Abstract: Cuminum cyminum fruit or Nigella sativa seed, a traditional medicine for treatment of various disorders, was fed to male Wistar rats at 6% of standard rat diet for 4 weeks. A 6% C. cyminum fruit or 6% N. sativa seed diet was not toxic to rats. Depression in growth, hepatotoxicity and nephrotoxicity were observed in rats that had been given paracetamol at 500 mg kg-1 per os for 4 weeks. These findings were accompanied by leucopenia, macrocytic normochromic anemia and alterations of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and concentrations of cholesterol, urea and other serum constituents. Serum bilirubin did not change. In rats given the mixture of paracetamol 500 mg kg-1 plus 6% C. cyminum fruit or 6% N. sativa seed for 4 weeks, the recovery of paracetamol hepatotoxicity was evidenced by increase in body weight, absence of hepatocellular fatty vacuolation and significant improvement of serbiochemical and hematological parameters. There was no evidence of any antinephrotoxic activity of plants used.
INTRODUCTION
Paracetamol (acetaminophen, N-acetyl-p-aminophenol) has been used increasingly as a substitute for other analgesics such as aspirin and phenacetin and its sales have in many countries exceeded those of aspirin. However, much publicity has been given to paracetamol poisoning in which the major target organ is the liver and the primary lesion is centrilobular hepatocellular necrosis (Mitchell, 1977; Prescott, 1983). Other reported complications of paracetamol poisoning include oliguric renal failure (Cobden et al., 1982), pancreatitis and myocarditis (Wakeel et al., 1987).
Severe hepatic necrosis was induced in mice, hamsters, cats and other species by paracetamol but species differences in susceptibility were observed and related to the extent of metabolic activation of the drug (Tee et al., 1987). It has been found that paracetamol is converted by cytochrome P450 dependent mixed function oxidase to a reactive arylating metabolite, known as N-acetyl-p- benzoquinone imine (NAPQI) that may cause hepatic necrosis (Dahlin et al., 1984) and a depletion of both mitochondrial and cytosolic pools of reduced glutathione (Tirmenstein and Nelson, 1989).
Cuminum cyminum L., a member of the family Umbelliferae, is locally known as Kamoon and its fruit is used in Sudan and other countries as antipyretic, antiseptic, antispasmodic, carminative, diuretic and emmenagogue and for the treatment of leprosy, leukoderma and scorpion stings due to the presence of volatile oil, flavonoids, monoterpenes, sesquiterpenes and probably other constituents (Haroun et al., 2002; Ishikawa et al., 2002).
Nigella sativa L. (Ranunculaceae) is also a multipurpose medicinal plant locally known as Black seed or Habat Al-Barakah and is used in traditional medicine as antiarthritic, antihelminthic, antiseptic, antispasmodic, appetizer, emmenagogue and nerve tonic and for the treatment of ascites, asthma and pustular dermatitis due to its content of alkaloid, volatile oil, saponins, sterols and quinones (Al-Yahya, 1986; Daba and Abdel Rahman, 1998). Nigella sativa seed was fed to 7 day old Hibro broiler chicks at 2 and 10% of standard diet for 7 weeks and was not found to adversely affect growth (Al-Homidan et al., 2002).
It is well known that a drug or plant may interact with another drug or plant and as a consequence modification in activity and/or toxicity can be observed. For example, the hepatoprotective effect of Kulthi, Dolichos biflorus on paracetamol induced liver toxicity was described in rats (Laskar et al., 1998). On the other hand, chronic administration of ethanol to mice and hamsters was found to enhance the hepatotoxic effect of paracetamol (Zimmerman, 1986).
Because of the common use of C. cyminum fruit and N.sativa seed in the treatment of various ailments as well as the lack of information on their possible interactions with paracetamol we investigated the effect of paracetamol alone or combined with low levels of dietary C. cyminum fruit or N.sativa seed on the growth, organ pathology, hematological and serobiochemical parameters of Wistar rats.
MATERIALS AND METHODS
Paracetamol and Plant Material
Paracetamol produced by Amipharma Laboratories, Sudan, was purchased in
a local pharmacy in Khartoum and used in this study.
Cuminum cyminum fruits and Nigella sativa seeds were purchased in a local market, ground separately to a fine powder and then mixed into a normal rat diet.
Study Design
Forty two 12 week old male Wistar rats were housed within the premises of
the Medicinal and Aromatic Plants Research Institute, National Center for Research,
Khartoum, under light/dark cycle with feed and drinking water provided ad
libitum.
The rats were randomly allotted to 7 groups of 6 rats each. Rats in group1 were the controls and given untreated diet and water. Groups 2 and 3 received drinking water containing paracetamol at 500 mg L-1 or diet containing paracetamol at 500 mg kg-1, respectively. Groups 4 and 5 received diet containing 6% ( w/w) of C. cyminum fruit or 6% (w/w) N. sativa seed, respectively. Groups 6 and 7 received a diet that contained a mixture of paracetamol at 500 mg kg-1 and 6% (w/w) of C. cyminum fruit or paracetamol at 500 mg kg-1 and 6% (w/w) of N. sativa seed, respectively.
