Liver disease is the major cause of serious health problem leading to morbidity and mortality worldwide and the problem has increased in search for hepatotherapeutic agents from plants. The present study was designed to compare the probable hepatoprotective activity of olive oil and N. sativa oil on CCl4 induced liver damage in male rats.
Methods
Forty males of a new model of albino rats (Wistar strain) (175–205 g) were divided into four groups. The 1st Group (G1) was the negative control group, the remaining rats were injected with CCl4 (1 ml/kg body weight) with equal amount of olive oil on the 1st and 4th day of every week for 4 weeks. The 2nd group (G2) was the positive control, the 3rd group (G3) and the fourth group (G4) were treated orally with N. sativa oil and olive oils using stomach tube.
Results
The positive control group showed an increase in hepatic enzymes, total bilirubin, creatinine, uric acid, lipid peroxide total cholesterol, triglyceride, low density lipoprotein, very low density lipoproteins, interleukin-6, and a decrease in antioxidant enzymes, high density lipoprotein cholesterol, a decrease in total protein and albumin an when compared with negative control group. Histology of the CCl4 treated group revealed inflammation and damage of liver cells. Treating the hepatotoxic rats with olive oil and N. sativa oil showed a significant improvement in all biochemical tests compared with the positive CCl4 control group. In addition, the liver tissues of olive oil treated group showed mild improvement in inflammatory infiltration and in N. sativa oil treated group showed normal hepatocytes with no evidence of inflammation.
Conclusion
This study revealed that olive oil and N. sativa oil have a protective effect against CCl4-induced hepatotoxicity in male rats. Nigella sativa oil was more effective than olive oil.
Abkürzungen
ALP
Serum alkaline phosphatase
ALT
Serum alanine aminotransferase
AST
Serum aspartate aminotransferase
B.W
Body weight
BWG
Body weight gain
BWG%
Body weight gain ratio (Percent)
Cat
Catalase
CCl4
Carbon tetrachloride
Crea
Creatine kinase-MB
CYP
Cytochrome P450
CYP-2E1
Cytochrome P450 2E1
FER
Food Efficiency ratio
FI
Food intake
G1
The first group, was untreated control group and was administered with (1 ml/kg body weight) olive oil (intraperitoneally injected)
G2
The positive CCl4 control group and received only CCl4 (1 ml/kg body weight): olive oil (1:1) on the 1st and 4th day of every week intraperitoneally injected for 4 weeks
G3
The 3rd group, was injected with CCl4 [(1 ml/kg body weight): olive oil (1:1)] intraperitoneally on the 1st and 4th day of every week and cotreated orally with olive oil (1 ml/kg bw) for 4 weeks using a stomach tube
G4
The 4th group, was injected with CCl4 [(1 ml/kg body weight): olive oil (1:1)] intraperitoneally injected and cotreated orally with (1 ml/kg b w) of Nigella sativa oil for 4 weeks using a stomach tube
GGT
Gamma-glutamyl transferase
GSH
Glutathione
GSST
Glutathione reductase
GSTs
Glutathione S-transferases
H&E
Hematoxylin and eosin
HDL
High density lipoprotein
LDL
Low density lipoprotein cholesterol
MDA
Malondialdehyde
N. sativa
Nigella sativa
ROS
Reactive oxygen species
S.IL-6
Serum interleukin-6
SOD
Superoxide dismutase
SPSS
Statistical program for sociology scientists
TC
Total cholesterol
TG
Triglyceride
TIL6
Tissue Tinterleukin-6
TQ
Thymoquinone
UA
Uric acid
VLDL
Very low density lipoproteins cholesterol
Background
Liver is a multipurpose organ of the body that controls internal chemical environment [1]. It handles the metabolism and excretion of drugs and other xenobiotics from the body thereby providing protection against foreign substances by detoxifying and eliminating them [2]. Liver purifies enthetic chemical molecules through oxidation, reduction and/or conjugation [3]. It is certainly affected by free radical and causes disease hepatitis, cirrhosis, liver cancer and other alcohol related disorders [4]. Liver disease is the major causes of serious health problem leading to morbidity and mortality worldwide and the problem has increased in search for hepatotherapeutic agents from plants [5, 6].
Liver injury or dysfunction is well known as a serious health problem [7] and can be produced by toxic chemicals, drugs, and virus infiltration from ingestion or infection [8]. Exposure of diverse environment pollutants and xenobiotics such as alcohol, paracetamol, carbon tetrachloride (CCl4), thioacetamide are the major cause of liver disorder, which damage the liver by producing reactive oxygen species [5] which are extremely toxic and produce injury in the tissue through covalent bond and oxidation in DNA base, lipid and protein, Also can change the functional activity of enzymes and structural proteins [9]. Carbontetrachloride (CCl4) is hepatotoxicant and has been commonly used for generating liver injury in rat model [10]. In hepatocytes CCl4 is metabolized by the cytochrome P450 to produce the highly reactive free radicals [11]. The key role in the pathogenesis of CCl4-induced hepatic injury is oxidative stress, which is a physiological status associated with unbalance between free radical [10] and antioxidant defenses [12]. In addition, CCl4 hepatotoxicity also causes increased blood flow and cytokine accumulation that are characteristic of tissue inflammation.
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For long time, plants were used in treatment of many diseases especially in the East region countries [13]. In addition, Lycium barbarum protects mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation by reducing the hepatic necrosis and the serum alanine aminotransferase (ALT) level induced by CCl4 intoxication, inhibiting cytochrome P450 2E1 expression, and restoring the expression levels of antioxidant enzymes and decreasing the level of nitric oxide metabolism and lipid peroxidation induced by CCl4 [14].
Gorinstein et al. [15] stated that olive oils improve lipid metabolism and increase antioxidant potential in rats fed diets containing cholesterol. Administration of olive oil may have a potential role as an antioxidant and in lowering the risk of malignant neoplasms, especially breast and stomach cancer; and also in ovary, colon and endometrium cancer [16]. The popularity of olive oil is increasing mostly attributed to its antioxidant and anti-inflammatory effects, which may help in preventing diseases in humans [17]. Diverse studies have exposed that the consumption of olive oil may have a potential role in decreasing the risk of malignant neoplasms, especially breast and stomach cancer; and also in ovary, colon and endometrium cancer [18, 19].
