Anti-Inflammatory Property of Plantago major Leaf Extract Reduces the Inflammatory Reaction in Experimental Acetaminophen-Induced Liver Injury

Abstract

Hepatic injury induces inflammatory process and cell necrosis.
Plantago major
is traditionally used for various diseases. This study aimed to determine the anti-inflammatory property of
P. major
leaf extracts on inflammatory reaction following acetaminophen (APAP) hepatotoxicity. Thirty male Sprague-Dawley rats were divided into 5 groups, namely, normal control (C), APAP, aqueous (APAP + AQ), methanol (APAP + MT), and ethanol (APAP + ET) extract treated groups. All APAP groups received oral APAP (2 g/kg) at day 0. Then, 1000 mg/kg dose of
P. major
extracts was given for six days. The levels of liver transaminases were measured at day 1 and day 7 after APAP induction. At day 7, the blood and liver tissue were collected to determine plasma cytokines and tissue 11
β
-HSD type 1 enzyme. The in vitro anti-inflammatory activities of methanol, ethanol, and aqueous extracts were 26.74 ± 1.6%, 21.69 ± 2.81%, and 12.23 ± 3.15%, respectively. The ALT and AST levels were significantly higher in the APAP groups at day 1 whereas the enzyme levels of all groups showed no significant difference at day 7. The extracts treatment significantly reduced the proinflammatory cytokine levels and significantly increased the 11
β
-HSD type 1 enzyme activity (

p
<
0.05

). In conclusion, the
P. major
extracts attenuate the inflammatory reaction following APAP-induced liver injury.

Full text

Research Article
Anti-Inflammatory Property of Plantago major
Leaf Extract Reduces the Inflammatory Reaction in
Experimental Acetaminophen-Induced Liver Injury
Farida Hussan,1Adila Sofea Mansor,2Siti Nazihahasma Hassan,2
Tg. Nurul Tasnim Tengku Nor Effendy Kamaruddin,2
Siti Balkis Budin,2and Faizah Othman1
1Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak,
Cheras, 56000 Kuala Lumpur, Malaysia
2Department of Biomedical Science, Faculty of Health Sciences, Universiti Kebangsaan Malaysia,
JalanRajaMudaAbdulAziz,50300KualaLumpur,Malaysia
Correspondence should be addressed to Farida Hussan; khinpapah@gmail.com
Received  April ; Revised  July ; Accepted  July 
Academic Editor: Sonia Piacente
Copyright ©  Farida Hussan 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.
Hepatic injury induces inammatory process and cell necrosis. Plantago major is traditionally used for various diseases. is study
aimed to determine the anti-inammatory property of P. major leaf extracts on inammatory reaction following acetaminophen
(APAP) hepatotoxicity. irty male Sprague-Dawley rats were divided into  groups, namely, normal control (C), APAP, aqueous
(APAP + AQ), methanol (APAP + MT), and ethanol (APAP + ET) extract treated groups. All APAP groups received oral APAP
( g/kg) at day . en,  mg/kg dose of P. major extracts was given for six days. e levels of liver transaminases were measured
at day  and day  aer APAP induction. At day , the blood and liver tissue were collected to determine plasma cytokines and
tissue 𝛽-HSD type  enzyme. e in vitro anti-inammatory activities of methanol, ethanol, and aqueous extracts were . ±
.%, . ±.%, and . ±.%, respectively. e ALT and AST levels were signicantly higher in the APAP groups at day 
whereas the enzyme levels of all groups showed no signicant dierence at day . e extracts treatment signicantly reduced the
proinammatory cytokine levels and signicantly increased the 𝛽-HSD type  enzyme activity (𝑝 < 0.05). In conclusion, the P.
major extracts attenuate the inammatory reaction following APAP-induced liver injury.
1. Introduction
Inammation is a body homeostasis in response to any
type of tissue injury. It is a complex phenomenon which
involves innate and adaptive immune responses. It initi-
ates leucocytes migration, compliment system stimulation,
macrophages activation, and cytokines production from
activated macrophages and neutrophils. Cytokines such as
tumour necrosis factor-𝛼(TNF-𝛼)andinterleukins(IL)play
important roles in recruitment of neutrophils and activation
of macrophages to accelerate tissue repair process.
Drug-induced liver injury causes acute liver failure [].
Acetaminophen (APAP) is one of the drugs which induce
liver damage []. APAP-induced liver injury is due to its
toxic metabolites N-acetyl-p-benzoquinone imine (NAPQI).
Excessive production of NAPQI in APAP toxicity leads to
glutathione (GSH) depletion, resulting in binding of the
NAPQItocellularproteinwhichtriggerscellinjury[]. e
liver injury develops within three to ve hours following
exposure of APAP toxic dose and reaches the peak at  hours
[]. Cell injury elicits inammatory reaction in the liver [].
𝛽-Hydroxysteroid dehydrogenase type  (𝛽-HSD type
) enzyme is a native enzyme in liver and interconverts
inactive glucocorticoids (cortisone) to active cortisol which
has anti-inammatory action []. Anti-inammatory action
of cortisol suppresses cellular immunity and potentiating of
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 347861, 7 pages
http://dx.doi.org/10.1155/2015/347861

