Elettaria cardamomum Essential Oil Rescues Paracetamol-Induced Hepatorenal Damage via Modulating Oxidative Stress in Rats

Abstract

Hepatoprotective efficacy of cardamom (Elettaria cardamomum) seeds crude essential oil was evaluated in
experimentally paracetamol (PCM) hepatotoxic rats. Sixty-five male Sprague Dawley rats divided into six groups.
First group was kept as control negative, while the 2nd group was given PCM (500 mg/kg bw). The 3rd and 4th groups
were orally administered silymarin + PCM (500mg/kg bw + 500 mg/kg bw), and cardamom oil + PCM (100 mg/kg
bw + 500 mg/kg bw), respectively. The 5th and 6th groups were treated with silymarin (500 mg/kg bw) and cardamom
oil (100 mg/kg bw), respectively in normal rats. All the previous dosages lasted for two weeks. Blood samples collected
weekly from all tested groups. Assessment of hemogram, some serum biochemical parameters and histopathology of
liver and kidney were carried out. Chemical compositions of cardamom oil was established by gas chromatographymass
spectrometry (GC-MS), revealed that the oil was rich in monoterpenoids, while the monoterpene hydrocarbon,
was existing with comparatively small percent. Results of PCM-intoxicated rats revealed significant decrease in total
protein and albumin with significant hyperglycemia and elevation in serum transaminases, urea and creatinine which
were significantly ameliorated in groups receiving cardamom oil or silymarin. Administration of cardamom essential
oil significantly ameliorates the hepato-renal profiles and elevates total antioxidant capacity. Moreover, the oil protects
the liver and kidneys from histopathological alterations in PCM-intoxicated rats. Neither cardamom oil nor silymarin
induce any alterations in serum biochemistry and total antioxidant activity in normal rats. The present results proved
the ameliorative effect of cardamom essential oil against hepato-renal changes in PCM-intoxicated rats reasonably
due to its potent antioxidant effect.
Keywords: Cardamom Oil, Paracetamol, Gas Chromatography-Mass Spectrometry, Antioxidant, Hepato-renal, Rats

Full text

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INTRODUCTION
Liver is considered one of the most important organs
in the body as it’s responsible for many vital functions
(Mayuren et al., 2010). So, liver must be kept in healthy
condition for keeping normal physiological function. Drugs
are the principal cause of hepatic injury that resulting in
substantial clinical problem all over the world (Lakshmi
et al., 2018). Paracetamol, which known as acetaminophen
is considered one of the most used analgesics (Sheen et
al., 2002). Paracetamol when taken in a therapeutic dose is
known to be relatively nontoxic (Siemionow et al., 2016).
Its toxic eect is resulted when taken in a single or repeat-
ed high dose, or after chronic ingestion (Tittarelli et al.,
2017). Hepato- renal dysfunction is considered as one of
the numerous problems in drug therapy (Cepa et al., 2018).
Paracetamol-induced hepatotoxicity has been studied for
several years (Hinson et al., 2010; Ahmed et al., 2019).
Research Article
Abstract | Hepatoprotective ecacy of cardamom (Elettaria cardamomum) seeds crude essential oil was evaluated in
experimentally paracetamol (PCM) hepatotoxic rats. Sixty-ve male Sprague Dawley rats divided into six groups.
First group was kept as control negative, while the 2nd group was given PCM (500 mg/kg bw). e 3rd and 4th groups
were orally administered silymarin + PCM (500mg/kg bw + 500 mg/kg bw), and cardamom oil + PCM (100 mg/kg
bw + 500 mg/kg bw), respectively. e 5th and 6th groups were treated with silymarin (500 mg/kg bw) and cardamom
oil (100 mg/kg bw), respectively in normal rats. All the previous dosages lasted for two weeks. Blood samples collected
weekly from all tested groups. Assessment of hemogram, some serum biochemical parameters and histopathology of
liver and kidney were carried out. Chemical compositions of cardamom oil was established by gas chromatography-
mass spectrometry (GC-MS), revealed that the oil was rich in monoterpenoids, while the monoterpene hydrocarbon,
was existing with comparatively small percent. Results of PCM-intoxicated rats revealed signicant decrease in total
protein and albumin with signicant hyperglycemia and elevation in serum transaminases, urea and creatinine which
were signicantly ameliorated in groups receiving cardamom oil or silymarin. Administration of cardamom essential
oil significantly ameliorates the hepato-renal proles and elevates total antioxidant capacity. Moreover, the oil protects
the liver and kidneys from histopathological alterations in PCM-intoxicated rats. Neither cardamom oil nor silymarin
induce any alterations in serum biochemistry and total antioxidant activity in normal rats. e present results proved
the ameliorative eect of cardamom essential oil against hepato-renal changes in PCM-intoxicated rats reasonably
due to its potent antioxidant eect.