Nigella sativa L. (is also known as black seed or black cumin) seeds have curative potential as described in the Old Testament and in Islamic culture [20]. Black seed oil is traditionally used for enhancing immunity and combating inflammatory and respiratory diseases, among many disorders [21]. Thymoquinone, present in N. sativa oil, has growth inhibitory effects against a variety of cancerous cells through the inhibition of DNA synthesis and the induction of cell cycle arrest [22, 23]. Nigella sativa anti-inflammatory potential accounts for the observed analgesic, antidiabetic, and antihistaminic effects, and ability to alleviate diabetes, respiratory diseases, rheumatoid arthritis, multiple sclerosis, and Parkinson’s disease [24, 25].
The aim of this study is to compare the protective and curative effects of olive oil and Nigella sativa oil on CCl4 induced liver damage in male rats.
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Methods
Animal
Forty males of a new model of albino rats (Wistar strain) weighing about 175–205 g were obtained from King Fahd Medical Research Center (KFMRC), King Abdulaziz University, Jeddah, Saudi Arabia. All the animal experiments were carried out under protocols approved by the Institutional Animal House of the University of King Abdulaziz at Jeddah, Saudi Arabia. The plan of our study specifically was approved by our institutional ethics committee at King Abdulaziz University (KAU-1435). The rats were housed in standard laboratory conditions at a temperature of (25 ± 3 °C), relative humidity (50–55 %) and a 12 h light/dark cycle (five rats / cage) 2 weeks before the start of the experiment. Cages, bedding, and glass water bottles (equipped with stainless steel sipper tubes) were replaced twice per week. Stainless steel feed containers were changed once a week. All animals fed standard nutritionally balanced diet and drinking water ad libitum.
Conventional animal basal diet
Standard nutritionally balanced diet consisted of the following ingredients; 20.0 % protein, 4.0 % fat, 5.0 % fiber, 1.0 % vitamin mix, 3.50 % mineral mix, 0.25 % choline chloride and 66.25 % corn starch. Its energy equals 2850 kcal/kg. The diet manufactured by a Grain Silos and Flour Mills, in Jeddah, KSA. Basal diet food was stored in a dry place out of direct sunlight.
Carbon Tetrachloride (CCl4) were obtained from Merck Ltd., Coimbatore, Tamilnadu (India).
The olive oil was an extra virgin oil, produced from a local olive oil factory in Jeddah, Saudi Arabia. The quality of the used olive oil was tested to be sure that it is true extra virgin olive oil spectrophotometrically by having a stronger absorbance. Nigella sativa oil were purchased from a local herbal medicine shop in Jeddah, Saudi Arabia.
Design of the experiment
Forty rats were divided randomly into four groups, each consists of ten rats as follows:
1.
Group 1)G1(: The first group is untreated control group and was administered with olive oil (intraperitoneally injected at 8.00 Am in the 1st and 4th day of every week until the last day of the experiment) which was used as vehicle, and fed normal basal diet and water for 4 weeks.
CCl4 (1 ml/kg body weight) was administered to animals of all the remaining groups at 8.00 Am in the 1st and 4th day of every week until the last day of the experiment by intraperitoneal injection with equal amount of olive oil.
2.
Group 2 (G2) was the positive CCl4 control group and received only CCl4 (1 ml/kg body weight): olive oil (1:1) at 8.00 Am in the 1st and 4th day of every week intraperitoneally injected for 4 weeks.
3.
Group 3 (G3) was injected with CCl4 [(1 ml/kg body weight): olive oil (1:1)] at 8.00 Am in the 1st and 4th day of every week intraperitoneally and cotreated daily with olive oil (1 ml/kg bw, for 4 weeks) orally using a stomach tube, at 8.00 Am. This group represents the positive control treated with olive oil which is well known with it is hepatoprotective effect.
4.
Group 4 (G4) was injected with CCl4 [(1 ml/kg body weight): olive oil (1:1)] intraperitoneally injected and cotreated daily with (1 ml/kg b w) of Nigella sativa oil for 4 weeks orally using a stomach tube, at 8.00 Am.
Samples collection and organs weight
At the end of the experiment rats were anesthetized using diethyl ether then, blood samples were collected from the heart of rat under anesthesia with diethyl ether. Serum was separated by centrifugation at 7000 rpm for 15 min at 4 °C. After collection of blood, anaesthetized animals were scarified by cervical dislocation. The abdomen was opened and the organs (Heart, liver, kidney sand testes) were rapidly dissected out, weighed and kept in saline. Apiece of liver was washed in sterile saline and fixed in 10 % buffered formalin for histopathological studies. A piece of liver was kept at ice-cold temperature to prepare to prepare liver tissue homogenate for antioxidant enzymes, lipid peroxide and interleukin-6 estimation.
Food intake and water consumption
Food intake per cage was recorded once per week.
Weight gain (g), body weight gain ratio (BWG%) and food efficiency ratio (FER)
Body weight gain (g), body weight gain ratio (BWG%) and food efficiency ratio (FER) were calculated as follows:
A piece of the liver tissue was cut into small pieces and washed with phosphate-buffered saline and then grinded in a homogenization buffer consisting of 0.05 M Tris-HCl pH 7.9, 25 % glycerol, 0.1 mM EDTA, and 0.32 M (NH4)2SO4 and containing a protease inhibitor tablet from Roche (Germany). The lysates mix was homogenized on ice using a Polytron homogenizer. The mix was sonicated in an ice bath to prevent overheating for 15 s followed by 5 min centrifugation at 12,000 rpm and 4∘C. The supernatant was aliquoted and stored at −80∘C.
