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Ethanolic Extract of Citrus Sinensis Peel Markedly Mitigate Paracetamol-

Induced Hepatotoxicity in Rats

Article in Journal of Health and Rehabilitation Research · December 2023

DOI: 10.61919/jhrr.v3i2.270

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Original Article
Ethanolic Extract of Citrus Sinensis Peel Markedly Mitigate
Paracetamol-Induced Hepatotoxicity in Rats
Hafiz Muhammad Mazhar Asjad1*, Muhammad Shoaib Akhtar2, Maryam Khan2, Nabeela Tabassum Sial3, Imran Sohail4, Laiba Arshad1
1Forman Christian College University Lahore.
2University of Sargodha.
3Lahore College for Women University Lahore.
4
Government College University Lahore.
*Corresponding Author: Hafiz Muhammad Mazhar Asjad; Email: [email protected]

Conflict of Interest: None.

Asjad HMM., et al. (2023). 3(2): DOI: https://doi.org/10.61919/jhrr.v3i2.270

ABSTRACT
Background: Herbal medicines have historically been crucial for enhancing human health. Previous studies have highlighted the
antioxidant properties of various parts of Citrus sinensis. In light of widespread reliance on plant-based remedies, this study focuses
on a particular herbal medicine, Citrus sinensis, known for its rich content of vitamin C, pectins, phenolic compounds, and flavonoids.

Objective: This research aims to investigate the hepatoprotective potential of the ethanolic extract of Citrus sinensis peel in vivo,
particularly focusing on its antioxidant capabilities.

Methods: The study utilized biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT),
alkaline phosphatase (ALP), bilirubin, and total proteins to assess the hepatoprotective action of the Citrus sinensis peel extract in
serum of Wistar albino rats. The rats were divided into five groups, with varying treatments including a control group, a paracetamol-
induced hepatotoxicity group, and groups receiving different doses of the Citrus sinensis extract.

Results: Treatment with the ethanolic extract of Citrus sinensis peel at doses of 200 mg/kg and 400 mg/kg significantly mitigated
the paracetamol-induced elevation of serum levels of AST, ALT, ALP, bilirubin, and total proteins. These improvements were
comparable to those observed with the standard drug, silymarin.

Conclusion: The study confirms the hepatoprotective effects of Citrus sinensis peel extract against paracetamol-induced liver
damage. This protection was evident both biochemically and histopathologically, suggesting the potential therapeutic utility of Citrus
sinensis in liver disorders.

Keywords: Alanine aminotransferase, Antioxidant, Aspartate aminotransferase, Bilirubin, Citrus sinensis, Hepatoprotective, Liver
markers, Paracetamol, Silymarin, Total proteins.

INTRODUCTION
The use of herbal remedies has significantly improved human health. Eighty percent of people worldwide rely on traditional
therapies, primarily plant-based remedies (1). Herbal medicines have a long history of use in treating liver disorders and are the
most widely used strategy in many Western polls (2). However, only 5–15% of the estimated 250,000 species of higher plants
recorded have been examined for their potential therapeutic value (3). These practices, replacing enduring beliefs, have been
transmitted from generation to generation through oral history and/or restricted literature. Standardization remains a challenge,
and clinical trials are lacking to confirm efficacy. Some plant extracts used for digestive or biliary disorders may be harmful due to
potent, liver-toxic alkaloids. Molecules like phyllanthin, glycyrrhizin, picroside, silibinin, and baicalein—derived from Phyllanthus
amarus, licorice root, Picrorhiza kurroa, milk thistle, and sho-saiko-to—have proven antifibrotic, antioxidative, anticarcinogenic,
and/or antiviral properties (4). They hold the potential to be foundational molecules for developing novel hepatoprotective drugs
(5). Although herbal treatments are effective for many disorders, their misuse is frequent. Therefore, thorough research of these
natural medicines is required in light of current knowledge (6).
Vitamin C, pectins, phenolic compounds, and flavonoids are abundant in Citrus sinensis. The main flavonoids in citrus fruits are
narirutin, hesperidin, eriocitrin, and naringin (7). One orange provides 116% of the recommended daily intake of vitamin C, a major