Keywords: Cardamom Oil, Paracetamol, Gas Chromatography-Mass Spectrometry, Antioxidant, Hepato-renal, Rats
AymAn A. KhAttAb1, AzzA m. tAwfeK2, KhAled Abo-el-Sooud3*, KAwKAb A. Ahmed4,
Abd el nASSer el-Gendy5, A.r. Ahmed2
Elettaria cardamomum Essential Oil Rescues Paracetamol-Induced
Hepatorenal Damage via Modulating Oxidative Stress in Rats
Received | June 26, 2020; Accepted | July 05, 2020; Published | September 20, 2020
*Correspondence | Khaled Abo-EL-Sooud, Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza,12211, Egypt; Email:
kasooud@cu.edu.eg
Citation | Khattab AA, Taweek AM, Abo-EL-Sooud K, Ahmed KA, El-Gendy AN, Ahmed AR (2020). Elettaria cardamomum essential oil rescues paraceta-
mol-induced hepatorenal damage via modulating oxidative stress in rats. Adv. Anim. Vet. Sci. 8(s2): 24-33.
DOI | http://dx.doi.org/10.17582/journal.aavs/2020/8.s2.24.33
ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331
Copyright © 2020 Abo-EL-Sooud et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
1Egypt Center for Research and Regenerative Medicine, Military Forces, Cairo, Egypt; 2Department of Clinical
Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt; 3Department of Pharmacology, Faculty
of Veterinary Medicine, Cairo University, Giza,12211, Egypt; 4Department of Pathology, Faculty of Veterinary
Medicine, Cairo University, Giza, Egypt; 5Medicinal and Aromatic plants Research department, National Research
Centre, Dokki, Giza, Egypt

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e metabolism of the ingested therapeutic dose of PCM
is mainly accomplished by cytochrome P450 followed by
glucuronidation or sulfation whereas N-acetyl-p-benzo-
quinone imine (NAPQI) is conjugated with reduced glu-
tathione (GSH) levels are associated with liver damage
(Hinson et al., 2010). us, excessive drugs accumulations
become accessible for biotransformation and metabolism
by cytochrome P450, which result in GSH depletion. Ex-
hausted GSH levels allow NAPQI free to bind with other
targeted cellular proteins which evoke cellular oxidative
stress and indulge in the cellular necrosis process (Hamza
and Al-Harbi, 2015). Hence, the use of antioxidants could
oer protective and preventive eects against drug-in-
duced hepatic injury.
Numerous herbal medicines were evaluated both in vitro
and in vivo for their hepatoprotective ecacy all over the
world. Moreover, several polyherbal formulations were de-
veloped and proved to be more eective than the known
synthetic hepatoprotective drugs (Lakshmi et al., 2018).
Application of essential oils (EOs) as a substitute to syn-
thetic chemicals is an increasing claim nowadays. Medici-
nal plants including cardamom, which belong to the family
of Zingiberaceae, has benecial eects especially against
human diseases due to their bioactive compounds (Mut-
lu-Ingok and Karbancioglu-Guler, 2017).
Cardamom is widely used for avoring purposes in food,
and in medicine it is used to treat gastrointestinal disor-
ders (Jamal et al., 2006). Although, commonly known with
their avoring properties, their antibacterial, and antifun-
gal properties have recently been of great interest. It has
been reported that the antimicrobial activity of essential
oil is generally due to phenolic and terpenoid compounds
as well as aliphatic compounds (Lv et al., 2011). Little in-
formation has been reported on the antioxidant activity of
aromatic oily liquid which can be obtained by steam distil-
lation from native cardamom seeds.
In conduction with the previous literature, the present work
was performed to investigate the anticipated hepatoprotec-
tive activity of cardamom oil as compared to silymarin as
standard drug against PCM-induced hepato-renal toxicity
in adult male albino rats. Such task was achieved through-
out the assessment of hematological, serum biochemical
and histopathological changes associated with the admin-
istration of PCM and/or the herbal extracts to albino rats.
MATERIALS AND METHODS
AnimAlS
Sixty-ve male Sprague Dawley rats weighing 180 ± 20 g
(12~14 weeks old), purchased from the Laboratory Ani-
mals Breeding Unit, Faculty of Veterinary medicine, Cairo
university were used. Rats were acclimatized for one week
before the beginning of the experimental study. e ani-
mals were housed in separate cages and fed balanced com-
mercial diet and oered water ad libitum. Experimental
protocol was in accordance with the guidelines approved
by the National Institute of Health for the Care and Use
of Laboratory Animals and were comforted by Ethics of
animal use in research Committee, Cairo University:
(Approval ID: CU/ ΙΙ/ F/ 72/ 18).
ChemiCAlS
Paracetamol powder was purchased from (Sigma pharma-
ceuticals, Egypt) and were applied in a dose of 500 mg/kg
(Bektur et al., 2013).
Silymarin was purchased from local market pharmacy
(SEDICO, Egypt), and applied in a dose of 500 mg/kg.
(Juma’a et al., 2009). All chemical and solvents were ana-
lytical authentic grade.
PlAnt mAteriAlS
Cardamom seeds were purchased from local market. e
seeds were identied and authenticated and the voucher
specimen of was deposited in the Herbarium of the de-
partment of ora, ministry of agriculture.
eSSentiAl oil extrACtion:
e essential oil was obtained by hydro distillation using
Clevenger apparatus for 2 hrs. e oils were dried over so-
dium sulfate and stored in an amber ask at 4ºC according
to Guenther (1961) and expressed as ml/100g, e extract-
ed essential oil was dehydrated over anhydrous sodium sul-
phate and stored at freezer till used for Gas Chromatogra-
phy-Mass Spectrometry (GC-MS) analysis.