Liver enzymes
Serum alanine aminotransferase (ALT) was estimated according to the method of Schumann and Klauke [26] using human kit (Germany), serum aspartate transaminase (AST) was estimated according to the method of Bergmeyer et al. [27], using Swemed diagnostics kit (India) and serum alkaline phosphatase (ALP) was estimated according to the method of Rick [28] using Human Kit (Germany). Estimation was done according to the instruction of the supplier.
Total proteins
Total protein and albumin were measured using commercial kits according to the instruction of the supplier. Total protein was quantified according to the method of Cannon et al. [29] using a Total protein kit Sigma-Aldrich (USA). Serum Albumins were estimated according to the method of Lee [30] using Sigma-Aldrich (USA) according to the instruction of the supplier.
Total bilirubin
Total bilirubin was estimated according to the method of Balistreri and Shaw [31] using Human Kit (Germany) according to the instruction of the supplier.
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Kidney functions
Kidney functions parameters; creatinine, uric acid, blood urea were measured using commercial kits according to the instruction of the manufacturer as follows: i- Serum urea and uric acid were estimated according to the methods of Fawcett and Scott [32], Fossati et al. [33], respectively using Human kit (Germany), ii- Serum creatinine was estimated according to the method of Tietz [34] using Moody International creatinine kit (UKAS, Germany).
Assessment of lipid profile
Lipid profile was determined by assessing serum TG, cholesterol, VLDL, LDL and HDL levels using commercial kits, following manufacturer’s instructions. Serum total cholesterol (TC), serum high density lipoprotein (HDL) and serum triglyceride (TG) were estimated according to the method of Young [35] using Spinreact kit (Spain) according to the instruction of the supplier. The value of serum low density lipoprotein (LDL) and serum very low density lipoproteins (VLDL) was calculated according to the equation of Srivastava [36] as follows:
i-
LDL = TC – (HDL + TG\5).
ii-
VLDL = TC-(LDL+ HDL).
Antioxidants and lipid peroxide
Antioxidant enzymes (catalase, glutathione-S-transferase), and lipid peroxide were assayed in the serum and liver tissue homogenate colorimetrically using Biodiagnostic kit (Egypt), according to the instruction of the manufacturer. The calculations of catalase activity, glutathione-S-transferase activity and lipid peroxide concentration were estimated by the suitable equation of the kit.
Interleukin-6
The proinflammatory cytokines IL-6 was estimated in the serum and liver tissue homogenate according to the method of Hirano [37] using R&D Systems Inc (United States) kits according to the instruction of manufacturer.
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Histopathological investigations
A piece of liver was fixed in 10 % formalin, dehydrated in gradual ethanol (50–99 %), cleared in xylene, and embedded in paraffin. Sections were prepared and then stained with hematoxylin and eosin dye for microscopic investigation [38].
Statistical analysis
Data were analyzed using SPSS program. T-test and the mean ± SD were calculated, and then the data were analyzed using one way analysis of variance (ANOVA, p < 0.05) using the protected least significant difference (LSD) test using SAS software.
Results
Food intake
Table 1 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on food intake. The mean values of food intake (FI) were not significantly changed in the 1st, 2nd and 4th week as a result of CCl4 induced liver hepatotoxicity. Whereas in the 3rd week, the mean values of FI in the olive oil treated group (G3) and the N. sativa treated group (G4) were lower than that of the negative control. The differences were significant at 1 % (P < 0.05).
Table 1
Effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on food intake
Food Intake g/day
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
N. sativa oil
1st week
Mean ± SE
14.50 ± 0.22 a
14.50 ± 0.223 a
14.66 ± 0.21 a
14.50 ± 0.23 a
LSD 0.05 = 0.719
T-test
-
0.00NS
−0.41NS
0.01NS
2nd week
Mean ± SE
16.16 ± 0.16 a
15.83 ± 0.16 a
15.50 ± 0.22 a
15.50 ± 0.22 a
LSD 0.05 = 0.730
T-test
-
1.58 NS
1.58 NS
1.58 NS
3rd week
Mean ± SE
19.16 ± 0.54 a
18.16 ± 0.40 ab
17.00 ± 0.44b
17.16 ± 0.40 b
LSD 0.05 = 1.128
T-test
-
3.87**
2.90*
2.23*
4th week
Mean ± SE
20.01 ± 0.00 a
20.00 ± 0.00 a
20.02 ± 0.00 a
20.03 ± 0.00 a
LSD 0.05 = 0.251
T-test
-
0.00 NS
0.02NS
0.01NS
Data are represented as mean ± SE. T-test values *: significant at P < 0.05,**: significant at P < 0.01. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P> 0.05. LSD least significant difference, N.S non significant
Water consumption
Table 2 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on the weekly water consumption. In the positive control group, water consumption in the 1st and 2nd weeks was similar to that of the negative control, whereas in the 3rd and 4th weeks it was significantly higher than that of the negative control. In G3 and G4, water consumption in 1st and 2nd weeks was significantly (in the 1st week for G3 and G4 and the 2nd week for G4) or non significantly (in G3 in the second week) higher than that of the positive control group. While, in the 3rd and 4th weeks the mean values of water consumption was highly significant (in the 3rd week for G3 and G4) or non significant (in the 4th week) compared with the positive control group.
