Journal of Ethnopharmacology 155 (2014) 736–743

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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: Received 24 January 2014 Received in revised form 18 April 2014 Accepted 6 June 2014 Available online 16 June 2014

Ethnopharmacological relevance: In traditional Egyptian medicine, Phoenix dactylifera L. (date palm) seeds are listed in folk remedies for the management of diabetes, liver diseases and gastrointestinal disorders. The present study was conducted to investigate the protective effect of Phoenix dactylifera L. seeds aqueous suspension against the chemically-induced hepatic injury in rats. 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 (1.0 g/kg) were administered orally in a daily manner. Results: Our results demonstrated that Phoenix dactylifera seeds significantly improved the CCl4-induced alterations in liver function parameters (AST, ALT, ALP and albumin). Moreover, the CCl4-induced oxidative stress, represented by elevated thiobarbituric acid reactive substance (TBARS), nitric oxide and 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 CCl4induced liver intoxication, and this hepatoprotective effect might be attributed to the antioxidant and free radical scavenging activities. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Phoenix dactylifera seeds Carbon tetrachloride Hepatoprotective Antioxidants Oxidative stress

1. Introduction 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: [email protected] (D.H.A. Abdelaziz), [email protected] (S.A. Ali). 1 Tel.: þ20 1005288921; fax: þ20 25541601. http://dx.doi.org/10.1016/j.jep.2014.06.026 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

species and free radicals generated during its metabolism (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 hepatoprotective 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

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 caffeinefree 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 polyphenolic 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 Governorate, 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,

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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 Institutional 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.

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2.8. Determination of lipid peroxide level Lipid peroxidation level in the liver homogenate was determined as thiobarbituric acid reactive substances (TBARS) level spectrophotometrically 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 dihydrochloric 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).

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 hematoxylineosin (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 significant. 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).

2.13. Alkaline comet assay

3.2. Effect of Phoenix dactylifera seeds on body weight gain and relative liver weight

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 electrophoresis conditions were 2 V/cm for 2 min and 100 mA. Staining was

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

Table 1 Effect of Phoenix dactylifera seeds (PDS) and silymarin on the growth parameters in CCl4-intoxicated rats. Parameter

Control

CCl4

Silymarin

Raw PDS

Roasted PDS

Weight gain (g) Relative liver weight (g/100 g BW)

90.7 79.6 4.34 70.11

22.8 7 10.3### 4.8 7 0.14#

41.6 7 6.16## 4.6 7 0.12

55.4 7 11.18 4.4 7 0.063

607 7.08n 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

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 relative liver weight compared to the CCl4 group (P o0.05).

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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

3.3. Effect of Phoenix dactylifera seeds on serum hepatic markers 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 control 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 treatment was equivalent to that of silymarin-treated group (Fig. 1).

To investigate the ability of Phoenix dactylifera seeds to enhance the antioxidant capacity of liver, we assessed superoxide dismutase (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).

3.4. 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). Silymarintreated group showed significant decline in the level of TBARS and

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;

120

6 *

5

*

*

80

##

4

###

100

3

60

2

40

1

20

0

0

500

500

**

400

400

300

300 ***

***

**

***

200

***

*** 100

100

0 CCl4 Silymarin Raw PDS Roasted PDS

***

###

###

200

***

0

-

+ -

+ + -

+ + -

+ +

-

+ -

+ + -

+ + -

+ +

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.

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TBARS

30

0.25 0.2 0.15 0.1 0.05

20 15 10

0

SOD

120

GST

16 14

(U/min/mg protein)

100

(U/mg protein)

25

5

0

80 60 40 20

12 10 8 6 4 2

0

CCL4 Silymarin Raw PDS Roasted PDS

NO

35

(µmol/mg protein)

(nmol/mg protein)

0.3

0

-

+ -

+ + -

+ + -

+ +

-

+ -

+ + -

+ + -

+ +

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 represented 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 significantly 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 hepatocytes, 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, treatment with Phoenix dactylifera seeds, either raw or roasted, ameliorated the histological manifestations of liver injury as confirmed by less cytoplasmic vacuolization of hepatocytes, decreased fibrosis and apoptosis (Table 2) (Fig. 4; E–G). Furthermore, the improvement of liver histology exerted by roasted Phoenix dactylifera 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 Phoenix 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. Moreover, a recent study has reported that Phoenix dactylifera seeds supplementation has in vivo antioxidant activity which represented by diminishing the lipid peroxidation product (malondialdahyde) 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 hepatoprotective drug. In addition, in the current study we used an aqueous suspension of Phoenix dactylifera seeds powder to get the advantages 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

7

Tail DNA %

6 5 4 3 2 1 0 35

Tail Moment (U)

30 25 20 15 10 5 0

CCL4 Silymarin Raw PDS Roasted PDS

-

+ -

+ + -

+ + -

+ +

Fig. 3. Effect of Phoenix dactylifera seeds on DNA damage induced by CCl4. The 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 dactylifera seeds (1 g/kg) for both raw and roasted, silymarin (50 mg/kg). Data are presented as mean 7 SD, n¼ 7. ###Po 0.001 as compared with the control group. n P o0.05, nnPo 0.01, nnnPo 0.001 compared with CCl4-treated.