GAS ChromAtoGrAPhy-mASS SPeCtrometry
AnAlySiS
e GC-MS analysis of the essential oil of cardamom
seeds was carried out using an instrument stands at the
Department of Medicinal and Aromatic Plants Research,
National Research Center with the following specica-
tions. Instrument: a TRACE GC Ultra Gas Chromato-
graphs (THERMO Scientic Corp., USA), coupled with
a THERMO mass spectrometer detector (ISQ Single
Quadruple Mass Spectrometer). e GC-MS system was
equipped with a TR-5 MS column (30 m x 0.32 mm i.d.,
0.25 μm lm thickness). Analyses were carried out using
helium as carrier gas at a ow rate of 1.0 mL/min and
a split ratio of 1:10 using the following temperature pro-
gram: 60oC for 1 min; rising at 4.0 oC/min to 240 oC
and held for 1 min. e injector and detector were held at
210oC. Diluted samples (1:10 hexane, v/v) of 1 μL of the
mixtures were always injected. Mass spectra were obtained
by electron ionization (EI) at 70 eV, using a spectral

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Table 1: e chemical composition of cardamom oil by GC-MS
No Compounds RT Peak Area (%)
1 L-Phellandrene 4.24 0.05
2α-Pinene 4.43 0.46
3 Sabinene 5.40 1.13
4 2-α-PINENE 5.56 0.15
5α-Myrcene 5.81 0.59
6 2,3-Dehydro-1,8-Cineole 5.91 0.10
7 Octanal (CAS) 6.42 0.07
8α-Terpinene 6.68 0.32
9 D-Limonene 7.07 1.66
10 1,8-Cineole 7.22 27.10
11 α-Ocimene 7.62 0.12
12 λ-Terpinene 8.05 0.56
13 Trans Sabinene Hydrate 8.57 0.18
14 α-Terpinolene 8.98 0.38
15 Linalool 9.63 6.61
16 1-Terpineol 10.63 0.08
17 Linalyl Propionate 12.57 0.19
18 L-4-Terpineol 12.90 1.99
19 α-Terpineol 13.60 4.62
20 N-Octyl Acetate 14.11 0.05
21 Nerol (CAS) 14.77 0.06
22 Z-Citral 15.43 0.14
23 Linalyl Acetate 15.59 2.09
24 Geraniol 15.87 1.37
25 E-Citral 16.75 0.21
26 Ocimenyl Acetate 18.35 0.16
27 α-Terpinenyl acetate 19.82 47.40
28 Neryl Acetate 20.33 0.13
29 Geranyl Acetate 21.19 0.75
30 α-Terpinyl Propionate 23.34 0.12
31 Á-Selinene (CAS) 25.43 0.08
32 λ-Muurolene 26.42 0.08
33 Nerolidol 28.44 0.92
34 Trans-Farnesol 28.82 0.05
range of m/z 40-450 17. e identication of the chemical
constituents of the EO were deconvoluted using AMDIS
software (www.amdis.net) and identied by its retention
indices (relative to n-alkanes C8-C22), mass spectrum
matching to authentic standards (when available), and
Wiley spectral library collection and NIST library database.
Most of the compounds were identied using two dierent
analytical methods: (a) KI, Kovats indices in reference to
n-alkanes (C9-C22) (National Institute of Standards and
Technology); and (b) mass spectra (authentic chemicals,
Wiley spectral library collection and NSIT library).
exPerimentAl deSiGn
Rats were divided into six groups. Group I (control nega-
tive group): rats that fed only standard diet. Group II (con-
trol positive group) (n=15): rats treated with 500 mg/kg
bw. PCM orally. Group III (n=15): rats treated with sily-
marin+ PCM (500 mg/kg bw for both), Group IV (n=15)
rats treated with cardamoms+ PCM (100 mg/kg bw +500
mg/kg bw, respectively). Group V (n=5): rats treated with
silymarin (500 mg/kg bw). Group VI (n=5): rats treated
with cardamom (100 mg/kg bw.). All treatments were ad-
ministered orally for 14 days.

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Table 2: Eect of oral administration of cardamom oil on erythrogram parameters in paracetamol-induced hepatotox-
icity in rats.
