% sulfanilamide in was scored according to an arbitrary scale (between to þ þ þ).
5% phosphoric acid and 0.1% naphthyl-ethylenediamine dihydro-
chloric acid in water) and measuring the absorbance at 543 nm. 2.15. Statistics
Nitrite level was calculated using a standard curve for sodium
nitrite and its level was expressed as mmol/mg protein (Green All data were expressed as mean7SEM for seven rats in each
et al., 1982). group. Statistical analysis was performed by one-way analysis
of variance (ANOVA) followed by Tukey–Kramer test for multiple
2.11. Superoxide dismutase (SOD) activity comparisons using GraphPad Instat (Graph software Inc., V 3.05, Ralf
Stahlman, Purdue Univ.). Po0.05 was considered statistically signifi-
Superoxide dismutase (SOD) activity in liver homogenate was cant. Appropriate graphs were plotted using Microsoft Excel 2007.
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 3. Results
enzymes required to inhibit the rate of pyrogallol oxidation by 50%
at 25 1C. The activity was expressed as units/mg protein. 3.1. Phytochemical constituents of Phoenix dactylifera seeds
2.12. Determination of tissue protein content The preliminary phytochemical analysis revealed that Phoenix
dactylifera seeds contained significant amounts of total phenolics
Protein content in liver homogenate was determined according (38.8 mg gallic acid equivalent g 1) and total flavonoids (87.86 mg
to Lowry's method using bovine serum albumin (BSA) as a rutin equivalent g 1).
standard (Lowry et al., 1951).
3.2. Effect of Phoenix dactylifera seeds on body weight gain and
2.13. Alkaline comet assay relative liver weight
The Comet assay was performed essentially as described by The growth performance of the studied groups was assessed
Singh et al., (1988). Liver tissue (0.5 g) was crushed and transferred using the increase in body weight at the end of the experiment.
to 1 ml ice-cold PBS (phosphate buffer saline). This suspension was The body weight gain and the relative liver weights of all studied
stirred for 5 min and filtered. Cell suspension (100 ml) was mixed groups are shown in Table 1. The administration of CCl4 for
with 600 ml of low melting agarose (0.8% in PBS). The mixture 4 weeks attenuated the weight gain significantly (22.8 710.3 vs.
(100 ml) was spread on pre-coated slides. The coated slides were 90.77 9.6, P o0.001) compared to the control group. The group
immersed in lyses buffer for 15 min. The slides were placed in treated with silymarin gained more weight than CCl4 group but
electrophoresis chamber containing TBE buffer. The electrophor- still significantly less than the control group (Po 0.01). Treatment
esis conditions were 2 V/cm for 2 min and 100 mA. Staining was with raw or roasted Phoenix dactylifera seeds markedly improved
Table 1
Effect of Phoenix dactylifera seeds (PDS) and silymarin on the growth parameters in CCl4-intoxicated rats.
Weight gain (g) 90.7 79.6 22.8 7 10.3### 41.6 7 6.16## 55.4 7 11.18 607 7.08n
Relative liver weight (g/100 g BW) 4.34 70.11 4.8 7 0.14# 4.6 7 0.12 4.4 7 0.063 4.3 7 0.068n
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.
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 NO (P o0.001 and Po0.01, respectively) compared to CCl4 group.
Phoenix dactylifera seeds showed significant weight increment Furthermore, treatment with Phoenix dactylifera seeds, either raw
compared to CCl4 group (P o0.05). Furthermore, CCl4 intoxication or roasted, caused efficient attenuation of TBARS and NO levels
increased the relative liver weights significantly compared to the compared to CCl4 group (Po 0.001 and P o0.01, respectively).
control group (P o0.05). Although the other groups showed Despite the marked decrease in NO levels in both raw and roasted
decrease in the relative liver weights, only the group treated with Phoenix dactylifera seeds-treated groups, they did not restore the
roasted Phoenix dactylifera seeds showed significantly lower rela- liver NO values to the control values (P o0.05).
tive liver weight compared to the CCl4 group (P o0.05).