 Evidence-Based Complementary and Alternative Medicine
humoral immunity []. erefore, the activity and expression
of 𝛽-HSD type  enzyme might involve in inammatory
reaction following hepatocytes injury.
Plantago major (P. m a j o r ) of Plantaginaceae family is
commonly known as broadleaf plantain []anditispop-
ular in traditional medicine for wound healing as well
as treating diseases related to skin, respiratory organs,
digestive organs, reproduction, circulation, cancer, infection,
and pain []. Besides the traditional applications, many
researches have been done to prove its medicinal properties
such as antiulcerogenic, anti-inammatory, and immune-
modulating activities and antioxidant, antiviral, and anti-
carcinogenic activities []. In Malaysia, P. m a j o r has been
used as a diuretic, tonic, and cough mixture []andto
treat urinary calculus [] and diabetes []. P. ma j o r leaves
possess numerous bioactive compounds such as avonoids,
terpenoids, pectin, iridoid glycosides, and tannins which
express anti-inammatory and antioxidant activities [–].
e present study was aimed at determining the eect of P.
major onthechangesinplasmacytokinessuchasTNF-𝛼,
IL-, IL-, and IL- following APAP-induced liver injury.
Furthermore, the changes of 𝛽-HSD type  enzyme in liver
tissuewouldbedeterminedandtheroleofthisenzyme
against inammatory reaction in response to liver injury
would be discussed.
2. Materials and Methods
2.1. Experimental Animals. irty male Sprague-Dawley rats
(– g) were obtained from the institutional animal
resource unit. e rats were reared in stainless steel cages
with a room temperature of 27 ± 2∘C with  hours light
and dark cycle. All rats were allowed to access food and tap
water ad libitum.Alltheanimalhandlingprocedureswere
in accordance with the ethical guideline with the approval
number UKMAEC: FP/ANAT//FARIDA/-SEPT./-
OCT.--SEPT.-.
2.2. Extract Preparation. e P. m a j o r plant is collected from
Cameron Highland, Malaysia. e leaves were plucked and
cleaned with tab water. Air-dried ground leaves powder of
P. m a j o r ( g for each solvent) was macerated in three
dierent solvents such as % methanol, % ethanol, and
deionized water, respectively. e solutions were kept for 
days in a dark room at 22±3∘C. e mixture was then ltered
and the supernatant was collected. e process was repeated
three times. e three batches of supernatant were mixed.
e aqueous extract was freeze-dried whereas the methanol
and ethanol extracts were concentrated in rotary evaporator
at –∘C.en,theconcentrateduidwaslaterfreeze-
dried into powder. e extract powder was then sent to the
Forest Research Institute of Malaysia (FRIM) to evaluate anti-
inammatory activity with lipoxygenase assays.
2.3. Lipoxygenase Assay. is assay was done according
to the method developed by Malik et al. []withslight
modication. Soybean lipoxygenase (...) type I-B and
linoleic acid were purchased from Sigma (St. Louis, MO,
USA). In assay protocol,  𝜇L of  mM sodium phosphate
buer (pH .),  𝜇L of test-compound solution, and  𝜇L
of lipoxidase enzyme solution were mixed and incubated for
 min at ∘C. e reaction was then initiated by the addition
of  𝜇Llinoleicacid(substrate)solution,withtheformation
of (Z, E)-(S)--hydroperoxyoctadeca-,-dienoate and
thechangeinabsorbancewasmeasuredatnm.Test
compounds and the positive control were dissolved in DMSO.
All the reactions were performed in triplicate in -well
microplate in Tecan Innite M Microplate Reader (Tecan,
Austria). e IC values were then calculated using the
GraphPad Prism Analysis.
2.4. Experimental Design. e rats were acclimatised for one
week prior to administering any test agents and then divided
into two groups. Normal control group (C, 𝑛=6)received
oral .% normal saline (NS) throughout the experiment
whereasAPAPgroup(𝑛=24) which was subdivided into
APAP, APAP + AQ, APAP + MT, and APAP + ET received
oral APAP ( g/kg). e subgroups received .% NS and
aqueous, methanol, and ethanol extracts, respectively, for 
days. e extract dose ( mg/kg) was chosen based on our
preliminary study. At day  which was  hours aer the APAP
induction, the blood was collected from the retroorbital space
to determine the plasma liver enzyme. At day , the rats
wereanaesthetisedandthebloodwascollectedviacardiac
puncture. en, the rats were sacriced and the liver was
harvested.
2.5. Biochemical Analysis. e collected blood was put in
EDTA tube and centrifuged at  ×gin
∘Cforminutes.
e plasma was collected and stored at −∘Cuntilfurther
analysis. Liver enzyme levels at days  and  were determined
by semiautomatic method, using Bioanalyzer, semiautomatic
BTS-, BioSystems S.A., Spain. e plasma cytokine levels
at day  were tested using ProcartaPlex cytokine assay kits
(eBioscience, USA). Liver tissue was rinsed with phosphate
buer saline (PBS) and prepared for tissue homogenisa-
tion and immunostaining. e activity of 𝛽-HSD type 
enzyme was determined in liver tissue using enzyme-linked
immunosorbent assay (ELISA) kit (Uscn, USA).
2.6. Immunohistochemical Staining. e formalin xed liver
tissue was incubated in pH  citrate buers at ∘Cfor
 minutes and .% hydrogen peroxidase for  min-
utes at room temperature. e primary antibody used was
rMR--D and anti-rabbit IgG was used as a secondary
antibody. e presence of 𝛽-HSD type  enzyme was
detected using the streptavidin-peroxidase system and ,󸀠-
diaminobenzidine (DAB). Lastly, it was counterstained with
Harris haematoxylin.
2.7. Statistical Analysis. e data was presented as mean ±
standard error of mean (SEM). e normally distributed
data were analyzed using parametric analysis of variance
(ANOVA) test. e data that were not normally distributed
were analyzed using nonparametric tests, Mann-Whitney 𝑈
test. e signicant value was set as 𝑝 < 0.05. All mentioned