Group Weeks RBCs x 106 /ul Hb g/dl PCV % MCV  MCH pg MCHC %
Control
1st 5.01±0.17a15.70±1.14a28.83±3.70a57.77±9.49a31.17±2.46a54.87±6.63a
2nd 5.04±0.18a15.60±1.12a28.01±3.40a55.19±8.33a31.02±2.53a54.23±6.78a
3rd 5.01±0.17a15.70±1.14a28.83±3.70a57.77±9.49a31.17±2.46a54.87±6.63a
Paracetamol
1st 3.97±0.26b12.60±0.82b22.27±2.02b56.07±1.62a31.70±0.44a54.67±1.50a
2nd 4.45±0.52a13.93±0.31a25.40±0.46a57.63±8.01a31.50±3.20a54.87±2.15a
3rd 4.96±0.15a14.97±0.75a28.83±0.75a58.10±0.66a30.17±1.29a51.90±1.65a
Paracetamol +
silymarin 1st 4.99±0.09a15.60±0.75a27.47±1.24a55.03±1.64a31.27±2.06a56.93±4.96a
2nd 5.03±0.11a15.60±0.92a27.93±1.31a55.50±2.19a30.97±1.30a55.93±4.40a
3rd 4.99±0.10a14.67±0.15a29.03±1.70a58.13±2.25a29.43±0.76a50.63±3.23a
Paracetamol +
cardamom
1st 5.01±0.11a15.63±0.74a27.30±0.46a54.50±2.08a31.20±1.85a57.27±1.97a
2nd 5.03±0.13a15.50±0.40a27.27±0.29a54.27±1.94a30.87±1.42a56.83±1.00a
3rd 5.07±0.08a16.00±0.26a32.37±0.40a63.83±0.40a31.57±0.12a49.43±0.25a
Values represent mean ± SD
Means with dierent superscripts (a and b) within the same column are signicantly dierent at P value ≤ 0.05
CliniCoPAtholoGiCAl exAminAtionS
Blood samples were collected weekly for 3 weeks from the
rst 4 groups (5 rats from each group) and after 2 weeks
from 5th and 6th groups. Blood samples were collected from
retro-orbital venous plexus of rats under light ether anes-
thesia. Each sample was separated into 2 portions. e 1st
portion was received on dipotassium EDTA for the stand-
ard hemogram (CBC) utilizing Animal Blood cell Coun-
ter (ABC Vet, France) according to Feldman et al. (2000).
e 2nd portion of blood was collected in plain tubes for
serum separation. Serum samples were analyzed for total
antioxidant capacity (TAC) (Koracevic et al., 2001), glu-
cose, total protein &albumin (Doumas and Biggs, 1972),
serum creatinine (Tabacco et al., 1979), blood urea (Fabiny
and Ertingshausen, 1971), as well as activities of aspartate
aminotransferase (AST) (Tietz, 1986)., and alanine ami-
notransferase (ALT) (Reitman and Frankel, 1957). All the
biochemical analyses of serum were estimated spectropho-
tometrically utilizing commercial test kits supplied by Stan
Bio Laboratories incorporation, USA.
hiStoPAtholoGiCAl exAminAtionS
Liver and kidneys specimens from all groups (3 rats from
each group) at last week of experiment were collected and
xed in neutral buered formalin 10%, routinely processed
and embedded in paran wax. Paran blocks were sec-
tioned at 4-5 μm thickness and stained with Hematoxylin
and Eosin (Bancroft and Stevens, 2008) for histopatho-
logical examination by light microscope (Olympus BX50,
Japan).
StAtiStiCAl AnAlySiS
Data are expressed as mean ± SD. Variables were statisti-
cally analyzed by one-way ANOVA followed by a Tukey
post hoc test, was used to compare multiple groups, and all
comparisons were signicant when p ≤ 0.05 using software
COSTAT (version 6.400, Cohort software, USA).
RESULTS
ChemiCAl ComPoSitionS of CArdAmom eSSentiAl
oil
e chemical composition of cardamom essential oil was
analyzed by GC-MS showed that the monoterpene hy-
drocarbon, was present with relatively low concentrations
but the oil was rich in monoterpenoids. e main constit-
uents of the oil were α-terpinenyl acetate (47.40%), 1,8-ci-
neole (29.2%), α-terpineol (4.62%) linalyl acetate (2.09%)
and D-limonene (1.66%) in cardamom (Table 1).
hemoGrAm reSultS
Data of erythrogram and leucogram in PCM-intoxicated
are illustrated in Tables (2 & 3) respectively, all the he-
matological parameters were found to be not signicantly
dierent from control group. Paracetamol induced nor-
mocytic normochromic anemia during the 1st week of ad-
ministration. Moreover, neither cardamom nor silymarin
in normal rats induce any alterations in erythrogram and
leucogram in normal rats (Table 4).
bioChemiCAl reSultS
Assessment of changes in protein prole, activities of
ALT, AST, glucose in addition to some renal biomarkers
(urea and creatinine) are presented in Table 5. Results of
PCM-intoxicated rats revealed signicant decrease in to-
tal protein and albumin in with signicant hyperglycemia
and elevation in serum transaminases urea and creatinine
which were signicantly ameliorated in groups receiving

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Table 3: Eect of oral administration of cardamom oil on leucogram parameters in paracetamol-induced hepatotoxicity
in rats.