3.5. Effect of Phoenix dactylifera seeds on antioxidant enzymes in
3.3. Effect of Phoenix dactylifera seeds on serum hepatic markers liver tissue
The liver function parameters analyzed in this study are shown To investigate the ability of Phoenix dactylifera seeds to enhance
in Fig. 1. Treatment with CCl4 for 4 weeks induced abnormal liver the antioxidant capacity of liver, we assessed superoxide dismu-
function parameters as shown by elevation of serum levels of tase (SOD) and glutathione S-transferase (GST) in liver tissue
hepatic enzymes AST, ALT and ALP (P o0.001) whereas, serum homogenate. We found that 4 weeks of CCl4 treatment induced
albumin level significantly decreased (P o0.01) compared to con- significant decline in SOD and GST enzymes activities in the liver
trol group. Administration of Phoenix dactylifera seeds (raw or homogenates compared to the control group (Po 0.05 and
roasted) restored normal levels of ALT, AST and ALP (P o0.001) and Po 0.01, respectively). The liver homogenates of groups treated
caused significant increase in serum albumin level (P o0.05) with silymarin, raw and roasted Phoenix dactylifera seeds showed
compared to CCl4 group. Furthermore, the improvement of liver higher levels of both SOD and GST. Yet, only the group treated
function parameters achieved by Phoenix dactylifera seeds treat- with roasted Phoenix dactylifera seeds demonstrated statistically
ment was equivalent to that of silymarin-treated group (Fig. 1). significant augmentation in the levels of both enzymes (P o0.05
for both) compared to the CCl4 group (Fig. 2).
3.4. Potency of Phoenix dactylifera seeds as a free radical scavenger
3.6. Effect of Phoenix dactylifera seeds on oxidative DNA damage
After four weeks of CCl4 exposure, levels of TBARS (marker of in liver tissue
lipid peroxidation) and NO in liver were significantly elevated
compared to the control group (P o0.001) (Fig. 2). Silymarin- The comet assay (Single-cell gel electrophoresis) is a sensitive
treated group showed significant decline in the level of TBARS and 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
(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 apoptosis) and this effect was superior to that achieved by
detect oxidative DNA breaks (Collins et al., 1995; Pfuhler and Wolf, silymarin (Fig. 4; D) and (Table 2).
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 4. Discussion
head, in microns, multiplied by the percentage of DNA in the tail
(Bowden et al., 2003). In the current study, dosing rats with CCl4 The present study was undertaken to investigate the protective
markedly damages DNA as shown by significant increase in tail effect of aqueous suspension of Phoenix dactylifera L. seeds against
DNA% and tail moment (P o0.001 for both). However, treatment the CCl4-induced hepatic injury in Wistar rats. To our knowledge,
with Phoenix dactylifera seeds, either raw or roasted, significantly the data published regarding the hepatoprotective effect of Phoe-
decreased the tail moment (Po 0.001 for both) and the tail DNA% nix dactylifera seeds are scanty and limited to a study by Al-Qarawi
(P o0.05 and P o0.01, respectively). In addition, silymarin signifi- et al. (2004) that demonstrated that both Phoenix dactylifera seed
cantly diminished the DNA breaks induced by CCl4 as shown and fruit have hepatoprotective effect on CCl4 treated rats. More-
in Fig. 3. 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-
3.7. Histopathology of liver dahyde) in the serum and the liver of normal rats (Habib and
Ibrahim, 2011).
To confirm our findings on the sera and liver homogenates, we It is noteworthy that the powder of roasted Phoenix dactylifera
investigated the pathologic changes in liver microscopically. The seeds is used in making caffeine-free drinks in Arab countries with
examination of the liver histology of CCl4 group revealed sever a common conception that it is effective against gastric upset and
hepatic injury represented by cytoplasmic vacuolization of hepa- indigestion. In addition, Phoenix dactylifera seeds have been used
tocytes, sever fibrosis in portal tract (Table 2) associated with in the Egyptian folk medicine for management of liver diseases for
inflammatory cells infiltration, lipidosis of hepatocytes as well as many years without scientific evidences (Duke, 1992). Accordingly,
sever apoptosis of hepatocytes (Fig. 4; B and C). However, treat- we used both raw (unroasted) and roasted Phoenix dactylifera
ment with Phoenix dactylifera seeds, either raw or roasted, ame- seeds in order to compare their effect with reference hepatopro-
liorated the histological manifestations of liver injury as confirmed tective drug. In addition, in the current study we used an aqueous
by less cytoplasmic vacuolization of hepatocytes, decreased fibro- suspension of Phoenix dactylifera seeds powder to get the advan-
sis and apoptosis (Table 2) (Fig. 4; E–G). Furthermore, the tages of all antioxidant contents.
improvement of liver histology exerted by roasted Phoenix dacty- There is a solid body of literature which has demonstrated that
lifera seeds treatment was significant ( mild to no fibrosis and CCl4 treatment induces hepatic damage in experimental animals
D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743 741
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.
