Hepatoprotective Effect of Niclosamide on Paracetamol-Induced

Liver Toxicity in Rats

Ali M. Roshdy

and Ismail A. Youssef

2,*

School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University,

Tianjin 300072, P.R. China

Bahauddin Zakariya University, Multan, Punjab 60000, Pakistan

Keywords:

Liver Fibrosis, Niclosamide, Paracetamol-Induced Hepatoxicity.

Abstract: Liver diseases is considered one of the leading causes of death and an important barrier to increasing life

expectancy. From among, liver fibrosis which is a result of a chronic damage to the liver leading to liver

cirrhosis. Niclosamide is a potent anti-helminthic drug which has been used in treating the tapeworm

infections. It also showed a hepatoprotective effect in induced liver toxicity models. The present study was

conducted to identify the hepatoprotective effect of niclosamide in a paracetamol-induced liver toxicity. Rats

were divided into five groups (6 rats per group): control group, disease group, 5 mg niclosamide group, 10

mg niclosamide group and 15 mg niclosamide group. Three doses of niclosamide (5 mg/kg, 10 mg/kg and 15

mg/kg) were injected intraperitoneal (i.p) for 4 weeks. Assessments included hepatic enzymes (AST, ALT,

ALP and GGT), oxidative stress (MDA, GSH and SOD) and inflammatory markers (IL-6, TNF- α and NF-

kB). The three doses of niclosamide (5 mg/kg, 10 mg/kg and 15 mg/kg) had significantly reduced the hepatic

enzymes with the prominent effect at dose 15 mg/kg. There was also a significant decrease in the MDA

activity, while significant increase in the GSH and SOD activity. Moreover, there was significant reduction

in the levels of IL-6, TNF- α and NF-kB with the use of niclosamide (5 mg/kg, 10 mg/kg and 15 mg/kg).

Therefore, in the current study, it presents the niclosamide as a promising hepatoprotective agent.

1 INTRODUCTION

Liver diseases have become a major health concern

globally ranks as one of the leading causes of death

and an important barrier to increasing life expectancy.

From among, liver fibrosis which is a result of a

chronic damage to the liver leading to the

accumulation of the extracellular matrix (ECM)

proteins (Friedman, 2003). The accumulation of

ECM proteins will cause the formation of fibrous

scar, changes in the hepatic architecture and

eventually leading to liver cirrhosis (Ginès, 2004).

Viral infection, autoimmune disease, alcohol intake,

Correspondence: All correspondence should be

addressed to

Dr. Ismail A.Youssef

Lecturer of Pharmacology and Toxicology

Department of Pharmacology & Toxicology

Faculty of Pharmacy, Bahauddin Zakariya University,

Multan, Pakistan

Mobile: +2-01118420848

Fax: +202-

22751038

E-mail: ismail.a.youssef@gmail.com

drug-induced and metabolic disorder are the most

common etiologies of chronic liver disease,

consequently, leads to liver fibrosis and cirrhosis

(Friedman, 2004). Drug-induced liver toxicity

manifestations can be ranging from increase in the

hepatic enzymes to liver cirrhosis. An overdose of

paracetamol can cause sever liver toxicity in

accordance to pervious study (Vermeulen, 1992).

Such toxicity arises from the generation of the toxic

metabolite NAPQI (N-acetyl-p-benzoquinoneimine),

which is produced from the metabolism of the

paracetamol by the liver due to the oxidation through

the cytochrome P450 (Cyp450) (Vermeulen, 1992;

Roshdy, A. and Youssef, I.

Hepatoprotective Effect of Niclosamide on Paracetamol-Induced Liver Toxicity in Rats.

DOI: 10.5220/0012013200003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 61-68

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

Cohen, 1997). Although, the toxic effect of NAPQI is

neutralized by glutathione (GSH) under normal

conditions, the overproduction of it will eventually

deplete the GSH stores and accumulate reactive

oxygen species (ROS) causing liver damage due to

oxidative stress (Guo, 2016; Wang, 2017; Salem,

2018). Paracetamol induced oxidative stress also

stimuli the production of inflammatory markers

interleukin-6 (IL6), tumor necrosis factor-alpha

(TNF-α) and nuclear factor-kappa B (NF-kB) (Liao,

2016).