Group Weeks TLCx 103 /µL Neutrophil % Lymphocyte% Monocyte % Eosinophil %
Control
1st 9.27±1.01a36.67±10.26a54.00±6.00a4.67±2.31a4.67±2.31a
2nd 9.21±1.02a36.14±10.47a53.00±6.00a4.61±2.34a4.61±2.33a
3rd 9.27±1.01a36.67±10.26a54.00±6.00a4.67±2.31a4.67±2.31a
Paracetamol
1st 8.93±0.47a37.33±4.62a56.67±2.08a3.67±1.53a2.33±1.53a
2nd 9.43±0.57a36.00±5.29a54.33±5.51a6.00±0.00a3.67±1.53a
3rd 9.20±0.50a35.33±6.11a54.33±5.51a6.00±0.00a4.33±1.53a
Paracetamol +
silymarin 1st 8.77±0.40a34.00±2.00a57.33±2.08a5.67±0.58a3.33±0.58a
2nd 9.20±0.44a33.33±4.16a59.33±0.58a4.33±2.08a3.00±1.73a
3rd 8.90±0.44a36.00±2.00a52.67±2.08a6.00±0.00a5.33±0.58a
Paracetamol +
cardamom 1st 8.63±8.90a36.00±36.00a58.00±56.50a3.33±4.33a2.67±3.25a
2nd 8.73±9.16a40.00±36.50a52.67±55.08a5.33±5.08a2.00±3.33a
3rd 9.20±9.14a36.67±36.17a55.00±54.00a5.33±5.50a3.00±4.33a
Values represent mean ± SD
Means with dierent superscripts (a and b) within the same column are signicantly dierent at P value ≤ 0.05
Table 4: Eect of oral administration of cardamom oil and silymarin on Complete Blood Count in normal rats
Group RBCs
x 106
/ul
Hb
g/dl PCV
%MCV
MCH
pg MCHC
%TLC
x
103 /μL
Neutrophil
%Lympho-
cyte % Mono-
cyte % Eosino-
phil %
Control 5.01
±0.17a
15.7
±1.14a
28.83
±3.70a
57.77
±9.49a
31.17
±2.46a
54.87
±6.63a
9.27
±1.01a
36.67
±10.26a
54.00
±6.00a
4.67
±2.31a
4.67
±2.31a
Silymarin 5.07
±0.15a
14.53
±0.35 a
28.30
±1.31a
55.87
±1.04a
28.70
±0.70a
51.43
±1.66a
9.47
±0.57a
31.33
±5.03a
60.00
±3.00a
5.67
±0.58a
3.00
±1.73a
Cardamom 5.10
±0.20a
14.83
±0.40a
29.53
±2.65a
57.80
±2.90a
29.10
±0.36a
50.40
±3.15a
9.13
±0.25a
32.67
±3.06a
56.33
±2.08a
6.00
±0.00a
5.00
±1.00a
Values represents means ± SD
Means with dierent letters within the same column are signicantly dierent p ≤ 0.05
Table 5: Eect of oral administration of cardamom oil on some biochemical parameters in paracetamol-induced
hepatotoxicity in rats.
Group Weeks Total pro-
tein (g/dl) Albumin
(g/d) Globulin
g/dl) A/G
Ratio ALT
(U/l) AST
(U/l) Glucose
(mg/dl) Urea
(mg/dl) Creatinine
(mg/dl)
Control
1st 8.43
±0.35a
3.33
±0.21a
5.10
±0.56a
0.66
±0.11a
39.40
±4.51d
138.90
±1.39c
94.60
±1.59bc
39.63
±1.10d
0.42
±0.03b
2nd 7.17
±0.35a
3.17
±0.15a
4.00
±0.35a
0.80
±0.09a
73.20
±5.37b
186.83
±5.08b
80.90
±2.72b
42.67
±1.29b
0.65
±0.01b
3rd 7.17
±0.35a
3.17
±0.15a
4.00
±0.35a
0.80
±0.09a
73.20
±5.37b
186.83
±5.08b
80.90
±2.72b
42.67
±1.29b
0.42
±0.03b
Paracetamol
1st 7.43
±0.35b
2.73
±0.21b
4.70
±0.26a
0.66
±0.11a
103.73
±1.10a
236.95
±3.6a
142.13
±13.70a
59.73
±1.99a
0.50
±0.03a
2nd 6.43
±0.21b
2.40
±0.20b
4.37
±0.57a
0.8 0
±0.09a
95.37
±6.70a
262.40
±0.69a
104.77
±9.47a
49.73
±1.31a
0.73
±0.05a
3rd 7.17
±0.15a
3.13
±0.06a
4.03
±0.12a
0.80
±0.09a
71.87
±1.54a
162.67
±8.50a
100.57
±4.45a
45.63
±1.16b
0.45
±0.01a
Paracetamol
+
silymarin
1st 8.20
±0.53ab
3.33
±0.15a
4.87
±0.67a
0.70
±0.13a
80.77
±1.33b
170.70
±5.00b
109.80
±6.24b
49.20
±0.56b
0.49
±0.02a
2nd 7.23
±0.21a
3.27
±0.06a
3.97
±0.23a
0.82
±0.06a
84.73
±4.21ab
168.03
±3.69c
93.63
±3.20ab
38.80
±2.82c
0.58
±0.02c

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3rd 7.10
±0.85a
3.07
±0.12a
4.03
±0.87a
0.78
±0.16a
58.27
±1.63b
143.67
±1.53b
104.90
±3.80a
48.27
±1.57a
0.45
±0.02a
Paracetamol
+
cardamom
1st 8.23
±0.23ab
3.23
±0.21ab
5.00
±0.17a
0.65
±0.05a
55.70
±3.12c
134.47
±4.15c
82.47
±6.57c
46.30
±0.95c
0.41
±0.04b
2nd 6.90
±0.10ab
3.23
±0.15a
3.60
±0.10a
0.90
±0.06a
87.83
±5.95ab
167.03
±2.19c
88.00
±1.39b
44.73
±0.81b
0.56
±0.05c
3rd 7.20
±0.30a
3.30
±0.10a
3.90
±0.36a
0.85
±0.10a
67.53
±3.58a
130.77
±4.20c
90.97
±6.18a
44.93
±0.75bc
0.46
±0.05a
Values represent mean ± SD
Means with dierent superscripts (a and b) within the same column are signicantly dierent at P value ≤ 0.05
cardamom oil or silymarin at p ≤ 0.05.