Table 2
Effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on the weekly water consumption
Water consumed ml/day
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
N. sativa oil
1st week
Mean ± SE
33.33 ± 1.05 ab
32.50 ± 1.11 b
36.33 ± 0.88 a
36.33 ± 0.88 a
LSD 0.05 = 3.257
T-test
-
0.41 NS
−4.60***
−2.49*
2nd week
Mean ± SE
33.66 ± 0.88 b
32.50 ± 1.11 b
34.83 ± 0.90 b
37.16 ± 0.79 a
LSD 0.05 = 2.308
T-test
-
1.40 NS
−1.68 NS
−3.97**
3rd week
Mean ± SE
27.50 ± 1.11 b
38.00 ± 0.96 a
28.00 ± 1.00 b
29.16 ± 1.53 b
LSD 0.05 = 3.766
T-test
-
−11.38***
5.59***
6.30***
4th week
Mean ± SE
27.50 ± 1.11a
32.00 ± 1.00 ab
30.00 ± 1.29 b
29.00 ± 2.08 b
LSD 0.05 = 3.235
T-test
-
−2.18*
1.22 NS
1.04 NS
Data are represented as mean ± SE. T-test values *: significant at P < 0.05,**: significant at P < 0.01, ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P> 0.05. LSD least significant difference, N.S non significant
Total body weight
Results in Table 3 show the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on total body weight. In G2, (the rats with CCl4 induced hepatotoxicity, the positive control) the total body weight was significantly (p < 0.001) higher than that of the negative control in all weeks. In G3 the total body week in the first week was significantly (p < 0.001) higher than that of the positive control, whereas in the other weeks the increase in total body weight was non significant. In G4, the total body weight in the 1st and 4th weeks was significantly (p < 0.05) higher than that of the positive control, whereas in the 2nd and 3rd weeks the increase in total body weight was non significant compared with that of the positive control group.
Table 3
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on total body weight
Total body weight (g)
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
N. sativa oil
1st week
Mean ± SE
178.66 ± 1.08 d
201.33 ± 0.88 a
190.16 ± 2.10 c
197.50 ± 1.11 b
LSD 0.05 = 3.504
T-test
-
−19.79***
4.85***
2.49*
2nd week
Mean ± SE
181.66 ± 1.30 c
198.50 ± 0.95 ab
196.00 ± 3.10 b
200.83 ± 1.44 a
LSD 0.05 = 4.444
T-test
-
−11.82***
0.88 NS
−1.71 NS
3rd week
Mean ± SE
186.00 ± 1.29 c
201.16 ± 0.74 ab
199.50 ± 3.33 b
204.16 ± 1.74 a
LSD 0.05 = 4.875
T-test
-
−10.48***
0.56 NS
−1.56 NS
4th week
Mean ± SE
189.00 ± 1.50 c
203.83 ± 0.54 b
202.66 ± 3.06 b
208.50 ± 1.66 a
LSD 0.05 = 4.450
T-test
-
−8.34***
0.39 NS
−2.49*
Data are represented as mean ± SE. T-test values *: significant at P < 0.05, ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P> 0.05. LSD least significant difference, N.S non significant
Weight of organs
Table 4 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on organ weight. There were no significant difference between the weight of heart, liver, right kidney, left kidney, right testis and left testis in the positive control group and the negative control group.
Table 4
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on organs weight
Organs weight g
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
Nigella sativa oil
Heart
Mean ± SE
0.433 ± 0.091 b
0.450 ± 0.018 b
0.650 ± 0.022 a
0.566 ± 0.033 ab
LSD 0.05 = 0.164
T-test
-
−0.18 NS
−6.92***
−5.53***
Liver
Mean ± SE
4.100 ± 0.821ab
3.550 ± 0.172 b
4.600 ± 0.177 ab
5.133 ± 0.049 a
LSD 0.05 = 1.277
T-test
-
0.73 NS
−3.50**
−9.89***
Right kidney
Mean ± SE
0.483 ± 0.101 b
0.633 ± 0.021 ab
0.683 ± 0.040 a
0.600 ± 0.000 ab
LSD 0.05 = 0.160
T-test
-
−1.62 NS
−0.88 NS
1.58 NS
Left kidney
Mean ± SE
0.516 ± 0.104 b
0.683 ± 0.016 ab
0.700 ± 0.025 a
0.633 ± 0.021 ab
LSD 0.05 = 0.169
T-test
-
−1.53 NS
−0.54 NS
1.46 NS
Right testis
Mean ± SE
0.933 ± 0.194 a
0.965 ± 0.020 a
1.166 ± 0.033 a
1.166 ± 0.033 a
LSD 0.05 = 0.317
T-test
-
−0.15 NS
−5.38***
−7.79***
Left testis
Mean ± SE
0.966 ± 0.201 a
0.956 ± 0.019 a
1.2000 ± 0.025 a
1.200 ± 0.025 a
LSD 0.05 = 0.316
T-test
-
0.04 NS
−7.30***
−7.52***
Data are represented as mean ± SE. T-test values **: significant at P < 0.01, ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference, N.S non significant
Treating the hepatotoxic rats with olive oil and N. sativa oil in G3 and G4, respectively significantly (P < 0.01) increased the weight of heart, liver, right testis and left testis when compared with the positive control group, whereas the weight of right and left kidneys were non significantly changed.
Physiological evaluations (body weight gain, body weight ratio and food efficiency ratio)
Table 5 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on physiological evaluations (body weight, BWG %, BWG% and FER). BWG, BWG%, FER and FER% in G2 were very high significantly decreased as a result of liver damage compared with the negative control group (G1). Treating the hepatotoxicity in G3 and G4 with olive oil and N. sativa oil, respectively significantly (P < 0.001) increased these parameters compared with the positive control group.
Table 5
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on body weight, BWG %, BWG% and FER
Biological evaluation
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
Nigella sativa oil
BWG g /4 week
Mean ± SE
10.333 ± 1.282 a
2.500 ± 1.231 d
12.500 ± 1.765 a
11.000 ± 0.730 a
LSD 0.05 = 3.678
T.test
-
4.832***
−5.175***
−7.059***
BWG %
Mean ± SE
5.788 ± 0.721 a
1.254 ± 0.612 b
6.565 ± 0.914 a
5.564 ± 0.349 a
LSD 0.05 = 1.962
T.test
-
5.285***
−5.332***
−7.323***
FER g/day
Mean ± SE
0.020 ± 0.002 a
0.005 ± 0.002 b
0.026 ± 0.003 a
0.023 ± 0.001 a
LSD 0.05 = 0.012
T.test
-
4.832***
−5.367***
−7.416***
FER %
Mean ± SE
2.087 ± 0.259 a
0.518 ± 0.255 b
2.660 ± 0.375 a
2.341 ± 0.155 a
LSD 0.05 = 0.839
T.test
-
4.734***
−5.251***
−7.274***
Data are represented as mean ± SE. T-test values ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P> 0.05. LSD least significant difference
Liver enzymes, total protein, albumin and total bilirubin
Table 6 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on liver enzymes, total protein, albumin and total bilirubin. CCl4 induced hepatotoxicity in rats of the positive control significantly (P < 0.001) increased the mean values of ALT, AST and ALP, whereas decreased the mean values of total protein, albumin and total bilirubin compared with that of the negative control group. Treating the CCl4 induced hepatotoxicity in rats with olive oil and N. sativa oil in G3 and G4, respectively significantly (P < 0.001) decreased the mean values of liver enzymes (ALT, AST and ALP) and increased total protein, albumin and total bilirubin compared with that of the negative control. Treating the CCl4 induced hepatotoxicity in rats with N. sativa in G4 was more efficient than treating with olive oil in G3.