References
5. Conclusions
Al-Farsi, M., Alasalvar, C., Al-Abid, M., Al-Shoaily, K., Al-Amry, M., Al-Rawahy, F.,
Our results demonstrate that aqueous Phoenix dactylifera seeds 2007. Compositional and functional characteristics of dates, syrups, and their
suspension (raw or roasted), at a dose of 1 g/kg, was able to by-products. Food Chemistry 104, 943–947.
Al-Farsi, M.A., Lee, C.Y., 2008. Nutritional and functional properties of dates: a
attenuate the pathological consequences of CCl4 treatment. This review. Critical Reviews in Food Science and Nutrition 48, 877–887.
was revealed by mitigation of DNA damage, decrease of lipid Ali-Mohamed, A.Y., Khamis, A.S.H., 2004. Mineral ion content of the seeds of six
peroxidation, less fibrotic changes in liver besides the normalization cultivars of Bahraini date palm (Phoenix dactylifera). Journal of Agricultural and
Food Chemistry 52, 6522–6525.
of serum levels of hepatic markers (AST, ALT, ALP and albumin) in a Al-Qarawi, A., Mousa, H., Ali, B., Abdel-Rahman, H., El-Mougy, S., 2004. Protective
way comparable to that of silymarin. Moreover, roasted Phoenix effect of extracts from Dates (Phoenix dactylifera L.) on carbon tetrachloride–
dactylifera seeds suspension showed superior effect on boosting the induced hepatotoxicity in rats. The International Journal of Applied Research in
Veterinary Medicine 2, 176–180.
antioxidant capacity of liver cells. The protective ability of Phoenix Al-Qarawi, A.A., Abdel-Rahman, H., Ali, B.H., Mousa, H.M., El-Mougy, S.A., 2005. The
dactylifera seeds can be explained, at least partially, by the high ameliorative effect of dates (Phoenix dactylifera L.) on ethanol-induced gastric
content of antioxidants (polyphenols and flavonoids) which act as ulcer in rats. Journal of Ethnopharmacology 98, 313–317.
Bowden, R.D., Buckwalter, M.R., McBride, J.F., Johnson, D.A., Murray, B.K., O'Neill, K.
free radical scavengers. Therefore, Phoenix dactylifera seeds (either
L., 2003. Tail profile: a more accurate system for analyzing DNA damage using
raw or roasted) may be a promising hepatoprotective agent against the Comet assay. Mutation Research 537, 1–9.
chemically-induced liver damage in vivo. However, further studies Collins, A.R., Ma, A.G., Duthie, S.J., 1995. The kinetics of repair of oxidative DNA
are required to investigate the active constituents responsible for damage (strand breaks and oxidised pyrimidines) in human cells. Mutation
Research 336, 69–77.
the effect of Phoenix dactylifera seeds and to explore the other Comporti, M., 1985. Lipid peroxidation and cellular damage in toxic liver injury.
mechanisms implicated in this protective effect. Laboratory Investigation 53, 599–623.
D.H.A. Abdelaziz, S.A. Ali / Journal of Ethnopharmacology 155 (2014) 736–743 743
Dang, S.-S., Wang, B.-F., Cheng, Y.-A., Song, P., Liu, Z.-G., Li, Z.-F., 2007. Inhibitory Mihara, M., Uchiyama, M., 1978. Determination of malonaldehyde precursor in
effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats. World tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278.
Journal of Gastroenterology 13, 557–563. Mourelle, M., Muriel, P., Favari, L., Franco, T., 1989. Prevention of CCL4-induced liver
Duke, J.A., 1992. Handbook of Phytochemical Constituents of GRAS Herbs and Other cirrhosis by silymarin. Fundamental & Clinical Pharmacology 3, 183–191.