Evidence-Based Complementary and Alternative Medicine 
0
0.5
1
1.5
2
2.5
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
IL-1𝛼 (pg/mL)
∗
∗
∗
∗
(a)
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
∗∗
0
0.2
0.4
0.6
0.8
1
1.2
1.4
IL-1𝛽 (pg/mL)
(b)
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
∗∗∗
0
2
4
6
8
10
12
14
16
18
TNF-𝛼(pg/mL)
(c)
F : Eects of three dierent P. major extracts on the level of proinammatory cytokines in APAP-induced rats: (a) IL-𝛼, (b) IL-𝛽,and
(c) TNF-𝛼.∗Signicant dierence from the APAP group (𝑝 < 0.05).
statistical analyses were conducted using Statistical Product
and Service Solutions (SPSS) soware, version .
3. Results
e lipoxygenase inhibition of the extracts was found to
be . ±.%, . ±.%, and . ±.% in the
methanol, ethanol, and aqueous extracts, respectively. e
results were presented as mean ±SEM (% inhibition).
e standard, nonselective lipoxygenase inhibitor, nordihy-
droguaiaretic acid (NDGA), showed . ±.% inhibition
at the nal concentration of  𝜇g/mL. e methanol and
ethanol extract showed higher antilipoxygenase activity than
the aqueous extract.
3.1. Changes in Liver Enzymes. e liver enzymes such as ala-
nine aminotransferase (ALT) and aspartate aminotransferase
(AST) were signicantly increased in APAP groups compared
tothecontrolgroupatdaywhichwashoursaerthe
APAP ( g/kg) induction. e liver enzyme levels in all the
APAP groups at day  were signicantly reduced compared
today(𝑝 < 0.05). At day , there was no signicant change
of AST and ALT levels in all groups except the aqueous extract
treated group which revealed signicant lower ALT level than
the APAP group of the same day (𝑝 < 0.05). e results were
shown in Table .
3.2. Changes of Inammatory Cytokines. e mean levels of
IL-𝛼,IL-𝛽,andTNF-𝛼in all groups at day  were shown in
Figure .eIL-𝛼level in the APAP group was signicantly
higher than that of the control group (𝑝 < 0.05)(Figure(a)).
However, there was no signicant dierence in IL-𝛽and
TNF-𝛼between the control and the APAP group (Figures
(b) and (c)). e level of IL-𝛼,IL-𝛽,andTNF-𝛼in the
APAP group was signicantly higher compared to the treated
groups (𝑝 < 0.05)whichindicatedthatP. m a j o r treatment
signicantly reduced the proinammatory cytokines. e
mean levels of IL- and IL- in all groups were shown in
Figure . e levels of IL- and IL- in all groups showed
no signicant dierence (𝑝 > 0.05).
3.3. Changes of 11𝛽-HSD Type 1 Enzyme Activity. e activity
of 𝛽-HSD type  enzyme in all groups was shown in Figure .