Niclosamide is a potent anti-helminthic drug

which has been used for long period in treating the

tapeworm infections (Al-Hadiya, 2005). Niclosamide

mechanism of action can be illustrated by its ability

to uncouple oxidative phosphorylation in

mitochondria, such mechanism protects the

mitochondria by reducing ROS production (Alasadi,

2018; Al-Gareeb, 2017). In a previous study,

niclosamide showed a hepatoprotective effect against

methotrexate-induced liver toxicity (Zeki, 2021).

Therefore, in the present study, we investigate the

hepatoprotective effect of niclosamide against

paracetamol-induced liver toxicity using in vivo

model (albino Wistar rats).

2 MATERIALS AND METHODS

2.1 Drugs and Chemicals

Niclosamide was obtained from Sigma-Aldrich. The

selection of niclosamide doses was according to a

previous study (Boyapally, 2019). Dimethyl

sulfoxide (DMSO) and chlorpromazine were

purchased from Thermo Fisher Scientific. Normal

saline solution, Polyethylene glycol (PEG) and

ketamine were purchased from Sigma-Aldrich. The

vehicle used for injection of niclosamide was DMSO

and PEG 200 with ratio 1:4 in normal saline. The

concentration of DMSO in normal saline was 1%.

The chemicals useful for these studies were almost of

analytical rank.

2.2 Animals

Experiments were conducted using Male albino

Wistar rats (250-300 g. n=30), procured from the

Holding company for biological products and

Vaccines (VACCERA, Egypt). The animals were

maintained at temperature of 25 ◦C and with 12/12 h

light/dark cycle. They were housed for 2 weeks prior

to the pharmacological experiments to adapt the

laboratory conditions. The study has been approved

by recommendations approved by the Egyptian

Russian University Research Ethics Committee

(REC-ERU), Egypt.

2.3 Paracetamol Induced Toxicity

Paracetamol was used for the induction of acute

hepatotoxicity according to the liver damage model

(Sreedevi, 2009). The paracetamol was suspended in

0.5% tween-80 and the dose administered was 2

gm/kg per oral.

2.4 Experimental Design

Animals were divided into five groups with 6 rats per

each group. Group 1: was the control group, rats

received vehicle only. Group 2: was the disease

group, rats injected with paracetamol (2 g/kg, p.o.).

Group 3: was the 5 mg niclosamide group, rats were

injected with paracetamol (2 g/kg, p.o.) and 5

mg/kg/day niclosamide i.p. for 4 weeks. Group 4: was

the 10 mg niclosamide group, rats were injected with

paracetamol (2 g/kg, p.o.) and 10 mg/kg/day

niclosamide i.p. for 4 weeks. Group 5: was the 15 mg

niclosamide group, rats were injected with

paracetamol (2 g/kg, p.o.) and 15 mg/kg/day

niclosamide i.p. for 4 weeks. The injection of

niclosamide and vehicle was started at the same day

of surgery.

After 4 weeks, the animals were anesthetized

using ketamine (100 mg/kg) and chlorpromazine i.p.

(0.75 mg/kg), then sacrificed. The blood samples

were collected by puncturing the retro-orbital plexus

for analysis of biochemical markers, and the liver was

removed for examination of oxidative stress and

inflammatory markers.

2.5 Biochemical Spectrophotometric

Analysis of

2.5.1 Biomarkers for Liver Function

A colorimetric method was used to assess Serum

aspartate transaminase (AST) and Alanine

transaminase (ALT) according to the instructions of

manufacturer’s (TECO DIAG- NOSTICS, 1268 N.

Lakeview Ave, Anaheim, U.S.A.). Modifications has

been carried on the original methods by Doumas and

Briggs, and Reitman and Frankel respectively

(Doumas, 1969; Reitmen, 1957).