Economic Plants. Herbal Reference Library. CRC Press, Florida. Naziroğlu, M., Cay, M., Ustündağ, B., Aksakal, M., Yekeler, H., 1999. Protective effects
Endoh, D., Okui, T., Ozawa, S., Yamato, O., Kon, Y., Arikawa, J., Hayashi, M., 2002. of vitamin E on carbon tetrachloride-induced liver damage in rats. Cell
Protective effect of a lignan-containing flaxseed extract against CCl(4)-induced Biochemistry and Function 17, 253–259.
hepatic injury. Journal of Veterinary Medical Science 64, 761–765. Okuda, M., Li, K., Beard, M.R., Showalter, L.A., Scholle, F., Lemon, S.M., Weinman, S.
Fairbairn, D.W., Olive, P.L., O'Neill, K.L., 1995. The comet assay: a comprehensive A., 2002. Mitochondrial injury, oxidative stress, and antioxidant gene expres-
review. Mutation Research 339, 37–59. sion are induced by hepatitis C virus core protein. Gastroenterology 122,
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S. 366–375.
R., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Oliveira, C.P.M.S., da Costa Gayotto, L.C., Tatai, C., Della Bina, B.I., Janiszewski, M.,
Analytical Biochemistry 126, 131–138. Lima, E.S., Abdalla, D.S.P., Lopasso, F.P., Laurindo, F.R.M., Laudanna, A.A., 2002.
Habib, H.M., Ibrahim, W.H., 2011. Effect of date seeds on oxidative damage and Oxidative stress in the pathogenesis of nonalcoholic fatty liver disease, in rats
antioxidant status in vivo. Journal of the Science of Food and Agriculture 91, fed with a choline-deficient diet. Journal of Cellular and Molecular Medicine 6,
1674–1679. 399–406.
Habib, H.M., Platat, C., Meudec, E., Cheynier, V., Ibrahim, W.H., 2014. Polyphenolic Ozturk, I.C., Ozturk, F., Gul, M., Ates, B., Cetin, A., 2009. Protective effects of ascorbic
compounds in date fruit seed (Phoenix dactylifera): characterisation and acid on hepatotoxicity and oxidative stress caused by carbon tetrachloride in
quantification by using UPLC-DAD-ESI-MS. Journal of the Science of Food and the liver of Wistar rats. Cell Biochemistry & Function 27, 309–315.
Agriculture 94, 1084–1089. Paranthaman, R., Praveen Kumar, P., Kumaravel, S., 2012. HPLC and HPTLC deter-
Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first mination of caffeine in raw and roasted date seeds (Phoenix Dactylifera L.). The
enzymatic step in mercapturic acid formation. Journal of Biological Chemistry Journal of Chromatography and Separation Techniques 1, 249–253.
249, 7130–7139. Parola, M., Leonarduzzi, G., Biasi, F., Albano, E., Biocca, M.E., Poli, G., Dianzani, M.U.,
Halim, A.B., el-Ahmady, O., Hassab-Allah, S., Abdel-Galil, F., Hafez, Y., Darwish, A., 1992. Vitamin E dietary supplementation protects against carbon tetrachloride-
1997. Biochemical effect of antioxidants on lipids and liver function in experi- induced chronic liver damage and cirrhosis. Hepatology 16, 1014–1021.
mentally-induced liver damage. Annals of Clinical Biochemistry 34 (Pt 6), Parola, M., Pinzani, M., Casini, A., Albano, E., Poli, G., Gentilini, A., Gentilini, P.,
Dianzani, M.U., 1993. Stimulation of lipid peroxidation or 4-hydroxynonenal
656–663.
treatment increases procollagen alpha 1 (I) gene expression in human liver fat-
Juhaimi, F. Al, Ghafoor, K., Özcan, M.M., 2012. Physical and chemical properties,
storing cells. Biochemical and Biophysical Research Communications 194,
antioxidant activity, total phenol and mineral profile of seeds of seven different
1044–1050.
date fruit (Phoenix dactylifera L.) varieties. International Journal of Food Science
Pfuhler, S., Wolf, H.U., 1996. Detection of DNA-crosslinking agents with the alkaline
and Nutrition 63, 84–89.
comet assay. Environmental and Molecular Mutagenesis 27, 196–201.
