Effect of Nigella Sativa (Black Cumin), Vitamin E and their Combination on Metoprolol Induced Hepatotoxicity in Rabbits - Open Journal Systems
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Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 1059
Effect of Nigella Sativa (Black Cumin), Vitamin E and their
Combination on Metoprolol Induced Hepatotoxicity in Rabbits
Fatima Hussein1, Jawad Ahmed2
1
Post Graduate Ministry of Health, Basrah Health Directorate, Iraq, 2Assistant Professor, Department of
Pharmacology, College of Medicine, University of Basrah, Iraq
Abstract
Background: Metoprolol (MET) is a selective Beta-1 blocker. It is metabolized in the liver. Unlike most
Betablockers, Metoprolol is unique in causing liver toxicity, which is infrequent, ranging from raised liver
enzymes to severe liver damage. Aim: hepatotoxicity was induced in rabbits, and protective effects of
Nigella sativa (NS), Vitamin E (Vit E), and a combination of Nigella sativa + Vitamin E were investigated
on Metoprolol-induced hepatotoxicity.
Methods: 36 male rabbits were divided into six groups and treated as follow: Metoprolol (20mg/kg),
Nigella sativa (2ml/kg), control group, Metoprolol + Nigella sativa, Metoprolol + Vitamin E ( 175mg/
kg/day), Metoprolol + Nigella sativa + Vitamin E. Treatments were given orally for two weeks. Liver
enzymes, malondialdehyde, glutathione, and interleukins (IL-6, IL-10) were measured in serum and liver
homogenates. Histopathological examinations were done for two animals in each group.
Results: Elevated liver enzymes and histopathological changes were seen in all Metoprolol-treated animals.
Nigella sativa, Vitamin E, and Nigella sativa + Vitamin E reduced Metoprolol-induced rise in liver enzymes.
Metoprolol increased malondialdehyde concentration in serum and liver homogenate. Treatment with
Nigella sativa, Nigella sativa + Vitamin E insignificantly decreased these levels. Neither Metoprolol nor
Metoprolol and Nigella sativa, Vitamin E, and their combination changed the levels of IL-6 or IL-10.
Conclusion: Metoprolol increased liver enzymes, and treatment with Nigella sativa, Vitamin E, and Nigella
sativa + Vitamin E significantly modified rise in liver enzymes. Metoprolol and other treatments had little
effect on IL-6 or IL-10.
Keywords: Metoprolol, hepatotoxicity, Nigella Sativa, vitamin E, antioxidants, hepatoprotective agents,
interleukins.
Introduction or oxidative stress may be involved.6 NS (black cumin),
herbal medication is known for its antioxidants, and
MET is an indispensable selective β1 receptor
liver-protective potentials7 was chosen in the present
blocker drug used to treat hypertension, angina pectoris,
study to examine its hepatoprotective effect either alone
heart failure, and arrhythmias.1 Since its approval by
or in combination with Vit E, which is also known for its
the WHO in 1982, MET was at the top list of essential
antioxidants effects8 against MET-induced liver injury
medicines and considered the safest and most effective
in a rabbit model.
medication for health care. Despite being well-tolerated,
Beta-blockers have many mild side effects, including
Materials and Methods
sleep disturbances, fatigue, cold extremities, weight loss,
bradycardia, and depression2; however, hepatotoxicity Animal handling
or elevation of liver enzymes are frequently reported
The study was carried out between November
notably with labetalol and MET.3,4,5 The underlying
2019 and July 2020. The protocol was approved by
mechanism of this toxicity is not well understood, but
the Ethical Committee at Basrah College of Medicine.
it is thought that it could have an immunological basis1060 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
Thirty-six fertile male rabbits weighing between 1 to 2 by making a small hole in the capsule. The content of
kg purchased from a local market in Basrah and included the capsule was squeezed into a small container, and this
were used. The rabbits were kept in separate cages in procedure was repeated for five capsules. The obtained
the animal house of the College for adaptation with free dose of Vit E is 1340 mg. Then one milliliter of tin corn
access to food in the form of trefoil and water. Room oil was added to dilute Vit E.
temperature was set at 22 ± 2°C, and a light/dark cycle
A conversion equation was used to calculate a dose
was 12/12 hours.
in mg from a dose in IU.