Table 6
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on liver enzymes, total proteins, albumin and total bilirubin
Liver enzymes U/l
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
Nigella sativa oil
AIT
Mean ± SE
25.16 ± 1.35d
71.00 ± 1.41a
58.83 ± 0.47b
52.16 ± 1.13c
LSD0.05 = 3.275
T-test
-
−21.00***
7.15***
39.46***
AST
Mean ± SE
26.50 ± 0.76d
81.16 ± 1.66a
65.50 ± 0.99b
50.50 ± 1.11c
LSD0.05 = 2.501
T-test
-
−44.47***
10.95***
23.91***
ALP
Mean ± SE
156.66 ± 2.17d
286.16 ± 2.82a
228.50 ± 2.50b
185.50 ± 2.43c
LSD 0.05 = 8.122
T-test
-
−37.51***
14.90***
19.55***
Total protein
Mean ± SE
7.65 ± 0.04a
4.81 ± 0.09d
5.66 ± 0.04c
6.80 ± 0.05b
LSD 0.05 = 0.201
T-test
-
22.55***
−10.04***
−16.22***
Albumin
Mean ± SE
4.50 ± 0.05a
2.56 ± 0.09d
3.10 ± 0.04c
3.85 ± 0.04b
LSD 0.05 = 0.203
T-test
-
15.72***
−4.78***
−16.19***
Total Bilirubin mg/dl
Mean ± SE
0.42 ± 0.00d
1.38 ± 0.03a
0.84 ± 0.00b
0.76 ± 0.04c
LSD 0.05 = 0.077
T-test
-
−28.02***
15.58***
15.11***
Data are represented as mean ± SE. T-test values ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference
Renal function
Table 7 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on urea, creatinine and uric acid. CCl4 induced hepatotoxicity in rats of the positive control significantly at (P < 0.001) increased the mean values of urea, creatinine and uric acid compared with that of the negative control. Treating the CCl4 induced hepatotoxicity in rats with olive oil and N. sativa oil in G3 and G4, respectively significantly (P < 0.001) decreased the mean values of urea, creatinine and uric acid, when compared to that of the negative control. Treating the CCl4 induced hepatotoxicity in rats with N. sativa in G4 was more efficient than treating with olive oil in G3.
Table 7
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks renal functions
Parameters mg/dl
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
Nigella sativa oil
Urea
Mean ± SE
24.00 ± 0.57 d
55.50 ± 1.25 a
43.83 ± 1.13b
32.66 ± 1.33 c
LSD 0.05 = 3.250
T-test
-
−21.65***
8.43***
16.30***
Creatinine
Mean ± SE
0.53 ± 0.02 d
1.70 ± 0.05 a
1.15 ± 0.04 b
0.79 ± 0.01 c
LSD 0.05 = 0.117
T-test
-
−16.34***
11.00***
15.72***
Uric acid
Mean ± SE
4.18 ± 0.07 d
6.61 ± 0.09 a
6.01 ± 0.08 b
5.10 ± 0.07 c
LSD 0.05 = 0.278
T-test
-
−15.56***
8.21***
10.48***
Data are represented as mean ± SE. T-test values ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference
Lipid profile
Table 8 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on serum lipid profile. The mean values of TC, TG, LDL-C and VLDL-C in the positive control were significantly (P < 0.001) higher than that of the negative control. In contrast, the mean values of HDL in the positive control were significantly (P < 0.001) lower than that of the negative control (30.00 ± 0.57 and 48.33 ± 0.42 mg/dl, respectively).
Table 8
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on serum lipid profile levels
C
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
Nigella sativa oil
TG mg/dl
Mean ± SE
115.00 ± 4.64d
244.83 ± 4.77a
218.33 ± 15.12b
181.33 ± 1.97c
LSD 0.05 = 26.170
T-test
-
−15.21***
1.50NS
9.80***
TC mg %
Mean ± SE
161.00 ± 3.06d
278.00±3.63a
227.00±2.06b
189.00±3.26c
LSD 0.05 = 8.802
T-test
-
-22.16***
15.23***
16.90***
HDL mg/dl
Mean±SE
48.33±0.42a
30.00±0.57d
40.83±0.47b
36.50±0.42c
LSD 0.05=1.554
T-test
-
24.11***
−18.02***
−7.67***
LDL mg/dl
Mean ± SE
89.33 ± 3.33d
198.50 ± 4.13a
145.50 ± 2.12b
115.83 ± 3.41c
LSD 0.05 = 9.502
T-test
-
−17.86***
15.38***
14.68***
VLDL mg/dl
Mean ± SE
23.73 ± 1.56c
48.96 ± 0.95a
43.66 ± 3.02c
36.26 ± 0.39b
LSD 0.05 = 5.595
T-test
-
−11.67***
1.50 ***
9.80***
Data are represented as mean ± SE. T-test values *: significant at P < 0.05,**: significant at P < 0.01, ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference, N.S. non significant
Treating the CCl4 induced hepatotoxicity in male rats with olive oil in G3 very high significantly (P < 0.001) decreased the mean values of TC, TG, LDL-C and VLDL-C compared with that of the positive control. In addition, the mean values of HDL in G3 were very high significantly (P < 0.001) higher than that of the positive control.