Paracetamol treated rats showed significant decrease in
TAC compared to control, in the meantime cardamom
and silymarin dosages significantly elevate total antioxi-
dant capacity at p ≤ 0.05 (Table 6). e ameliorative eects
of cardamom and silymarin against hepato-renal changes
in PCM-intoxicated rats was observed as their serum bio-
chemical parameters behaved rather similar to control neg-
ative group. Moreover, neither cardamom nor silymarin in
normal rats induce any alterations in serum biochemistry
and in TAC activity normal rats (Tables 7).
Table 6: Eect of oral administration of cardamom oil on
Total Antioxidant Capacity parameters in paracetamol-
induced hepatotoxicity in rats.
Group Weeks TAC (mmol/l)
Control 1st 1.87 ±0.06b
2nd 1.87 ±0.06b
3rd 1.38 ±0.02a
Paracetamol 1st 1.27 ±0.06c
2nd 1.37 ±0.06c
3rd 138 ±0.02a
Paracetamol + sily-
marin
1st 2.27 ±0.06a
2nd 2.00 ±0.00a
3rd 1.37 ±0.02a
Paracetamol +
cardamom
1st 2.17 ±0.06a
2nd 2.07 ±0.06a
3rd 1.36 ±0.03a
Values represent mean ± SD
Means with dierent superscripts (a and b) within the same
column are signicantly dierent at P value ≤ 0.05
hiStoPAtholoGiCAl findinGS
liver tiSSue
Liver of control rats revealed normal histological structure
of hepatic lobule from central vein surrounded by radiat-
ing hepatocytes forming hepatic cords (Fig. 1a). On the
contrary, liver of PCM treated rats revealed remarkable
histopathological alterations described by hepatocellular
vacuolization, sinusoidal leukocytosis (Fig. 1b) and hepa-
tocellular necrosis associated with inammatory cells in-
ltration (Fig. 1c). Moreover, Kuper cells activation, ap-
optosis of hepatocytes (Fig. 1d) and portal brosis (Fig.
1e) were also recorded in all examined sections. Marked
amelioration of the histopathological picture was noticed
in liver of rats co-treated with PCM+ silymarin, the exam-
ined sections showed no histopathological changes except
Kuper cells activation was noticed in some sections (Fig.
1f). Moreover, liver of rats treated with silymarin revealed
no histopathological alterations (Fig. 1g). Regression of
the histopathological lesions was noticed in liver of rats
co-treated with PCM + cardamom, as slight activation of
Kuper cells and binucleation of hepatocytes (Fig. 1h). No
histopathological alterations were recorded in liver of rats
treated with cardamom alone (Fig. 1i).
Figure 1: Liver of rat a) from control, normal group
showing the normal histological structure of hepatic lobule
from central vein surrounded by radiating hepatocytes.
b), c), d) and e) treated with paracetamol, b) showing
hepatocellular vacuolization (short arrow) and sinusoidal
leukocytosis (long arrow). c) showing focal hepatocellular
necrosis associated with inammatory cells inltration
(arrow). d) showing Kuper cells activation (short arrow)
and apoptosis of hepatocytes (long arrow). e) portal
brosis (arrow). f ) co-treated with paracetamol + silymarin
showing Kuper cells activation (arrow). g) treated with
silymarin showing no histopathological alterations. h).
co-treated with paracetamol + cardamom showing slight
activation of Kuper cells (short arrow) and binucleation
of hepatocytes (long arrow). i) treated with cardamom
showing no histopathological alterations. (H & E, scale
bar 25 um).