Preparation of NS oil, Vit E, and MET solution
(The equation is : 1 IU of Vitamin E = 0.67 mg).
The seeds of NS were purchased from a local shop
MET tablets (metoprolol succinate 50mg,
in Basrah city and authenticated by a special botanist in
AstraZeneca, Switzerland) were used for the study. Two
the city of Basrah. The oil of NS was obtained by cold
MET tablets were crushed using a mortar, dissolved in
press method using a special machine for this purpose.
10 ml of distilled water to obtain a suspension of 10 mg/
Each kilogram of NS seeds produced 250 milliliters of
ml. The suspension was placed in water-tight containers,
the oil (a yield of 25%). The oil is collected in a dark
shaken well to get a homogenous mixture. A dose of 20
bottle, stored in a cold and dry place.
mg/kg was used for oral dosing to the animals.
Vit E in the form of soft gel capsules (Nutri You,
Study design
UK) was purchased from local medicine stores in Basrah
city. The animals were divided randomly into six groups,
6 animals in each, and treated as shown in table 1
For calculation of the dose, Vit E oil was obtained
Table 1. Summary of the study design
Duration of
Groups Treatment
treatment
0 time 2 hours later
1st group Distilled water (2ml) oral - 14 days
2nd group MET (20mg/kg/day) oral - 14 days
3rd group NS (2ml/kg/day) oral - 14 days
4th group NS (2ml/kg/day) oral MET (20mg/kg/day) oral 14 days
5th group Vit E (175mg/kg/day) oral MET (20mg/kg/day) oral 14 days
NS (2ml/kg/day) +Vit E
6th group MET (20mg/kg/day) oral 14 days
(175mg/kg/day) oral
The doses were given using a (5 ml ) ordinary disposable syringe fixed to a nasogastric tube. The tube was
introduced to the animal’s stomach through a wooden tongue depressor with a hole in the middle fixed between the
teeth of the animal to prevent chewing the tube.Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 1061
Samples collection: 3. Serum and liver tissue Glutathione (GSH):
Estimating GSH in serum and hepatic tissue was
Blood samples (5-8 ml) were collected directly
measured by the competitive-ELISA detection method.
from the heart under light chloroform anesthesia. The
The GSH- ELISA kit sensitivity is 0.94 μg/ml, and the
samples were centrifuged, and serum was collected and
detection range is 1.56-100 μg/ml. ( Elabscience, USA).
stored at -80°C. The rabbits were then exposed to deep
chloroform anesthesia and sacrificed; the livers were 4. Serum and liver tissue Malondialdehyde
excised, washed with normal saline, and cut into two (MDA): Estimating MDA in serum and hepatic tissue
pieces. One gram was taken, homogenized mechanically was measured by the competitive-ELISA detection
using Heildolph mechanical homogenizer, Germany, method. MDA ELISA kit sensitivity is 4.688 ng/
centrifuged, and the supernatant was collected and stored ml and a detection range between 7.813-500ng/ml (
at -80°C. Another portion was transferred into a small MyBioSource, USA).
container containing 10% formalin for histopathological
Statistical analysis: SPSS (version 20) was used
examination.
for statistical analysis. A non-parametric Mann-Whitney
Laboratory measurements: test was used for comparing the means of different
parameters. P-value less than 0.05 is considered
1. Liver enzymes: The activities of aspartate
statistically significant.
aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), and total bilirubin in Results
serum were estimated by an autoanalyzer (Biochemistry
MET produced hepatotoxicity in all treated
Analyzer, Integra 400 Plus, Roche, Germany) by
animals as seen in liver enzymes and histopathological
selecting the already coded program in the analyzer for
examination. NS, Vit E, and NS + Vit E reduced the rise
all samples.