Similarly, treating the CCl4 induced hepatotoxicity in male rats with N. sativa oil very high significantly (P < 0.001) decreased the mean values of TC, TG, LDL-C and VLDL-C compared with the negative control values. Moreover, the mean values of HDL in G4 were significantly (P < 0.001) higher than that of the positive control. It is worthy to mention that N. sativa succeeded in lowering TC, TG, LDL-C and VLDL-C in G4 than olive oil in G3, whereas olive oil succeeded in increasing the levels of HDL than N. sativa.
Antioxidant enzymes and lipid peroxide
Table 9 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on serum and liver tissue homogenate antioxidant enzymes and lipid peroxide. CCl4 induced hepatotoxicity in male rats of the positive control group (G2) significantly (P < 0.001) lowered the mean values of catalase (CA), superoxide dismutase (SOD) and glutathione reductase (GSST) and increased lipid peroxide in serum and liver tissue homogenate as a result of liver damage compared with the negative control group.
Table 9
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on CAT, SOD and GSST in serum and liver tissue homogenate
Parameters
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
N. sativa oil
Serum Catalase (S.CAT) U/I
Mean ± SE
2.77 ± 0.01a
0.13 ± 0.00 c0
1.34 ± 0.05d
2.08 ± 0.06b
LSD 0.05 = 0.438
T-test
-
189.86***
−25.73***
−35.40***
Serum Superoxide dismutase (S.SOD) U/ml
Mean ± SE
626.11 ± 3.88a
215.31 ± 3.15d
427.01 ± 2.99c
544.50 ± 3.87b
LSD 0.05 = 9.935
T-test
-
91.91***
−58.51***
−66.38***
Serum Glutathione reductase (S.GSST) U/ml
Mean ± SE
717.81 ± 2.87a
246.68 ± 2.96d
518.28 ± 2.60c
631.68 ± 3.19b
LSD 0.05 = 9.663
T-test
-
99.95***
−65.26***
−88.89***
CAT U/g. Liver tissue
Mean ± SE
LSD 0.05 = 0.230
5.53 ± 0.13a
.18 ± 0.01 d0
2.38 ± 0.03c
4.58 ± 0.09b
T-test
-
42.97***
−64.97***
−44.43***
SOD U/g. Liver tissue
Mean ± SE
815.06 ± 3.27a
213.81 ± 2.40d
628.48 ± 2.09c
745.80 ± 2.89b
LSD 0.05 = 8.575
T-test
-
166.80***
−103.43***
−122.00***
GSST U/g. Liver tissue
Mean ± SE
734.76 ± 84.41a
373.33 ± 58.54d
669.95 ± 14.58c
739.01 ± 3.77b
LSD 0.05 = 10.833
T-test
-
2.53***
−6.69***
−6.46***
MDA nmol/ml
Mean ± SE
0.39 ± 0.01d
2.85 ± 0.01a
1.50 ± 0.03b
0.88 ± 0.02c
LSD 0.05 = 0.0817
T-test
-
−90.44***
40.04***
49.20***
MDA nmol/ g. Liver tissue
Mean ± SE
2.59 ± 0.12d
19.78 ± 0.37a
10.55 ± 0.37c
14.71 ± 0.17b
LSD 0.05 = 0.977
T-test
-
−45.43***
13.65***
10.84***
Data are represented as mean ± SE. T-test values ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference
Treating the CCl4 induced hepatotoxicity in male rats in G3 and G4 with olive oil and N. sativa oil, respectively significantly (P < 0.001) increased the catalase, superoxide dismutase and glutathione reductase and decreased lipid peroxide in the serum and kidney tissue homogenate compared with that of the positive control group. In addition, N. sativa was more efficient than olive oil in ameliorating the antioxidant enzymes under study in G4 and G3, respectively.
Interleukin-6
Data in Table 10 shows the effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on serum and tissue interleukin-6 (S.IL-6 and TIL6, respectively). The mean values of interleukin-6 (S.IL-6) of both serum and liver tissue homogenate in the positive control group were significantly (P < 0.001) higher than that of the negative control group which was in serum 21.96 ± 0.83 and 5.70 ± 0.21U/g, respectively and in tissue were 85.45 ± 3.11 and 43.05 ± 1.34 pg/ml, respectively. Treating the CCl4 induced hepatotoxicity in G3 rats with olive oil significantly (P < 0.001) lowered the mean values of S.IL-6 and T.IL-6 than that of the positive controls. Also, treating CCl4 induced hepatotoxicity in G4 with N. sativa oil significantly (P < 0.001) decreased the mean values of S.IL-6 and T.IL-6 compared with that of the positive control. N. sativa oil was more efficient than olive oil in ameliorating the levels of IL-6 in G4 and G3, respectively in both serum and liver tissue homogenate.
Table 10
The effect of treating CCl4 induced hepatotoxicity in male rats with olive oil and N. sativa oil for 4 weeks on serum and tissue tinterleukin-6 (S.IL-6 and TIL6)
Parameters
Statistics
G1
G2
G3
G4
N. Control
P. Control
Olive oil
N. sativa oil
Interleukin-6 (S.IL-6) pg/ml
Mean ± SE
5.70 ± 0.21d0
21.96 ± 0.83a
16.78 ± 0.44b
10.48 ± 0.57c
LSD 0.05 = 1.691
T-test
-
−18.38***
5.39***
10.98***
T.IL6 pg/g.tissue
Mean ± SE
43.05 ± 1.34d
85.45 ± 3.11a
64.80 ± 1.07b
56.10 ± 0.76c
LSD 0.05 = 5.463
T-test
-
−11.36***
6.08***
1.20***
Data are represented as mean ± SE. ***: significant at P < 0.001. ANOVA analysis: within each row, means with different superscript (a, b, c or d) are significantly different at P < 0.05, whereas means superscripts with the same letters mean that there is no significant difference at P > 0.05. LSD least significant difference
Histopathological investigation of the liver
Microscopically, liver of rats from G1 (negative control) showed normal architecture with portal tracts composed of normal bile duct, portal vein and hepatic artery. In addition, it showed normal kupffer cells (Fig. 1a). Whereas, photomicrography of the positive control representing CCl4 induced hepatotoxicity showed degeneration changes in hepatocytes with mild to moderate inflammation and congestion (Fig. 1b). In addition, disrupted hepatocytes and inflammatory cellular infiltration were also detected around the blood vessels as a result of induced hepatotoxicity. In Fig. 1c liver tissue of olive oil treated group showed improvement of the degeneration effect with minimal inflammatory infiltration. Figure 1d of N. sativa treated group showed nearly normal hepatocytes with no evidence of inflammation. This study indicates that olive oils and Nigella sativa has a protective effect against CCl4-induced impaired liver damage in male rats. N. sativa oil was more efficient than olive oil in ameliorating the liver tissues in G4 and G3, respectively.