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2020 | Volume 8 | Special Issue 2 | Page 30
Table 7: Eect of oral administration of cardamom oil and silymarin on biochemical parameters in normal rats
Group TAC
(mmol/l)
Total
protein
(g/dl)
Albumin
(g/d)
Globulin
g/dl)
A/G
Ratio
ALT
(U/l)
AST
(U/l)
Glucose
(mg/dl)
Urea
(mg/
dl)
Creatinine
(mg/dl)
Control 1.87
±0.06b
7.17
±0.35a
3.17
±0.15a
4.00
±0.35a
0.80
±0.09a
73.20
±5.37a
186.83
±5.08a
80.90
±2.72a
42.67
±1.29a
0.65
±0.01a
Silymarin 2.06
±0.12a
7.03
±0.15a
3.17
±0.06a
3.87
±0.15a
0.82
±0.04a
75.20
±4.13a
195.27
±6.02a
85.01
±1.50a
46.43
±4.29 a
0.66
±0.03a
Cardamom 2.03
±0.06a
7.33
±0.68a
3.27
±0.06a
4.07
±0.67a
0.82
±0.13a
78.23
±2.4a
194.67
±6.03a
84.67
±1.53a
48.4
±3.27a
0.67
±0.01a
Values represents means± SD
Means with dierent letters within the same column are signicantly dierent p ≤ 0.05
renAl tiSSue
Regarding kidneys, examined sections from control rats
revealed normalized histological structure of renal tissue
which consists from renal cortex and medulla (Fig. 2a).
Meanwhile, kidneys of rats treated with PCM showed var-
ia variable histopathological alterations which manifested
by vacuolar degeneration of renal tubular epithelium, slight
atrophy of glomerular tufts associated with distension of
Bowman’s space, thickening of the parietal layer of Bow-
man’s capsule and focal inammatory cells inltration (Fig.
2b). However, kidneys of rats co-treated with PCM + si-
lymarin showed restored histological structure and appar-
ent normal renal tissue (Fig. 2c). Moreover, kidneys of rats
treated with silymarin revealed no histopathological alter-
ations (Fig. 2d). On the other hand, improved histopatho-
logical picture was noticed in kidneys of rats co-treated
with PCM + cardamom. Slight vacuolization of epithelial
lining some renal tubules was the only histopathological
nding observed in sections from this group (Fig. 2e). In
addition, kidneys of rats treated with cardamom revealed
no histopathological alterations (Fig. 2f).
Figure 2: Kidney of rat a) from control, normal group
showing the normal histological structure of renal
parenchyma from renal cortex and medulla. b) Treated with
paracetamol showing thickening of the parietal layer of
Bowman’s capsule (short arrow) and focal inammatory cells
inltration (long arrow). c) Co-treated with paracetamol+
silymarin showing apparent normal renal tissue. d) Treated
with silymarin showing no histopathological alterations. e)
co-treated with paracetamol + cardamom showing slight
vacuolization of epithelial lining some renal tubules (arrow).
f) Treated with cardamom showing no histopathological
alterations. (H & E, scale bar 25 um).
DISCUSSION
Drugs are the major cause of liver failure that increasing
every year (Lakshmi et al., 2018). Hepato-renal dysfunc-
tion is considered one of the major inevitable drugs com-
plications (Cepa et al., 2018). Paracetamol is one of the
most popular pain killer (analgesic) that used as an alter-
native for NSAIDs, which is commonly used without a
prescription especially in children (Soha, 2017). It’s usu-
ally considered safe when used in its therapeutic dose, but
overdose is justly common and frequently associated with
hepatic and renal injury in both humans and experimental
animals (Samuel et al., 2015). e present study attempt-
ed to explore the hepatoprotective activity of cardamom
(Elettaria cardamomum) compared to silymarin against
PCM-induced hepato-renal toxicity in adult male albino
rats via the assessment of hematological, serum biochem-
ical and histopathological changes associated with the ad-
ministration of PCM and/or the herbal extracts to albino
rats.
e main constituents of the cardamom oil were α-terpi-
nenyl acetate (47.40%), 1,8-cineole (29.2%), α-terpineol
(4.62%) linalyl acetate (2.09%) and D-limonene (1.66%)
in cardamom. e results of GC-MS analysis indicated
that chemical composition proles obtained for cardamom
oil were very similar to the previous results of dierent
researchers with minor dierences (Savan and Kucukbay,
2013). ese ve constituents of the essential oil possessed
variable antioxidant activity when tested in the DPPH as-
say for non-specic hydrogen atom or electron donating
activity (Mutlu-Ingok and Karbancioglu-Guler, 2017).
Our data of present study strongly suggest that oral ad-
ministration of cardamom essential oil might protect the

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Advances in Animal and Veterinary Sciences
2020 | Volume 8 | Special Issue 2 | Page 31
liver against toxicity induced by PCM (500 mg/kg, p.o.) in
male Sprague Dawley rats. e hepatotoxicity induced by
PCM manifested biochemically by signicant elevation of
serum transaminases enzyme activities, urea and creatinine
concentrations and decrease in TAC level.
Regarding the hemogram, PCM toxicity revealed nor-
mocytic normochromic anemia during the 1st week which
may be related to destruction of mature RBC, reduction in
the rate of erythropoiesis or inhibition of erythropoietin
released from the kidney (Samuel et al., 2015).
Serum biochemical results illustrated that the daily dosage
of PCM (500 mg/kg) for two weeks caused elevations of
serum liver enzymes (ALT & AST) and signicant de-
crease in total protein, albumin in comparison to control
group. ese results are in agreement with the previous
experiments (Fatemi et al., 2010; Dadkhah et al., 2015).