Average body weight and body weight gain were estimated weekly for each group. After 2 weeks of treatment, 3 randomly selected rats from each group were killed under diethyl ether anesthesia. The remaining 3 rats/group were similarly killed after 4 weeks of treatment. Blood samples were collected from each of the killed rats for hematology and serum chemistry.
Blood Analyses
Erythrocytes (RBC), leukocytes (WBC), differential WBC counts, hemoglobin
(Hb) concentration, Packed Cell Volume (PCV), Mean Corpuscular Volume (MCV),
Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration
(MCHC) were evaluated (Schalm et al., 1975). Serum samples were analyzed
for the activities of aspartate aminotransferase (AST), alanine aminotransferase
(ALT) and alkaline phosphatase (ALP) and concentrations of total protein, albumin,
cholesterol, bilirubin and urea by commercial kits (Linear Chemicals, Barcelona,
Spain). Serum globulin was estimated by subtracting albumin from total protein
concentration.
Pathological Examinations
Post-mortem findings were recorded for all rats and specimens of intestines,
liver, spleen, kidneys and heart were fixed in 10% neutral buffered formalin,
embedded in paraffin wax, sectioned at 5 μm and stained with hematoxylin
and eosin (H and E).
Statistical Analysis
The significance of differences between means was compared at each time
point using. Duncan's multiple range tests after ANOVA for one-way classified
data (Snedecor and Cochran, 1989).
RESULTS
Changes in Growth
The rats given paracetamol in drinking water at 500 mg L-1 (group
2) or in the diet at 500 mg kg-1 (group 3) had the lowest (p<0.05)
body weight but none of the rats died during the 4-week period. The rats fed
diets containing mixture of paracetamol at 500 mg kg-1 plus 6% C.
cyminum (group 6) or paracetamol at 500 mg kg-1 plus 6% N.
sativa (group 7) had the highest (p<0.05) body weight.
Pathological Changes
After 2 weeks of treatment, no macroscopic or microscopic changes were observed
in the vital organs of the rats on the 6% C. cyminum (group 4), 6% N.
sativa (group 5), paracetamol at 500 mg kg-1 plus 6% C. cyminum
(group 6) and paracetamol at 500 mg kg-1 plus 6% N. sativa (group
7) or the control rats (group 1). Individual cell necrosis and small fatty vacuoles
in the centrilobular hepatocytes and degeneration of the epithelial cells of
scattered proximal convoluted tubules of the rats on paracetamol in drinking
water at 500 mg L-1 (group 2) or paracetamol at 500 mg kg-1
diet (group 3).
In groups 2 and 3, the main lesions observed at 4 weeks of treatment were necrosis and cytoplasmic fatty vacuolation of the centrilobular hepatocytes (Fig. 1), dilatation or necrosis of the renal proximal convoluted tubules (Fig. 2) and congestion of the cardiac blood vessels. Neither the spleen nor the intestine showed significant changes. No lesions were detected in the liver, heart, spleen, intestines or kidneys of groups 4 and 5. In groups 6 and 7, necrosis of the centrilobular hepatocytes was not seen and the intensity of cytoplasmic fatty vacuolation of the liver cell was reduced in some of the rats. In these groups, the renal lesions persisted and other vital organs showed no abnormality.
Hematological Changes
These data are summarized in Table 1. In the rats given
paracetamol in drinking water at 500 mg L-1 (group 2) or at similar
concentration in the diet (group 3) for 2 weeks, there were decreases in RBC,
PCV and neutrophils and increases in MCV, WBC and lymphocytes.
| Fig. 1: | Fatty vacuolation and necrosis of the centrilobular hepatocytes in a rat given paracetamol at 500 mg kg-1 diet for 4 weeks. H and E X200 |
| Fig. 2: | Dilatation of the proximal convoluted tubules and packing of the glomerular tufts in a rat given paracetamol at 500 mg kg-1 diet for 4 weeks. H and E X100 |
| Table 1: | Hematologic changes in rats given paracetamol alone or combined with C. cyminum fruit or N. sativa seed for 4 weeks |
| \Values are means+SE. Means within rows with no common letter(s) are significantly different (p<0.05) | |
After 4 weeks of treatment, the change in erythrocytic series persisted but WBC and neutrophils decreased and those of MCHC did not change. There were no adverse effects on the blood parameters of the rats fed diets containing 6% C. cyminum fruit (group 4) or 6% N. sativa seed (group 5) for 2 or 4 weeks. Although lymphocytosis was seen, no significant changes were observed in the rats fed the mixture of paracetamol at 500 mg kg-1 and 6% C. cyminum fruit (group 6) or of paracetamol at 500 mg kg-1 and 6% N. sativa seed diets (group 7) for 2 or 4 weeks. The control rats showed no hematologic changes.
| Table 2: | Serobiochemical changes in rats given paracetamol alone or combined with C. cyminum fruit or N. sativa seed for 4week |
| Values are means+SE. Means within rows with no common letter(s) are significantly different (p<0.05) | |
Serobiochemical Changes
These data are presented in Table 2. In groups 2 and 3,
there were increases in the activities of serum AST, ALT and ALP and in concentrations
of cholesterol and urea and decreases in the levels of total protein and albumin
over the 4-week period. Bilirubin concentration did not change. No significant
changes in serum constituents of rats in groups 4 and 5 were observed after
4 weeks of treatment. In groups 6 and 7 there were increases in enzyme activities,
urea and cholesterol concentrations without bilirubinemia during the 4-week
period. No significant serobiochemical changes were observed in the control
(group 1).