of liver enzymes produced by MET. MET increased
2. Serum and liver tissue interleukin-6 (IL- MDA levels in serum and homogenate, and treatment
6) and interleukin-10 (IL-10): estimation of serum with NS, NS+Vit E decreased these levels but not to
and hepatic tissue rabbit (IL-6) and (IL-10) used the significant levels. Neither MET nor MET and NS, Vit E,
quantitative sandwich enzyme immunoassay using and their combination changed the levels of IL-6 or IL-
ELISA kit (MyBioSource, USA). The rabbit IL-6 10. However, the level of serum IL-6 in the group treated
ELISA kit sensitivity is 1.0 pg/ml, rabbit IL-10 ELISA with Ns + Vit E and MET was found significantly higher
kit sensitivity is 9.375 pg/ml, and the detection range is than the level of IL-6 in MET treatment (P1062 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
CON: Control, MET: Metoprolol, NS: Nigella sativa, BIL: total bilirubin. (a): significantly different from the
Vit E: Vitamin E. ALT: alanine aminotransferase, AST: corresponding value of CON, (b): significantly different
aspartate aminotransferase, ALP: alkaline phosphatase, from the corresponding value of MET.
Table 3: The effect MET, NS, and combination of MET, NS, and Vit E on MDA and GSH in the serum and
liver homogenate in rabbits
Serum GSH (ug/ Liver GSH
Treatments Serum MDA (ng/ml) Liver MDA (ng/ml)
ml) (ug/ml)
CON 17.69 ± 17.21 7.94 ±4.9 14.55 ± 6.78 2.30 ± 1.13
MET 27.52 ± 17.42 21.36 ± 18.79 11.22 ± 1.88 2.35 ± 0.62
NS 20.38 ± 5.48 17.95 ± 11.27(a) 12.02 ± 2.25 2.07 ± 0.38
MET+NS 23.25 ± 10.78 12.32 ± 2.66 10.07 ± 4.85 2.26 ± 0.49
MET+Vit E 26.95 ± 3.97 16.72 ± 12.90 2.85 ± 1.32(b) 2.37 ± 0.31
MET+NS+Vit E 24.8 ± 21.20 17.69 ± 8.91 2.5 ± 1.70(b) 1.98± 0.60
CON: Control, MET: Metoprolol, NS: Nigella of blood cells. These histopathological changes were
sativa, Vit E: Vitamin E., MDA: malondialdehyde, modified in the group of rabbits treated with NS, Vit
GSH: glutathione (a): significantly different from the E, and NS and Vit E when given together with MET.
corresponding value of CON, (b): significantly different The slightest histopathological changes were observed
from the corresponding value of MET. in rabbits treated with NS and Vit E when given with
MET. These results were presented in figure 2.
Figure 1. represents the effect of MET on serum
IL-6 and IL-10 in MET-induced liver toxicity in Figure 2: showed the histopathological changes
rabbits and evaluates the protective effect of NS, Vit produced by various treatments: A (control group, X
E, and combination of NS and Vit E ON MET induced 40): no histopathological findings were seen; B (MET
hepatotoxicity. A and B showed the effect on IL-6 in group, X 40): moderate to severe inflammatory cells
liver homogenate and serum. C and D showed the effect infiltration, sinusoidal dilatation, vascular congestion,
on IL-10 in liver homogenate and serum. A significant and extravasation of blood cells, C (NS group, X 10):
difference was noticed between MET + NS + Vit E and mild inflammatory cells infiltration can be seen while
MET alone treated group (P=0.02). liver cells appeared normal, D (MET+NS group, X 40):
vascular congestion and dilatation of bile duct, mild to
CON: control, MET: Metoprolol, NS: Nigella sativa,
moderate inflammatory cells infiltration, E (MET+ Vit
Vit E: vitamin E, IL-6:interleukin 6, IL-10: interleukin
E group, X 40): vascular congestion and inflammatory
10.
cells infiltration, while liver cells appear normal, and F
Histopathological examination (MET+NS+Vit E group, X 10): minimal inflammatory
cells infiltration were seen while the rest of the liver cells
Histopathological examination confirmed the liver appear normal. CON: Control, MET: Metoprolol, NS:
enzyme changes produced by MET; these changes Nigella sativa, Vit E: Vitamin E.
included vascular congestion, inflammatory cell
infiltration, sinusoidal dilatation, and extravasationIndian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 1063
Discussion produced this protective effect through its antioxidant
effect.13 In one study, a hepatoprotective potential of NS
Drug-induced liver injury (DILI) is frequently
and Vit E has been investigated in chronic exposure to
encountered in medical practice. The extent of liver injury
malathion and proved very effective in preventing liver
ranged from mild liver enzyme changes to liver failure
injury.14 Unexpectedly, treatment with Vit E before MET
and death. Numerous published reports are documenting
reduced the concentration of serum GSH to levels that
DILI; some of these reports involved beta-blockers,
are very much lower than the control or other medicines.