 Evidence-Based Complementary and Alternative Medicine
T : Eects of three dierent P. major leaf extracts on aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes at
dayanddayaerAPAPinduction.
Groups AST at day 
U/L
AST at day 
U/L
ALT at day 
U/L
ALT at day 
U/L
Control . ±. . ±. . ±. . ±.
APAP . ±.a. ±.c. ±.a. ±.c
APAP + AQ . ±.a. ±.c. ±.a. ±.b
APAP + MT . ±.a. ±.c. ±.a. ±.c
APAP + ET . ±.a. ±.c. ±.a. ±.c
aSignicant dierence from the control group of the same day (𝑝 < 0.05).
bSignicant dierence from the APAP group of the same day (𝑝 < 0.05).
cSignicant dierence between the same group of day  and day  (𝑝 < 0.05).
0
0.5
1
1.5
2
2.5
IL-6 (pg/mL)
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
(a)
0
10
20
30
40
50
60
70
80
IL-10 (pg/mL)
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
(b)
F : Eects of three dierent P. major extracts on the level of anti-inammatory cytokines in APAP-induced rats: (a) IL- and (b) IL-
(no signicant dierence 𝑝 > 0.05).
0
50
100
150
200
250
300
11𝛽-HSD type 1activity (ng/mL)
Control
APAP
APAP + AQ
APAP + MT
APAP + ET
∗∗
F : Eects of three dierent P. major extracts on the activity
of 𝛽-HSD type  enzyme in APAP-induced liver tissue; ∗signicant
dierence from the APAP group (𝑝 < 0.05).
e enzyme activity between the control and APAP groups
showed no signicant dierence whereas the activity was
signicantly reduced in APAP group compared to the APAP
+ MT and APAP + ET groups (𝑝 < 0.05). erefore,
the methanol and ethanol extract were able to enhance the
enzyme activity following APAP-induced liver injury.
3.4. 11𝛽-HSD Type 1 Enzyme Expression. Figure showed
the expression of 𝛽-HSD type  enzyme in liver tissue. e
immunohistochemical staining of 𝛽-HSD type  enzyme
was used to determine the expression of enzyme. ere
was no expression of 𝛽-HSD type  in the APAP group
(Figure (b)). However, the greater intensity of expression
was found around the central vein area in both APAP + MT
and APAP + ET groups (Figures (d) and (e)).
4. Discussion
is study was aimed at determining the eect of P. m a j o r
extract treatment on inammatory reaction following APAP
toxicity. e APAP dose in the present study was proven to
produce liver toxicity in rats which showed increased liver
enzyme levels and liver necrosis []. e treatment dose of
 mg/kg of P. m a j o r extract was based on our preliminary
results.euseofhighdose(mg/kg)ofP. m a j o r showed
no toxic eect []. e minimum eective dose of this plant
is  mg/kg [].