© 2023 et al. Open access under Creative Commons by License. Free use and distribution with proper citation. Page 1058
Citrus Sinensis Peel Extract in Counteracting Paracetamol Hepatotoxicity
Asjad HMM., et al. (2023). 3(2): DOI: https://doi.org/10.61919/jhrr.v3i2.270

water-soluble antioxidant. Vitamin C inhibits the production of free radicals, thereby preventing tissue damage in aqueous
environments (8). Consumption of orange juice without added salt and sugar has been linked to reduced severity of inflammatory
diseases such as rheumatoid arthritis, osteoarthritis, and asthma (9, 10). Beta-cryptoxanthin, an orange-red carotenoid most
abundant in Citrus sinensis, is highly effective in reducing lung cancer risk (11, 12). Citrus sinensis also possesses antifungal, anti-
inflammatory, and anxiolytic properties (13, 14), while its peel extracts exhibit hypolipidemic, anti-inflammatory, and antioxidant
properties, attributable to bioflavonoids such as PMFs (polymethoxylated flavones) (15, 16). Researchers have demonstrated that
plant flavonoids are primarily responsible for antioxidant activity (17, 18). The present study investigates the in vivo hepatoprotective
effects of Citrus sinensis against paracetamol-induced liver injury.

MATERIAL AND METHODS

Preparation of Crude Ethanolic Extract
Citrus sinensis peels were dried in a shaded area for 5 to 7 days. The peels were then diced and ground into a fine powder. This
powder was placed in a sealed bottle, and ethanol was added until the material was completely immersed. The mixture was shaken
twice daily for 7 to 10 days. Subsequently, ethanol was evaporated using a rotary evaporator, yielding the crude extract as a semisolid
solution.
Chemicals and Drugs
Paracetamol was procured from M/S English Pharmaceuticals, Lahore. Silymarin (Siliver®) was obtained from a local pharmacy.
Ethanol was sourced from Labscan Asia Co., Ltd., Bangkok, Thailand. Serum diagnostic kits for biochemical assays were acquired
from DiaSys Diagnostic System GmbH, Holzheim, Germany. All solvents used in this study were of analytical grade.
Experimental Animals
Wistar rats of either sex, young and healthy, were acquired from the Islamia University of Bahawalpur (IUB). The animals were
housed in specially designed plastic cages with sipper bottles at the Department of Pharmacology, Faculty of Pharmacy, University
of Sargodha. They received humane care and were maintained at a constant room temperature (25-10°C) as per the guidelines of
the National Research Council (NRC, 1996). The rats were allowed a week to acclimate to their new environment and had unlimited
access to a standard laboratory diet and water. The Animal Ethics Committee of the University of Sargodha approved the study.
Additionally, the study protocol received approval from the Institutional Animal Ethics Committee of the Faculty of Pharmacy,
University of Sargodha.
Acute Toxicity Studies
The acute toxicity of the ethanolic extract of Citrus sinensis was investigated in Wistar albino rats. The animals were fasted overnight
prior to the test. The CPCSEA (Annexure-2d) methodology and the fixed dose OECD guideline No. 420 were employed for the study.
Each test dose was administered to a group of six rats, with the screening dose determined based on the LD50 value. The animals
were observed continuously for 24 hours.
Hepatoprotective Potential Evaluations
Assessment of the Hepatoprotective Effects in Paracetamol-induced Liver Damage
Procedure
Wistar albino rats were selected and divided into five groups, each consisting of six animals. Group I served as the vehicle control
group and received 1 ml/kg p.o. (per os, orally) of the vehicle. Group II was administered 640 mg/kg p.o. of paracetamol in 1% methyl
cellulose. Group III received a standard dose of 25 mg/kg of silymarin, while Group IV was given 200 mg/kg of the ethanolic extract
of Citrus sinensis dissolved in 1% Tween 80. Group V received 400 mg/kg p.o. of the ethanolic extract of Citrus sinensis suspended
in 1% Tween 80. Each group, except for Group I, received 640 mg/kg p.o. of paracetamol (in 1% methyl cellulose) at 0 hours. Silymarin
at a dose of 25 mg/kg was administered orally to Group III at intervals of 6, 12, and 18 hours. The ethanolic extract of Citrus sinensis
was administered to Groups IV and V at doses of 200 mg/kg and 400 mg/kg, respectively.
Twenty-four hours after the final dose of treatment, the animals were sacrificed. Blood (5 mL) was drawn from the brachial artery
under light sedation with ether. The serum was then separated from the blood by centrifugation for fifteen minutes at 3000 rpm.
This serum was used to determine the levels of ATP, AST, ALP, total bilirubin, and total protein using a biolyzer and diagnostic tools.
The liver was then excised for histopathological examination and placed in a 10% formalin solution. The data were presented as
mean ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA), followed by
Dunnett's test for post-hoc analysis. Values of P ≤ 0.05 were considered statistically significant.

