Nigella sativa seeds, commonly known as black seed or black cumin, belongs to the family Ranunculaceae, has been traditionally used in Arabian countries (Ali and Blunden, 2003) and Europe (Lautenbacher, 1997) for culinary and medicinal purposes as a natural remedy for a number of diseases and conditions.
There is a common Islamic belief that the black seed is a remedy for all aliments except aging or death.
The seeds are believed to have a carminative, stimulatory and diaphoretic property and are used in the treatment of allergic diseases (Kalus et al., 2003).
N. sativa seeds contain fixed oils, proteins, alkaloids, saponin and essential oils. Most of N. sativa activities have been attributed to volatile oils mainly thymoquinone and dithymoquione (Lautenbacher, 1997).
Thymoquinone was found to protect against biochemical and histological markers of liver damage (AL Gharably et al., 1997). El-Dakhakhny et al. (2000) found that pretreatment of rats with N. sativa for 4 weeks was effective in protecting against CCl4 and D-galactosamine induced hepatic damage.
Today in the ethanomedicinal practice there is search for treatment that stems from natural substances for curing liver disorders to replace the currently used drugs.
The present study aimed to evaluate the hepatoprotective role of N. sativa methanolic extract against a potent hepatotoxin, CCl4, induced liver damage in Wister albino rats.
MATERIALS AND METHODS
Preparation of Nigella sativa Methanolic Extract
Nigella sativa seeds were purchased from local market at Omdurman,
Sudan and purified. The seeds were dried and extracted according to Harborne
method (1976). Seventy gram of granulated seeds were inserted in soxhlet apparatus
(Quick Fit, Ex 5183). The oil have been separated using 100 mL of petroleum
ether (40-60%) overnight. The sample was unpacked and left to dry, then repacked
again with adding methanol (99.9%) as a solvent to get the polar constituents
of the plant. The extract was evaporated till dryness using a rotavapor.
Animals and Treatment
Twenty five healthy adult Wister albino rats of both sex were used in the
study in 2005. They were maintained in cages within the Department of Biochemistry,
Faculty of Veterinary Medicine, University of Khartoum. Water and feed were
provided ad libitum. They were kept for 7 days, as an adaptation period.
The rats were divided randomly into 5 groups, 5 rats each. They were treated daily for 10 days. Group A received liquid paraffin at dose rate of 0.2 mL kg-1 b.wt. intaperitoneally (i/p). Group B received dimethylsulfoxide I/p at a dose rate of 0.2 mL kg-1 b.wt. Group C was injected i/p with 0.2 mL kg-1 b.wt. of CCl4 dissolved in liquid paraffin at a concentration of one volume CCl4 to 9th volume liquid paraffin. Groups D and E were injected with CCl4 together with 250 and 500 mg kg-1 b.wt. of the N. sativa methanolic extract dissolved in dimethylsulfoxide per/os, respectively.
Clinical signs and mortality were recorded. Blood samples were collected from the orbital plexus by means of heparinized capillary glass tubes according to the method of Waynforth (1980). The blood was collected either in EDTA tubes for hematological studies, or in centrifuge tubes to separate serum. After 5 min of centrifugation of the blood at 3000 rpm, supernatant sera were immediately separated for biochemical analysis.
The hematological parameters were determined by sysmex automated hematology analyzer KX-21. The biochemical analysis was carried out on blood sera using Roche Diagnostic Hitachi 902 Analyzer. Serum Aspartate Amino Transferase (AST) and Alanine Amino Transferase (ALT) activities were determined according to Reitman and Frankel (1957). Alkaline phosphatase (ALP) was measured according to Chemie (1972). Total bilirubin concentration was determined according to Jendrassik and Grof (1938).
At the end of the experimental period, animals were necropsed. Livers and kidneys were excised immediately and weighed. Then small pieces of organs were fixed in neutral formalin, embedded in paraffin wax, sectioned at 5 μm and stained with hemotoxylin and eosin (Drury and Wallington, 1980).
Data was analyzed using complete randomized design in the experiment. General Linear Model's procedure of SAS (1998) software was used to run the data analysis at 0.05 level probabilities. Body weights and organ body weight ratio were compared using t-test procedure according to Mendenhall (1971).
RESULTS AND DISCUSSION
There were no clinical signs observed in rats of all groups except that two rats which received 250 mg kg-1 b.wt. of N. sativa methanolic extract looked dull after 5 days.
The control groups showed an increase in the body weight gain by 3.2%. However, the treated groups with CCl4, 250 and 500 mg kg-1 b.wt. of N. sativa methanolic extract showed loss in body weight by 10.3, 9.3 and 10.3%, respectively, which was not statistically significant compared to the control. On the other hand no significant changes in the relative liver and kidney weights (Table 1).