Evidence-Based Complementary and Alternative Medicine 
(a) (b)
(c) (d)
(e)
F : 𝛽-HSD type  enzyme expression in liver tissue (×; immunohistochemical stain): (a) control; (b) APAP; (c) APAP + AQ; (d)
APAP + MT; and (e) APAP + ET.
e in vivo anti-inammatory activity of P. m a j o r has
been well described []. e anti-inammatory activity
of the extracts was determined using lipoxygenase assay.
Lipoxygenase enzyme catalyses arachidonic acid to pro-
duce leukotrienes. Leukotrienes play role in inammatory
diseases. e plant extracts or the phytochemicals which
possess inhibitory eect on this enzyme have the potential
to be used in inammatory condition []. In the present
study, the highest anti-inammatory activity was found in the
methanol extract followed by the ethanol extract. e result
was similar to the nding of Beara et al. []. It is stated that its
anti-inammatory activity is contributed by avonoids such
as baicalein and hispidulin and iridoid glycosides such as
aucubin [].
Inammation is a body response to remove tissue debris
and to initiate tissue regeneration following injury. Per-
sistence inammatory reaction exaggerates tissue damage,
resulting in improper tissue repair process. Inammatory
reaction is initiated by the release of proinammatory
cytokines such as IL-, IL-, and TNF-𝛼[]. e proin-
ammatory cytokine in the circulation is a signal to recruit
neutrophils and leucocytes which subsequently remove the
cellular debris to promote tissue regeneration []. IL-
also acts as an anti-inammatory cytokine by inhibiting
the secretion of TNF-𝛼and IL- []. Extensive tissue
damage can be prevented by the action of anti-inammatory
cytokines including IL- or suppression of proinammatory
cytokines.

 Evidence-Based Complementary and Alternative Medicine
In this present study, the proinammatory cytokine (IL-
𝛼,IL-𝛽,andTNF-𝛼) levels were signicantly high in
the APAP group compared to the treated groups. e IL-
andTNF-𝛼which are released by activated macrophages
stimulate leukocyte adhesion to endothelial surfaces prior to
migration into tissues. IL- oen expresses synergistic action
with TNF-𝛼to initiate cell death []. e TNF-𝛼and IL-
levels in the plasma reect the severity of inammation [].
e inhibition of TNF-𝛼and IL- exhibits partial preven-
tion of APAP toxicity, including reduction of liver enzyme
in circulation []. e results of the present study showed
the lower level of TNF-𝛼and IL- in the P. m a j o r treated
groups. e eect was more pronounced in the methanol
extract treated group which was in line with the in vitro
anti-inammatory activities of P. m a j o r . It indicated that
the anti-inammatory property of the P. m a j o r was able to
prevent the inammatory reaction following APAP toxicity.
It has been documented that the natural products with anti-
inammatory properties decreased the levels of proinam-
matory cytokines [].
𝛽-HSD type  is present in liver and its role is to
interconvert inactive glucocorticoid to the active form [,
].Cortisolinhumanorcorticosteroneinrodentisanactive
form of glucocorticoid which has anti-inammatory proper-
ties. Glucocorticoids express its anti- inammatory action by
suppressing the nuclear transcription factors AP- and NF-
𝜅B which induce genes expression of all proinammatory
cytokines []. In the present study, the P. m a j o r extract
treated groups showed increased activity and expression of
𝛽-HSDtypeenzymeespeciallyinthemethanoland
ethanol extract treated groups. erefore, attenuation of
inammatory reaction might be related to the local tissue
production of active glucocorticoid. Furthermore, Dinarello
[] stated that glucocorticoid increases the transcription
of anti-inammatory proteins such as IL- and the IL-
type -decoy receptor. However, in the present study, the
anti-inammatory cytokine (such as IL- and IL-) levels
were not changed signicantly among all groups. Based on
our results, the glucocorticoid action activated by 𝛽-HSD
type  has potential role in suppression of proinamma-
tory molecules, rather than promoting anti-inammatory
cytokines.
5. Conclusion
In conclusion, the leaf extracts of P. m a j o r have the potential
in attenuation of the inammatory response by reducing the
levels of proinammatory cytokines. e potential mech-
anism of reduction in inammatory reaction by the P.
major extractscouldbeduetoproductionoflocaltissue
glucocorticoid. erefore, methanol and ethanol extracts
of P. m a j o r havepotentialtobeusedasanalternative
or adjunct treatment to reduce inammation-mediated cell
injury following APAP toxicity.
Conflict of Interests
e authors declare there is no conict of interests regarding
the publication of this paper.
Acknowledgments
e authors would like to express their gratitude to Universiti
Kebangsaan Malaysia for providing the research funding
(Grant no. DLP--) and the UKM Research Ethics
Committee, Forest Research Institute Malaysia (FRIM), and
the sta of the Anatomy Department, Faculty of Medicine,
UKM.
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