Table 2 Histopathological evaluations of effect of Phoenix dactylifera seeds (PDS) on the liver lesions of CCl4-intoxicated rats. Lesion

Control

CCl4

Silymarin

Raw PDS

Roasted PDS

Fibrosis Apoptosis





þþþ þþþ

þ /þ þ þþ

þ/ þ þ þ


/þ
/þ

CCl4 (0.5 ml of 10% CCl4 in olive oil) was given intraperitoneal twice a week for 4 weeks, PDS: Phoenix dactylifera seeds (1 g/kg), silymarin (50 mg/kg). Data are presented as (
) Normal; ( þ ) mild; ( þ þ) moderate; and ( þ þ þ) sever.

(Rechnagel and Glende, 1973; Tsukamoto et al., 1990). In our study, exposing Wistar rats to CCl4 for 4 weeks induced severe hepatic injury represented by elevated serum levels of ALT, AST and ALP. In addition, elevated oxidative stress, which is represented by high levels of TBARS and NO, was noticed in liver tissue of CCl4-treated rats compared to the control group. Furthermore, CCl4 treatment decreased the antioxidant capacity of liver tissue as shown by reduced activities of protective enzymes like superoxide dismutase (SOD) and glutathione S-transferase (GST). It is well established that the disturbance of the pro-oxidant/ antioxidant balance induced by CCl4 is attributed to the massive amount of reactive free radicals generated during its metabolism. These free radicals are thought to be responsible for lipid peroxidation of plasma membrane with subsequent cellular necrosis (Comporti, 1985; Weber et al., 2003). Moreover, CCl4 induces DNA damage, mainly due to the reactions of trichloromethyl and/or trichloromethyl peroxy radicals directly or indirectly via peroxidation products (Manibusan et al., 2007). Our comet results revealed massive DNA damage in CCl4 group which come in the same line with numerous reports (López-Diazguerrero et al., 2005;

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Kujawska et al. (2011)). In addition, lipid peroxidation is believed to be implicated in the induction of liver fibrosis and cirrhosis by upregulating collagen gene expression (Parola et al., 1993). Our histopathological results are consistent with these data; we detected fibrosis, apoptosis and vacuolization in liver tissue of CCl4-treated group which were severe in the centrilobular area. Several reports have demonstrated that the hepatic fibrosis can be attenuated if treated at an early stage and then the irreversible cirrhosis and hepatocellular carcinoma can be avoided (Dang et al., 2007; Lin et al., 2008). Numerous studies have shown that hepatotoxicity induced by CCl4 may be prevented by antioxidants supplementation which represents a rationale for using them in the treatment of liver disorders (Kamm et al., 1973; Naziroğlu et al., 1999; Ozturk et al., 2009). Our preliminary phytochemical analysis revealed that Phoenix dactylifera seeds possessed substantial amounts of total phenolics and flavonoids. This comes in accordance with several reports which stated that, Phoenix dactylifera seeds have vast array of antioxidants (phenolics and flavonoids) (Al-Farsi et al., 2007; Al-Farsi and Lee, 2008; Juhaimi et al., 2012). A recent study has revealed that Phoenix dactylifera seed constitutes one of the highest sources of the polyphenols, surpassing grapes, flaxseed, nut seeds and even date fruit (Habib et al., 2014). Notably, the concept of using natural products as antioxidants supplementation is based on synergistic effect achieved by presence of different types of antioxidant compounds in the same plant. In the current study, Phoenix dactylifera seeds (raw or roasted) enhanced the antioxidant capacity of liver tissues as represented by increased levels of SOD and GST. Interestingly, those enzymes reach values equivalent to that of the control group only in roasted Phoenix dactylifera seeds-treated group. This indicates that roasted Phoenix dactylifera seeds are more effective than the raw ones in boosting the intracellular antioxidant capabilities. A recent phytochemical report has revealed that the antioxidant flavonoid contents of roasted Phoenix dactylifera seeds are superior to that of raw ones (Paranthaman et al., 2012). On the other hand, the marker of lipid peroxidation, TBARS, decreased in the Phoenix dactylifera seeds treated group (both raw and roasted) to levels close to that of control. This finding is consistent with a recent study by Habib and Ibrahim (2011) which demonstrated that feeding normal rats with a diet containing 70 mg/kg Phoenix dactylifera seeds powder decreased the TBARS significantly in both serum and liver tissue. This denotes that Phoenix dactylifera seeds, with its antioxidant content, have the free radical scavenging activity that controls the CCl4-induced oxidative stress in liver tissues. In addition, the extent of oxidative DNA damage induced by CCl4 was mitigated when the animals were treated with raw or roasted Phoenix dactylifera seeds. Interestingly, the effect of roasted has surpassed that of raw ones and was comparable to that of silymarin. Our results come in accordance with previous studies showing that the extent of DNA damage was alleviated when the animals were treated with dietary antioxidants, flaxseed extract (Endoh et al., 2002), a carotenoid-producing algae (Vanitha et al., 2007) and cloudy apple juice (Kujawska et al., 2011). Our data revealed that treatment with Phoenix dactylifera seeds (either raw or roasted) significantly diminished the CCl4-induced elevations of liver function parameters (GOT, GPT and ALP) in serum. The reduction in these parameters toward the normal control values is a sign of the stabilization of plasma membranes as well as repair of liver injury. Accordingly, the anti-lipid peroxidation achieved by Phoenix dactylifera seeds halted the damaging effects of free radicals generated by CCl4 leading to healing of hepatic parenchyma and regeneration of hepatocytes. Moreover, the improvement of the hepatic integrity in the Phoenix dactylifera seeds-treated groups was very obvious in our

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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, vacuolization 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.

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 References

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.

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