There were no significant differences between the control and the treated groups in WBC, RBC, Hb, PCV, MCV and MCHC on day 10 (Table 2).
There was a statistically significant increase in ALT, AST and ALP on day 5 and 10 in the groups received CCl4 and N. sativa methanolic extract compared to control groups A and B. But the groups (D and E) which were given both CCl4 and N. sativa methanolic extract showed significant inhibition (p>0.05) of increased plasma levels compared to the group which given only CCl4 (Table 3). There were no significant differences in the total bilirubin concentration between the treated and control groups. However, the total bilirubin concentration raised from 0.2 on day 5 to 0.7 mg dL-1 on day 10 in the group received CCl4 compared to 0.6 and 0.4 in those treated with 250 and 500 mg kg-1 b.wt. of N. sativa methanolic extract, respectively (Table 3).
||Effects of Nigella sativa methanolic extract on body
weight and relative organ weight of Wister albino rats intoxicated with
|Means (±SD) within the same column having the same
superscript letter(s) are not significantly different at (p>0.05) based
||Effect of Nigella sativa methanolic extract on hematological
values of Wister albino rats intoxicated with CCl4 on day 10
of the experiment
|Means (±SD) within the same column having the same
superscript letter(s) are not significantly different at (p>0.05) level
based on SAS software method
||Effect of Nigella sativa methanolic extract on some
biochemical parameters of Wister albino rats intoxicated with CCl4
on day 5 and 10
|Means (±SD) within the same column having different
small superscript letter(s) are significantly different at (p>0.05) level
based on SAS software method
Histologically the livers of rats received CCl4, showed central vein congestion, centrilobular vacuolation and inflammatory cells infiltration compared to the normal livers of the control groups. These changes were less noticed in the treated groups.
The present study demonstrated a decrease in body weights in rats treated with CCl4 and N. sativa methanolic extract. This is in harmony with the previous findings of Zaoui et al. (2002) who stated a significant slow down of the body weights occurred in animals treated with N. sativa.
Meral and Kanter (2003) showed that N. sativa induced a significant increase for the reduced RBC, WBC, PCV and Hb levels. However, El-Sarha et al. (1997) reported a significant reduction in RBC and PCV in goats which were treated with N. sativa seeds while WBC was significantly increased. In our findings the WBC, RBC, PCV and Hb were unaffected but are in the lower range compared to the control. This indicates that CCl4 has no inhibitory effect in the synthesis of blood parameters as well as N. sativa do not affect the haempoietic system.
Ali and Blunden (2003) studied pharmacological and toxicological properties of Nigella sativa. They found that crude extracts of the seeds caused protection against nephrotoxicity and hepatotoxicity induced by either disease or chemicals. They stated that the seeds were characterized by a very low degree of toxicity.
In the present study, ALT, AST and ALP were significantly increased in rats received CCl4. This increase was improved when using N. sativa extract as evidenced by a decrease of the elevated serum levels of ALT, AST and ALP. These results are in line with that reported by Al Gmadi (2003) and Nevin and Dilara (2005) who stated a marked inhibition of increased enzyme plasma level in animals treated with Nigella sativa compared to those received CCl4. Mansour et al. (2001) stated that thymoquinone at a doe rate of 12.5 mg kg-1 b.wt. given intraperitoneally to mice caused significant reduction of elevated serum enzymes. In contrast, El Dakhakhny et al. (2000) and Zaoui et al. (2002) found that Nigella sativa fixed oil induced no change in hepatic enzymes level and serum bilirubin in treated rats after 4 and 12 weeks, respectively. Daba and Abdel Rahman (1998) tested thymoquinone and slybin in isolated hepatocytes against tetrabutylhydroxide induced liver toxicity and found less degree of protection by thymoquinone.
Histopathological studies provide evidence for the hepatoprotective effect of the plant as hepatic cells have potentiation of regeneration of cells and minimum changes were observed. The biochemical mechanisms involved in the development of CCl4 hepatotoxicity have long been investigated (Lee et al., 2003). It is generally believed that its toxicity is due to lipid peroxidation caused by carbon trichloromethyl radical. Inhibition of lipid peroxidation by Nigella sativa a meliorates CCl4 induced toxicity (Mansour et al., 2001). El Dakhakhny et al. (2000) claimed that hepatoprotective effect of thymoquinone is due to the presentation of intracellular glutathione.
In conclusion, based on the experimental findings it was suggested that administration of Nigella sativa play a role in protection of liver damage caused by CCl4. Thus it may be valuable as a co-therapeutic agent against conditions associated with liver injury. More detailed studies using different doses and covering longer period of observation are needed before reaching a clear cut conclusion about the future of liver damage treatment by Nigella sativa.