Colorimetric method was used to assess Gamma-

Glutamyl Transferase (GGT) using kit (XpressBio

503 Gateway Dr W Thurmont, MD 21788,). It

measures the level of the product p-nitroaniline

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(pNA) at 405 nm, which is produced from the

cleavage of the GGT substrate (©-glutamyl-p-

ntiroanilide) by the enzyme. The product pNA is

directly proportional to the GGT level. All the results

from AST, ALT and GGT assays expressed in U/L.

2.5.2 Biomarkers for Cholestasis

Colorimetric method was used to assess Serum

alkaline phosphate (ALP) according to the

instructions of manufacturer (BioAssay Systems.

3191 Corporate Place, Hayward, USA). It measures

the yellow-colored product at 405 nm, obtained from

the hydrolysis of the p-nitrophenyl phosphate by the

ALP. The yellow-colored product is directly

proportional to the ALP activity. The result value was

expressed as U/L.

2.5.3 Oxidative Stress Marker

Malondialdehyde (MDA)

Assessment of the tissue MDA was carried according

to the instructions of manufacturer (MyBioSource,

sunny Southern California, San Diego, USA).

Evaluation of the tissue MDA was based on the

reaction with thiobarbituric acid (TBA) to produce

thiobarbituric acid reactive substance (TBARS); pink

chromogen; which is measured at 532-535 nm.

2.6 Enzyme- Linked Immunosorbent

Assay (ELISA)

2.6.1 General Principle

This assay employs the quantitative sandwich

enzyme immunoassay technique. Antibodies specific

to the target antigens were used to pre-coat the ELISA

plates. For detection antibody, Biotin conjugated

antibody was used. Samples and biotin conjugated

antibody were added simultaneously to the wells and

washed using wash buffer. After using the wash

buffer to wash the wells, avidin conjugated

horseradish peroxidase (HRP) was added. To

visualize the HRP reaction, tetramethylbenzidine

(TMB) was used as substrate. The produced color

intensity was measured at 450 nm, which is

proportional to the amount of the antigen in the

sample.

2.6.2 Assessment of Oxidative Stress

Markers Superoxide Dismutase (SOD)

And Glutathione (GSH)

Assessment of the SOD and GSH tissue levels was

carried according to the instructions of manufacturer

(MyBioSource, sunny Southern California, San

Diego, USA), (Blue gene Biotech CO., LTD,

Shanghai, China), respectively. The results values

were expressed as mmol/mg protein.

2.6.3 Assessment of Inflammatory Markers

Assessment of the interleukin-6 (IL-6), tumor

necrosis factor-alpha (TNF-α) and nuclear factor-

kappa B (NF-kB) was carried according to the

instructions of the manufacturer (Cat no.

MBS175908, MyBioSource, sunny Southern

California, San Diego, USA), (Cat no. MBS9711597,

MyBioSource, sunny Southern California, San

Diego, USA) and (Cat no. MBS268833,

MyBioSource, sunny Southern California, San

Diego, USA), respectively. The result value of NF-

kB level was expressed as ng/mg protein, however,

the results values for both IL-6 and TNF-α were

expressed as pg/mg protein.

2.6.4 Statistical Analysis

The results were all expressed as mean ± S.D. To

compare between different groups, one-way analysis

of variance (ANOVA) was used followed by Tukey’s

test. The data considered statistically significant when

the P < 0.05. All of the statistical analysis and graphs

were done using GraphPad Prism version 5 software

(ISI Software, United States).

3 RESULTS

3.1 Effect of Niclosamide on Liver

Function and Cholestasis Markers

After 4 weeks, there was a significant increase in the

AST, ALT, ALP and GGT values in the disease group

by 5, 6, 5 and 8-fold respectively, when compared to

the control group. Treatment with niclosamide in

group 3 ( 5 mg/kg/day) cause a significant decrease in

the values of AST, ALT, ALP and GGT by 40%,

45%, 50% and 53% respectively, when compared to

the disease group. On the other hand, treatment with

niclosamide in group 4 ( 10 mg/kg/day) cause a

significant decrease in the values of AST, ALT, ALP

and GGT by 59%, 67%, 70% and 78% respectively,

when compared to the disease group. Furthermore,

treatment with niclosamide in group 5 ( 15

mg/kg/day) cause a significant decrease in the values

of AST, ALT, ALP and GGT by 63%, 70%, 73% and

80% respectively, when compared to the disease

group (Table 1).