RESULTS
Biochemical parameters:
© 2023 et al. Open access under Creative Commons by License. Free use and distribution with proper citation. Page 1059
Citrus Sinensis Peel Extract in Counteracting Paracetamol Hepatotoxicity
Asjad HMM., et al. (2023). 3(2): DOI: https://doi.org/10.61919/jhrr.v3i2.270

The hepatoprotective properties of silymarin (25 mg/kg) and the ethanolic fraction of Citrus sinensis (EFCS) (at doses of 200 mg/kg
and 400 mg/kg) were evident through their impact on total bilirubin, total proteins, as well as on AST, ALT, and ALP. Compared to the
vehicle-treated control group, the group treated with paracetamol exhibited significantly higher blood levels of ALT, AST, ALP, and
total bilirubin, along with significantly lower levels of total proteins and the albumin/globulin ratio (Table 1). Table 1 also summarizes
the effects of silymarin (25 mg/kg) and EFCS at doses of 200 mg/kg and 400 mg/kg on the paracetamol-induced increase in these
parameters. Notably, EFCS at doses of 200 mg/kg and 400 mg/kg significantly reduced the levels of serum enzymes and serum
bilirubin while significantly increasing the levels of serum total proteins and the albumin/globulin ratio, compared to the standard
medication, silymarin. These results demonstrate the hepatoprotective efficacy of the ethanolic extract of Citrus sinensis.

Table 1: Hepatoprotective activity of Citrus sinensis peel ethanol extract against paracetamol-induced hepatotoxicity: Serum
Enzymes, Bilirubin Total, and Total Proteins
Group Title ALT ASP ALP Bilirubin total Total Proteins Albumin Globulin A/G ratio
(U/L) (U/L) (U/L) (mg/dl) (g/dl) (g/dl) (g/dl) (%)

Vehicle 38** ± 41** ± 131** 5.000** ± 2.30** ± 2.170** ±

0.300** ± 0.010 7.300** ± 0.082
Control 16.2 6.18 ± 12.3 0.093 0.082 0.082
Paracetamol 302 ± 440 ± 380 ± 1.100 ± 3.00 ± 0.360 ±
5.000 ± 0.08 4.100 ± 0.165
Control 26.9 22.0 23.1 0.129 0.051 0.017
Silymarin 60** ± 107** 139** 4.800** ± 2.60** ± 1.817** ±
0.583** ± 0.021 7.000** ± 0.073
Standard 37.7 ± 9.1 ± 13.5 0.052 0.052 0.049
EFCS 200 99** ± 200** 280* ± 4.600** ± 2.700 ± 1.700** ±
0.613** ± 0.019 7.300** ± 0.144
mg/kg 45.5 ±50.3 28.6 0.144 0.144 0.144
EFCS 400 90** ± 139** 268* ± 4.600** ± 2.80** ± 1.640** ±
0.500** ± 0.010 7.000** ± 0.103
mg /kg 15.7 ± 26.2 32.2 0.103 0.030 0.103
Histopathological parameters

Histopathological examinations of the negative control mice revealed normal liver cells with a distinct nucleolus and nucleus, well-
conserved cytoplasm, and a healthy central vein (Fig A). Microscopic analysis of the paracetamol-treated animals revealed significant
necrosis, fatty alterations, degeneration, and ballooning. (Fig B). Liver slices from silymarin-treated animals revealed liver
architecture that was similar to that of untreated animals. (Fig C). According to Fig. D, animals given Citrus sinensis' ethanolic fraction
demonstrated a significant amount of hepatic protection against toxic substances.