which are widely used in practice for the treatment of
There is no direct explanation for this effect; however,
hypertension, angina, and many other cardiovascular
whether the high dose of Vit E used in the study is
diseases.9 The present study aimed at investigating the
blamed has not been yet confirmed.15 The significance of
effect of NS, Vit E, and a combination of NS with Vit
this effect needs further clarification. The combination
E on liver injury induced by MET. Rabbits were widely
of NS and Vit E modified MET toxicity apart from slight
used as a model for drug-induced hepatotoxicity.10,11
inflammatory changes in liver tissue, which still can be
The dose of MET used in the present study to cause liver
seen. Liver enzymes were also decreased. The obtained
damage was relatively high ( 20 mg/kg orally; the regular
effect is less than what is expected from the additive
dose for a human is up to 8 mg/kg). Treatment with MET
effects of two antioxidant agents (Vit E and NS). A
caused mild elevation in liver enzymes; this elevation was
similar pattern of a lack of effect of a combination of NS
observed in all measured enzymes, a finding frequently
and Vit E against cisplatin-induced renal toxicity was
reported by researchers.12,5 Elevation in liver enzymes
reported by Busari and colleagues (2018). In their study,
was associated with histopathological changes. In the
they showed that the combination of NS and Vit E,
control group, minimal or no histopathological changes
despite their antioxidant effects, did not show significant
were observed while, in the MET treated group, features
additive effects.16 On the contrary, the observed result of
of liver injury were observed, including congestion of
the combination was less than the effect observed with
the liver, extravasation, inflammatory cell infiltration,
each agent individually. NS may act differently and be
and sinus dilatation; these changes are highly suggestive
pro-oxidant rather than an antioxidant in particular cells
of liver injury. It was also observed in the group of MET
or organs.17 Thus, it can be speculated that NS may act as
treatment that the rabbits decreased food consumption
pro-oxidant in circumstances where interaction with Vit
with apparent weight loss; nevertheless, there were
E is involved so that such interaction may interfere with
no signs of yellow discoloration of the rabbit’s eyes
the combination’s protective effect. Vit E ( α-tocopherol),
suggestive of jaundice nor changes in urine color.
besides, whether at therapeutic doses or high doses, may
However, none of the rabbits treated with MET died
act as pro-oxidant, particularly in situations where the
within the 14 days study period.
antioxidant capacity is exhausted.18,19,20 Therefore, lack
NS mildly modified liver enzyme changes induced of effect of the combination, NS and Vit E on MET
by MET. The effect in restoring liver enzymes towards induced reduction in GSH could result from a pro-
control value was slight. MET-induced DILI was oxidant activity of NS and Vit E. Similarly, the observed
paralleled with elevation in serum MDA and also in rise in IL-6 in the group treated with the combination NS
liver tissue homogenate, and that NS treatment modified and Vit E compared to the control or MET treated group
the rise in MDA. This may suggest that NS may modify could be, as well, a result of the pro-oxidant potential of
MET-induced DILI through antioxidant effects. These the combinations.15
results were also supported by histopathological
It was noticed that the levels of interleukins (IL-6
examination, which reveals that the histopathological
and IL-10) are higher in the liver homogenate than in
changes seen with MET were reduced by NS treatment.7
the serum21; this result is in agreement with Norris and
The effect of Vit E on MET-induced liver injury was
colleagues, 2014.22 The result of this study showed that
similar to the effect produced by NS. The rise in liver
MET did not affect the level of IL-6 or IL-10; this result
enzymes by MET was moderately decreased by Vit E.
is in agreement with.23 NS alone also did not have an
Vit E decreased liver changes produced by MET but did
effect on IL-6, and neither MET + NS nor MET + Vit
not wholly prevented these changes. Most likely, Vit E
E changed the level of IL-6, but it was observed that the1064 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
level of IL-6 significantly increased in the group treated hepatotoxicity? Hepatology (Baltimore, Md.) 1989,
with MET + NS + Vit E. Further investigation of this 9:163-4.
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