Damaged structural integrity of the liver by PCM leads
to release of cytoplasmic enzymes or to changes in hepat-
ocyte membrane permeability and/or increased synthesis
or decreased catabolism of aminotransferases (Lebda et al.,
2013). Our results are conrmed by the histopathological
ndings which revealed remarkable hepatocellular vacuoli-
zation, sinusoidal leukocytosis and hepatocellular necrosis
associated with inammatory cells inltration. Moreover,
Kuper cells activation, apoptosis of hepatocytes (Fig. 1d)
and portal brosis were also recorded in all examined sec-
tions of liver in PCM treated rats. ese alterations may be
due to formation of large quantity of NAPQI, the PCM
metabolites that promotes histopathological alteration of
hepatocytes. ese results were consistent with that report-
ed by Hinson et al. (2010); Hamza and Al-Harbi, (2015).
Concerning protein prole our statistical analysis revealed
signicant decrease in both total protein and albumin in
PCM treated rats, this is agreed with Lebda et al. (2013)
and Hamza and Al-Harbi, (2015). ey ascribed this to
the decline in the number of cells responsible for albumin
synthesis in the liver through necrosis. On the other hand,
signicant hyperglycemia was noticed in rats treated with
acetaminophen. is is may be related to hepatotoxicity,
or due to direct eect of PCM toxic metabolites on pan-
creatic beta cell ultrastructure or due to body response to
stress enzymes (MacFie et al., 2009). Regarding TAC re-
sults PCM overdose leads to decreased TAC which may
resulted from generation for more NAPQI, which is con-
sequently adducted with mitochondrial proteins, leading
to ROS formation and oxidant stress (Du et al., 2016;
Ahmed et al., 2019). However, treatment with cardamom
and silymarin improved TAC capacity which may be due
to the ameliorating eect of both silymarin and cardamom
as both have the antioxidant eect (Madrigal-Santillán et
al., 2014; Aboubakr and Abdelazem, 2016; Ahmed et al.,
2019).
Rats treated with PCM showed signicant increase in se-
rum urea and creatinine comparable to control group and
this is supported by histopathological picture of the kid-
neys that showed vacuolar degeneration of renal tubular
epithelium, slight atrophy of glomerular tufts associated
with distension of Bowman’s space, thickening of the pa-
rietal layer of Bowman’s capsule and focal inammatory
cells inltration.
is may be due to nephrotoxicity that results from for-
mation of reactive oxygen species and increased oxidative
stress due to depletion of GSH levels (Canayakin et al.,
2016). On the other hand, the administration of carda-
mom ameliorated PCM induced acute liver and kidney in-
juries in rats, as evidenced by histologic, hematologic and
biochemical ndings. Similar protective eects were also
observed in rats receiving silymarin. e protective eects
of cardamom and silymarin in the restoration of normal
liver and kidney functions is conrmed by histopatholog-
ical ndings which revealed that the examined liver sec-
tions of rats co-treated with PCM + silymarin, showed no
histopathological changes except Kuper cells activation
was noticed in some sections. Regression of the histo-
pathological lesions was noticed in liver of rats co-treated
with PCM + cardamom, mild changes were observed as
slight activation of Kuper cells and binucleation of hepat-
ocytes. ese eects could be attributed to protection of
membrane integrity which may be due to the antioxidant
potential of cardamom and silymarin (El-Segaey et al.,
2007, Khare et al., 2012, Aboubakr and Abdelazem, 2016;
Ahmed, et al. 2019). Moreover, kidneys of rats co-treated
with PCM + silymarin showed restored histological struc-
ture and apparent normal renal tissue. On the other hand,
improved histopathological picture was noticed in kidneys
of rats co-treated with PCM + cardamom. Slight vacuoli-
zation of epithelial lining some renal tubules was the only
histopathological nding observed in sections from this
group. ese eects could be attributed to improvement
on glomerular function of kidney and maintaining posi-
tive nitrogen balance in addition to its antioxidant and free
radical scavenging properties (Bektur et al., 2013; Elkomy
et al., 2016).
CONCLUSION
e present research demonstrated that cardamom es-
sential oil diminished acute PCM-mediated hepatorenal
damage by inducing in vivo antioxidant activity and ame-
liorating the histopathological changes as well as biochem-
ical and hematological parameters in rats. It is conclusive
to use cardamom oil as a protective drug against oxida-
tive stress mediated toxicities in future. Further studies

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2020 | Volume 8 | Special Issue 2 | Page 32
are needed to explore the exact mechanism of antioxidant
properties of this promising oil.
ACKNOWLEDGEMENTS
e authors express sincere thanks to Dr Khaled Amer the
major general of Egypt Center for Research and Regenera-
tive Medicine, Military Forces, Cairo, Egypt. Many thanks
to departments of Clinical Pathology, Pharmacology, and
Pathology Faculty of Veterinary Medicine, Cairo Univer-
sity, and National Research Centre, Dokki, Giza, Egypt.
CONFLICT OF INTEREST
e authors declare no conicts of interest.
AUTHORS CONTRIBUTION
All authors contributed equally to the manuscript.
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