Hepatoprotective Effect of Niclosamide on Paracetamol-Induced Liver Toxicity in Rats

Table 1: Effect of Niclosamide (5, 10 and 15 mg/kg) on paracetamol induced changes in liver functions.

Groups AST (U/L) ALT (U/L) ALP (U/L) GGT (U/L)

Control 45.06 ± 5.312 32.64 ± 4.51 52.83 ± 4.13 23.95 ± 3.46

Disease 235.50 ± 5.39

198.01 ± 7.66

264.01 ± 5.81

188.0 ± 7.37

5 mg Niclosamide 138.50 ± 7.78

a, b

108.30 ± 7.36

a, b

132.7 ± 6.09

a, b

89.82 ± 4.93

a, b

10 mg Niclosamide 98.50 ± 3.85

a, b

66.42 ± 5.60

a, b

80.45 ± 5.08

a, b

42.82 ±4.21

a, b

15 mg Niclosamide 89.02 ± 4.63

a, b

60.76 ± 6.59

a, b

73.99 ± 6.53

a, b

37.27 ± 6.40

Data are represented as mean ± SD of 6 rats per group. a: significant difference from control group, and b: significant

difference from disease group at p < 0.05 using ANOVA followed by Tukey’s post-hoc test.

3.2 Effect of Niclosamide on Oxidative

Stress and Antioxidant Capacity

Comparing the level of the MDA in the control group

to the disease group. There was a significant increase

with around 6 folds in the disease group. Treatment

with niclosamide in the group 3 (5 mg/kg/day), group

4 (10 mg/kg/day) and group 5 (15 mg/kg/day) caused

a significant reduction by 40%, 64% and 69%

respectively, when compared to the disease group

(Figure 1). Moreover, GSH and SOD levels were

significantly decrease in the disease group by 75%

and 70% respectively, when compared to the control

group. Treatment with niclosamide in group 3 (5

mg/kg/day), group 4 (10 mg/kg/day) and group 5 (15

mg/kg/day) caused a significant increase in the levels

of GSH by 2 folds, 3.5 folds and 4 folds respectively,

when compared to the disease group (Figure 2).

While levels of SOD were increased with niclosamide

treatment in group 3 (5 mg/kg/day), group 4 (10

mg/kg/day) and group 5 (15 mg/kg/day) by 1.7 folds,

2.5 folds and 3 folds, respectively, when compared to

the disease group (Figure 3).

Figure 1: Effect of niclosamide on MDA. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

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Figure 2: Effect of niclosamide on GSH. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

Figure 3: Effect of niclosamide on SOD. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

3.3 Effect of Niclosamide on

Inflammatory Markers

There was a significant increase in the levels of IL-6,

TNF-α and NF-kB by 4.9, 5.6, 6.5 folds in the disease

group compared to the control group. On the other

hand, treatment using niclosamide in group 3 (5

mg/kg/day) caused significant reduction on IL-6,

TNF-α and NF-kB by 39%, 42% and 49%

respectively, when compared to the disease group.

Also, treatment using niclosamide in group 4 (10

mg/kg/day) caused a significant reduction on IL-6,

TNF-α and NF-kB by 58%, 60% and 71%

respectively, when compared to the disease group. In

addition, treatment using niclosamide in group 5 (15

mg/kg/day) caused a significant reduction on IL-6,

TNF-α and NF-kB by 63%, 67% and 76%

respectively, when compared to the disease group

(Figures 4, 5 and 6).

Hepatoprotective Effect of Niclosamide on Paracetamol-Induced Liver Toxicity in Rats

Figure 4: Effect of niclosamide on IL-6. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

Figure 5: Effect of niclosamide on TNF-α. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

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Figure 6: Effect of niclosamide on NF-kB. Data are presented as mean ± SD of 6 rats per group, a: significant difference from

control group, and b: significant difference from disease group at p < 0.05 using one-way ANOVA followed by Tukey’s post

hoc test. Con, control group; Dis, disease group; NCL5, 5 mg niclosamide group; NCL10, 10 mg niclosamide group; NCL15,

15 mg niclosamide group.