Fig. 1
a Liver of the negative control (G1) showing no histopathological alteration with normal hepatocytes (long arrow), b liver of rat from the positive control group showing congested hepatocytes (short arrow) (G2), c liver of rat from (G3) group treated with olive oil showing nearly normal hepatocytes (long arrow), d liver of rat from (G4) group Nigella sativa showing normal hepatocytes (long arrow) (h & e, ×200)
×
Discussion
In Saudi Arabia, the prevalence of liver diseases is relatively rising and the mortality and morbidity rates are significant [39, 40]. The present study was focused at studying the protective effect of olive oil and Nigella sativa oil to CCl4 induced hepatotoxicity in male rats. Liver injury can be induced directly from hepatic toxicity or indirectly from immune mediation by biological factors (e.g. hepatitis virus, bacteria, and parasite), environmental factors and chemical factors (e.g. medicine, industrial poisons and alcohol) [41].
In the current study, the positive control group rats given CCl4 showed a decrease in food intake. This result agrees with that of Wu et al. [42] and Tanaka et al. [43] who reported decreased food intake due to toxicity with CCl4. Moreover, decrease in food intake was detected after administration of olive oil and Nigella sativa to rats received CCl4. An increase in the total body weight in rats of the positive control group with induced CCl4 as compared with the negative control group was also encountered. Body weight loss is a distinctive feature of CCl4-induced hepatotoxicity [44]. One of the largest organ is liver that CCl4 administrated caused a rapid accumulation of triglycerides in the liver due to a block secretion of very low density lipoprotein by hepatocytes [45, 46]. Furthermore, increase in the total body weight was detected after administration of olive oil and N. Sativa oil to rats received CCl4.
In the current study, CCl4 induced liver damage in rats and consequently decreased BWG % which also accompanied with decreased FER compared with negative control group. The obtained results were in agreement with Fang et al. [17] and Khan et al. [47] who reported that CCl4 induced liver damage groups in rats showed significant reduction in body weight compared with rats non-injected with CCl4. Furthermore, increase in the body weight gain was detected after administration of olive oil. This result is consistent with that of Tufarelli et al. [48]. Furthermore, N. Sativa oil treated group (G4) showed very highly significant differences in BWG compared to the positive control group. This result is consistent with the study of Zaoui et al. [49]. Moreover, El-Sayed [50] discovered that the reduction seen in the body weight gain for 5 days after treatment with CCl4 that was alleviated by either N. sativa or thymoquione (one of the its major constituents) treatment on comparison with CCl4-treated animals. Our results showed that, no significant differences in heart, liver, kidneys and testes weight was observed after injection of CCl4 in rats for 28 daysays compared to the negative control. This agrees with the result of Kovalovich et al. [51].
The CCl4 injured liver functions showed significant increase in liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) compared with the negative control were observed after administration with CCl4 in the positive control as a result of hepatic cell damage. CCl4 induced hepatotoxicity and increased the aminotransferase and ALP activities and similar observation was found in group administrated with CCl4 and caused significant increase in ALT and AST enzymes in Wistar rats [52, 53]. Alanine aminotransferase is considered a highly sensitive and specific biomarker of hepatotoxicity. Elevation of ALP, a cell membrane enzyme is a primary marker of hepatobiliary effects and cholestasis [54]. Also, elevation in liver enzymes reflected liver cell damage and could be attributed to tissue breakdown, permitting the escape of intracellular enzymes from cytosol into the blood [52].
Treating the hepatotoxic rats in G3 and G4 with olive oil or N. Sativa, respectively reversed the activity of transaminases and restored them towards normal values indicating maintenance of functional integrity of hepatic cell membrane, however, they need a higher dose of Nigella sativa oil and olive oil to be restored to the normal levels. This agrees with our study which revealed that level of enzymes in CCl4 + olive oil group (G3) and CCl4 +N. Sativa oil group (G4) is lower than the CCl4 group (G2). These results agree with that of Krishnan and Muthukrishnan [55] who reported that AST, ALT and ALP elevated enzymatic levels were significantly returned toward normal levels by the 10 % aqueous extract of N. sativa.
On the other hand, renal function parameters in the present investigation showed a significant elevation in the level of uric acid (UA), urea and creatinine (CRE) when compared to that of the negative control. This indicates that the kidney was affected by CCl4 toxicity. UA and urea are the final product of nucleic acid or protein catabolism, respectively. The increased protein catabolism together with enhanced amino acid deamination for gluconeogenesis is possibly an acceptable postulate to understand the raised levels of urea. The elevated UA may be due to degradation of purines or to a rise of UA levels by either overproduction or inability of excretion. Moreover, 50 % of kidney function must be lost before an elevation in the serum CRE [56]. The current study shows that in spite of the ameliorative effect of both Nigella sativa oil and olive oil on kidney function parameters approaching the normal levels, they need a higher dose of Nigella sativa oil and olive oil to be restored to the normal levels.