DISCUSSION
The 7 day pre-trial period was aimed at achieving body weight uniformity, acclimatizing rats to the Medicinal and Aromatic Plants Research Institute environment and allowing unhealthy ones to be eliminated before the commencement of the experiment.
It is well known that the susceptibility of animals to feeding plant materials is dependent, at least, on the type of the active constituents and concentration in the amount added to the diet as well as on the rate of their metabolic conversion in the liver to metabolites and consequent excretion. For rats, the dietary levels (2 and 10%) represented non-toxic concentrations of plants exemplified by Thymus vulgaris (Haroun et al., 2002) and similarly for chickens by N. sativa (Al-Homidan et al., 2002). On the other hand, levels of 2% or more of dietary Francoeuria crispa, Artemisia abyssinica and Ammi visnaga have been toxic to rodents and chickens (Adam, 1998; Adam et al., 2000; Ibrahim et al., 2004).
In the present study, the differences in mean body weights between the rats on 6% C. cyminum and 6% N. sativa diets as compared to controls became larger with time over the 4 week period. The higher growth rate of C. cyminum and N. sativa-fed rats was most likely to have been due to the absence of adverse effects but may have been associated with a high protein and fat content of the plant materials. However, further work would be required to determine the amino acid and fatty acid profiles of the plant tissues.
The fact that body weights were decreased in rats given paracetamol alone suggests the impairment of growth and decrease in its rate of excretion by the injured liver and kidneys. The elevated activity of serum ALT, AST and ALP and cholesterol level and hypoproteinemia along with liver lesions, indicate paracetamol hepatotoxicity in rats. Nephrotoxicity was a feature of paracetamol toxicity in rats as evidenced by the presence of renal lesions and of an increase in urea concentration in serum. Hepatonephrotoxicity may account for the reduced growth rate in rats given paracetamol singly. Centrilobular hepatocellular necrosis produced by paracetamol in laboratory rodents was described by Wynne et al. (1987).
The damage to the centrilobular hepatocytes without significant bilirubinemia or icterus was observed in rats which had been fed Francoeuria crispa (Adam, 1998) or Artemisia abyssinica (Adam et al., 2000). On the other hand, bilirubinemia or icterus followed by damage to the periportal hepatocytes and proliferation of the bile ducts was detected in the rats on Rhazya stricta diet (Adam, 1999).
The increased MCV without effect on MCHC indicate macrocytic normochromic anemia. Previous studies showed macrocytic anemia in rats fed 10% A. abyssinica (Adam et al., 2000) and chicks fed 10% Cassia italica (Bakhiet and Adam, 1996). These findings suggest the involvement of plant constituents in derangement of the haematopoietic process. Leukopenia notable in the rats given paracetamol singly was due to neutropenia.
The centrilobular hepatocellular necrosis was less marked or unobservable in the rats given the mixture of paracetamol plus C. cyminum fruits or N. sativa seeds for 4 weeks. In some instances, cytoplasmic fatty vacuolation of the centrilobular hepatocytes was depicted in rats given the mixture of paracetamol plus C. cyminum fruits or N. sativa seeds for similar periods. Research would be required to verify the role in the antihepatotoxic activity of C. cyminum fruits or N. sativa seeds.
Although serum urea concentration was decreased, histopathological examination did not provide evidence of an antinephrotoxic effect of the plants. Laskar et al. (1998) described antihepatotoxic activity of the plant, Dolichos biflorus, against paracetamol induced hepatotoxicity in rats and suggested that biotransformation of paracetamol into a nephrotoxic compound, p-aminophenol, is responsible for elevation of blood urea nitrogen.
We conclude that the seeds of N. sativa and the fruits of C. cyminum were not toxic to Wistar rats at the concentration used in the test diets.
The hepatotoxicity of paracetamol in rats was evidenced by consistently lowered body weights, pathological changes and significant serobiochemical and hematological alterations. Nephrotoxicity of paracetamol was described in rats.
Antihepatotoxic activity of N. sativa seed and C. cyminum fruits against paracetamol hepatotoxicity was supported by improvement of body weights, less intense hepatocellular damage and correlation with serobiochemical and hematological changes.
Further investigations into the appropriate isolation, characterization and concentration of the active constituents in C. cyminum fruit and N. sativa seed are deemed necessary for elucidating their antihepatotoxic activity against paracetamol hepatotoxicity in rats.