4 DISCUSSION

Liver is an essential organ for regulation of the internal

body environment. Therefore, any damage affects the

liver due to any reasons can have a serious

consequence. One example of the causes behind liver

toxicity, is drug-induced toxicity by paracetamol.

Paracetamol is used as analgesic and antipyretic drug

at normal doses, however, in high doses it possesses a

hepatotoxic effect. Moreover, paracetamol-induced

hepatotoxicity was used as an experimental model for

evaluation of hepatoprotective agents.

In this study, liver fibrosis in rodents was induced

using paracetamol-induced hepatotoxicity model.

There was elevation in the hepatic markers AST, ALT,

ALP and GGT showing hepatic dysfunction

. Using

different doses of niclosamide ( 5, 10 and 15 mg/kg),

it displayed a significant decrease in the AST, ALT,

ALP and GGT enzymes activity. The prominent effect

was at dose 15 mg/kg, however, the effect of doses 10

mg/kg and 15 mg/kg was almost the same.

Paracetamol-induced liver toxicity showed before

an increase in the ROS and depletion in the antioxidant

reservoirs (Canayakin, 2016). Depletion of GSH

stores and decrease in its value in this study was in

accordance with a previously published results

(Kushwah, 2014). Treatment using niclosamide

different doses ( 5, 10 and 15 mg/kg) had a significant

effect on restoring the GSH stores displayed increase

of its values. Although there was a significant

difference in the effect on GSH between the dose 5

mg/kg and 10 mg/kg but comparing the effect of dose

10 mg/kg to 15 mg/kg, the values were closely related.

Same pattern was obvious with the SOD, where there

was a significant decrease in SOD value in the disease

group, correlated with the previous results (Madi,

2015). Co-treatment using niclosamide (5, 10 and 15

mg/kg) lead to the increase of SOD activity with the

prominent effect at 10 mg/kg and 15 mg/kg. Free

radicals cause tissue damage due to lipid peroxidation

(Esterbauer, 1991). One of the lipid peroxidation

products is MDA, which showed a surge in its values

in this study due to paracetamol-induced

hepatotoxicity in accordance with a recent study

(Rašković, 2017). Treatment using niclosamide (5, 10

and 15 mg/kg) displayed a decrease in MDA values.

Comparing the 3 different doses, the effect was the

most with 15 mg/kg and the dose 5 mg/kg showed the

lowest effect.

In addition, herein inflammatory markers such as

IL-6 and TNF-α was significantly increased with

paracetamol-induced liver toxicity. This elevation is

correlated to recent studies (Karakus, 2013; James,

2003). Co-treatment using niclosamide (5, 10 and 15

mg/kg) decrease the activity of these markers, doses

10 mg/kg and 15 mg/kg showed almost same pattern

with prominent effect if compared to dose 5 mg/kg.

Also, the inflammatory marker NF-kB was

significantly boosted with the paracetamol-induced

toxicity, in alignment with the recent study (Jiang,

2021). Using niclosamide various doses (5, 10 and 15

mg/kg) caused significant reduction in NF-kB

activity, such reduction was more obvious with the 10

mg/kg and 15 mg/kg doses.

Hepatoprotective Effect of Niclosamide on Paracetamol-Induced Liver Toxicity in Rats

5 CONCLUSION

The hepatoprotective effect of niclosamide against

liver toxicity induced by paracetamol for four-weeks

period was elevated by the author. Niclosamide

significantly cause reduction in the hepatic enzymes

AST, ALT, ALP and GGT. Moreover, niclosamide

had the ability to ameliorate oxidative stress by

significantly reduce MDA and increase SOD and

GSH. Nevertheless, niclosamide had also part in

inflammation reduction by significantly decrease the

inflammatory markers TNF-α, IL-6 and NF-kB.

Although further studies are needed for

understanding the exact mechanism of niclosamide in

healing liver toxicity, but niclosamide can be

considered a promising hepatoprotective drug.

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