The current findings indicated that there were a correlation between liver damage and kidney disease in this animal model that could be considered a novel study. On the other hand, the current results are consistent with other studies demonstrated a relationship between kidney disease and CCl4 liver toxicity [57‐59]. Treating the damaged liver rats with olive oil and N. sativa oil protected the liver and improved the kidney function. Olive oil has been shown to reduce the kidney induced toxicity by a different nephrotoxin that resulted in reduced urea, CRE and UA levels [25, 60, 61]. These previous studies are consistent with our present study that showed reduced urea, CRE and UA levels in olive oil and N. Sativa oil group when compared with the positive control group in rats with CCl4 induced liver and injury and the administration of in olive oil and N. Sativa could recover the injury.
In the present study, serum concentration of total protein and albumin decreased after the injection of CCl4 in rats of the positive control group due to hepatotoxicity. The present result is in agreement with previous studies [52, 62]. CC14 toxicity produced a significant decrease in plasma level of total protein and albumin. This may be as a result of releasing total protein and albumin from the cytoplasm into the blood quickly after cellular destruction and a reduction in forming hepatic protein [62]. Moreover, our results showed an increase in total protein and albumin after administration of olive oil and N. sativa oil to rats received CCl4. This result is also consistent with Al-Malki and El Rabey [25], Salem et al. [61] and Jin et al. [63].
In the current study, the CCl4 induced liver damage rats showed significant increase in total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and very low density lipoprotein (VLDL). In contrast, high density lipoprotein (HDL) was decreased compared with the negative control. This result is consistent with that of Hosseinzadeh et al. [64]. Treating these damaged-liver rats with olive oil and N. sativa oil significantly ameliorated the lipid profile parameters. The HDL was restored to the normal levels, whereas the other parameters need a higher dose of Nigella sativa oil and olive oil to be restored to the normal levels. This result is consistent with other studies [65, 66]. Oxidative stress due to CCl4 injection caused an increase in free fatty acid distribution to the liver and elevated hepatic TG accumulation and diet rich with olive oil and N. Sativa reduced the accumulation of TG in the liver [67].
In the current study, catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GSST) were decreased in the serum and liver tissue homogenate of G2 as a result of CCl4 injection compared with negative control group, whereas lipid peroxide (MDA) in serum and tissue levels were increased. In spite of restoring the antioxidant enzymes to the normal levels, lipid peroxide needs a higher dose of Nigella sativa oil and olive oil to restore it to the normal levels. This result is consistent with that of Fang et al. [17]. Similar to the other above mentioned parameters, treating the CCl4 induced hepatotoxicity with olive oil or N. Sativa oil in G3 and G4, respectively significantly increased CAT, SOD and GSST and reduced MDA compared to the positive control group. This result is consistent with Krishnan and Muthukrishnan [55] and İlhan and Seçkin [67].
In the present study, CCl4-induced liver toxicity in male rats showed significant increase in both serum interleukin-6 (S.IL-6) and tissue interleukin-6 (T.IL-6) which is a proinflammatory cytokines compared with that of the negative control as a result of the inflammation occurred in the liver due to toxicity. This result is consistent with the increase in liver enzyme in the blood stream as a result of liver cells damage. Moreover, a decrease in the S.IL-6 and T.IL-6 levels was detected after administration of olive oil and N. Sativa to rats received CCl4. This agrees with other previous results [22, 23]. Several studies revealed the benefits of medical plants like olive oil or Nigella sativa (N. Sativa) oil on mice, rat and a rabbit model which showed anti-steatotic, anti-inflammatory and antioxidant effect and also delay in the development of liver disease [68‐70].
Liver tissues showed many pathological changes as a result of CCl4 hepatotoxicity which is consistent with previous investigations [5, 8, 9]. Treating the CCl4 induced hepatotoxicity with olive oil or N. Sativa oil in G3 and G4, respectively significantly improved the liver tissues and nearly restored them to the normal. This result is consistent with other studies showed hepatoprotective role for both olive oil and N. sativa oil against pathological changes due to their higher content of antioxidant substance such as flavonoids and phenolic compounds [16, 17, 71]. To get full protection, the dose of Nigella sativa oil and olive oil may be increased to 1.5 ml /Kg body weight.
Conclusion
This work showed that CCl4 causes liver hepatotoxicity as revealed by the elevation of liver function parameters and the adverse histopathogical changes in the liver tissues of rats of the positive control group. Treating the hepatotoxic rats with olive oil and N. sativa oil protected the liver structure against CCl4 toxicity in the third and the fourth groups, respectively. This hepatoprotective activity may be attributed to the biologically active compounds exist in both olive oil and Nigella sativa which work to scavenge free radicals. So, the current study recommends that the doses of olive oil and N. sativa oil should exceed the doses used in this study. The proposed dose is 1.5 ml/Kg body weight, in order to protect livers from the ecological hazards of CCl4 toxicity. It is also worthy to mention that olive oil is used here as the positive treatment due to its protective action to the liver. In addition, N. sativa is more efficient than olive oil in protecting the liver against CCl4 toxicity.
Acknowledgements
The authors, therefore, acknowledge with thanks DSR technical and financial support.
Funding
The project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (416-35-أط).
Availability of data and materials
Data are all contained within the paper.
Authors’ contributions
MNE, HAE, MAZ and AMA designed and carried out the experimental work. MNE, HAE, MAZ and AMA analyzed the statistical data and interpretation of results. MNE, HAE and MAZ drafted and critically evaluated the manuscript. All authors read and approved the final manuscript.
Authors’ information
MNA is an associate professor of Biochemistry at Biochemistry Department, King Abdulaziz University, Saudi Arabia. HAE is a Professor of Molecular Biology at GEBR, Sadat City University, Egypt. He was A Professor of Molecular Biology at Biochemistry Department, King Abdulaziz University, Saudi Arabia during this research. MAZ is an assistant professor of Biochemistry at Biochemistry Department, King Abdulaziz University, Saudi Arabia. AMA was MSc student at Biochemistry Department, King Abdulaziz University, Saudi Arabia.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not relevant.
Ethics approval
The plan of our study specifically was approved by our institutional ethics committee at King Abdulaziz University (KAU-1435).
Financial disclosure
None.
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