Kamm, J.J., Dashman, T., Conney, A.H., Burns, J.J., 1973. Protective effect of ascorbic
Rechnagel, R.O., Glende, E.A., 1973. Carbon tetrachloride hepatotoxicity: an example
acid on hepatotoxicity caused by sodium nitrite plus aminopyrine. Proceedings
of lethal cleavage. CRC Critical Reviews in Toxicology 2, 263–297.
of the National Academy of Sciences of the United States of America 70,
Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum
747–749.
glutamic oxalacetic and glutamic pyruvic transaminases. American Journal of
Kanter, M., Coskun, O., Budancamanak, M., 2005. Hepatoprotective effects of Nigella
Clinical Pathology 28, 56–63.
sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems
Roth, E.F., Gilbert, H.S., 1984. The pyrogallol assay for superoxide dismutase:
and liver enzymes in carbon tetrachloride-treated rats. World Journal of absence of a glutathione artifact. Analytical Biochemistry 137, 50–53.
Gastroenterology 11, 6684–6688. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for
Kujawska, M., Ignatowicz, E., Ewertowska, M., Markowski, J., Jodynis-Liebert, J., quantitation of low levels of DNA damage in individual cells. Experimental Cell
2011. Cloudy apple juice protects against chemical-induced oxidative stress in Research 175, 184–191.
rat. European Journal of Nutrition 50, 53–60. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
Lee, Young Ik, Hwang, J.M., Im, J.H., Lee, Yoon Ik, Kim, N.S., Kim, D.G., Yu, D.Y., Moon, phosphomolybdic-phosphotungstic acid reagents. American Society for Enol-
H.B., Park, S.K., 2004. Human hepatitis B virus-X protein alters mitochondrial ogy and Viticulture 16, 144–158.
function and physiology in human liver cells. Journal of Biological Chemistry Tanikawa, K., Torimura, T., 2006. Studies on oxidative stress in liver diseases:
279, 15460–15471. important future trends in liver research. Medical Molecular Morphology 39,
Lin, H.-M., Tseng, H.-C., Wang, C.-J., Lin, J.-J., Lo, C.-W., Chou, F.-P., 2008. Hepato- 22–27.
protective effects of Solanum nigrum Linn extract against CCl(4)-induced Tsukamoto, H., Matsuoka, M., French, S.W., 1990. Experimental models of hepatic
oxidative damage in rats. Chemico-biological Interactions 171, 283–293. fibrosis: a review. Seminars in Liver Disease 10, 56–65.
Lin, X., Liu, X., Huang, Q., Zhang, S., Zheng, L., Wei, L., He, M., Jiao, Y., Huang, J., Fu, S., Tsukiyama-Kohara, K., 2012. Role of oxidative stress in hepatocarcinogenesis
Chen, Z., Li, Y., Zhuo, L., Huang, R., 2012. Hepatoprotective effects of the induced by hepatitis C virus. International Journal of Molecular Sciences 13,
polysaccharide isolated from Tarphochlamys affinis (Acanthaceae) against 15271–15278.
CCl4-induced hepatic injury. Biological & Pharmaceutical Bulletin 35, Vanitha, A., Murthy, K.N.C., Kumar, V., Sakthivelu, G., Veigas, J.M., Saibaba, P.,
1574–1580. Ravishankar, G.A., 2007. Effect of the carotenoid-producing alga, Dunaliella
López-Diazguerrero, N.E., Luna-López, A., Gutiérrez-Ruiz, M.C., Zentella, A., Königs- bardawil, on CCl4-induced toxicity in rats. The International Journal of
berg, M., 2005. Susceptibility of DNA to oxidative stressors in young and aging Toxicology 26, 159–167.
mice. Life Sciences 77, 2840–2854. Weber, L.W.D., Boll, M., Stampfl, A., 2003. Hepatotoxicity and mechanism of action
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement of haloalkanes: carbon tetrachloride as a toxicological model. Critical Reviews
with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. in Toxicology 33, 105–136.
Manibusan, M.K., Odin, M., Eastmond, D.A., 2007. Postulated carbon tetrachloride Zhishen, J, Mengceng, T, Iianming, W, 1999. The determination of flavonoid
mode of action: a review. Journal of Environmental Science and Health. Part C: contents in mulberry and their scavenging effects on superoxide radicals. Food
Environmental Carcinogenesis & Ecotoxicology Reviews 25, 185–209. chemistry 64, 555–559.