Fig. 1: A typical liver histogram showed untreated rat liver cells

with prominent nuclei, well-conserved cytoplasm, and a
healthy central vein. B: The liver of a paracetamol-impaired rat
showed significant necrosis, fatty alterations, degeneration,
and ballooning. C: Rat liver histograms treated with silymarin
showed normal liver cells with distinct nuclei and nucleoli,
well-conserved cytoplasm, and a healthy central vasculature.
D: Rat liver histogram treated with an ethanolic extract of
citrus sinensis peel that was similar to normal.

© 2023 et al. Open access under Creative Commons by License. Free use and distribution with proper citation. Page 1060
Citrus Sinensis Peel Extract in Counteracting Paracetamol Hepatotoxicity
Asjad HMM., et al. (2023). 3(2): DOI: https://doi.org/10.61919/jhrr.v3i2.270

DISCUSSION
In the current research, paracetamol served as a toxic agent to evaluate the protective action of Citrus sinensis against hepatic injury.
The extent of toxicity was assessed using histopathological analyses and biochemical enzyme markers, including ATP, AST, ALP, serum
bilirubin, and protein levels. Adherence to OECD guidelines facilitated the determination of acute oral toxicity (19). Citrus sinensis
was deemed safe at doses up to 2000 mg/kg, as determined by the LD50 method. However, a dose of 400 mg/kg was selected for
its hepatoprotective action. Paracetamol at a dosage of 650 mg/kg body weight induced hepatotoxicity. The liver's metabolism of
paracetamol via the cytochrome p450 enzyme system, leading to the production of the minor but potent alkylating metabolite
NAPQI, was central to this process. NAPQI initiates its harmful effects by covalently bonding to cellular proteins and depleting
glutathione, disrupting calcium homeostasis.
Supporting these observations, biochemical parameters in the current study showed a significant increase in the paracetamol control
groups. Histopathological profiles corroborated the presence of noticeable damage in the same group. This indicates that injury is
caused by mechanisms such as lipid peroxidation, depletion of glutathione reserves, or generation of free radicals. Notably, the
administration of 400 mg/kg of Citrus sinensis peel ethanolic extract significantly reduced serum enzyme and bilirubin levels.
Furthermore, histopathological studies confirmed the restoration of normal liver histology, thus supporting the organ's protection.
This study's findings align with those of Lagha-Benamrouche & Madani (2013) (20), reinforcing the hepatoprotective potential of
Citrus sinensis. The strengths of this research include its comprehensive approach in assessing both biochemical and
histopathological changes. However, limitations exist in the scope of the study, such as the need for further research to isolate and
identify the specific active constituents responsible for the hepatoprotective effect. While this study provides valuable insights, it
also highlights the necessity for continued research to fully understand the mechanisms underlying the hepatoprotective properties
of Citrus sinensis.

CONCLUSION
The findings of this study demonstrate that an ethanolic extract of Citrus sinensis peel significantly reduced serum levels of ALT, AST,
ALP, and bilirubin. Furthermore, histopathological examination indicated that Citrus sinensis peel might possess the capacity to
mitigate liver damage, restoring liver function towards a near-normal state. These hepatoprotective effects are likely attributed to
the plant's reputed antioxidant properties. Future research should focus on isolating, characterizing, and purifying more active
ingredients from Citrus sinensis. Additionally, further experimental studies are warranted to elucidate the precise mechanisms
through which this plant exerts its hepatoprotective action.

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