Effect of Emilia sonchifolia (Linn.)DC on alcohol– induced oxidative stress in pancreas of male albino rats

Abstract

To explore the efficacy of n-hexane extract of Emilia sonchifolia (E. sonchifobia) against ethanol induced pancreatic dysfunction in the young Wistar albino rats.
The rats were divided into four groups. Control rats in group received distilled water orally, group received oral administration of 20% (w/v) ethanol dissolved in drinking water, group received oral administration of 20% (w/v) ethanol in distilled water+n-hexane extract of E. sonchifolia (250 mg/kg body weight), and group received oral administration of n-hexane extract of E. sonchifolia (250 mg/kg body weight) alone. Liver marker enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), pancreatic enzymatic antioxidants superoxide dismutase, lipid peroxidation, catalase, glutathione peroxidase, non-enzymatic antioxidants glutathione and vitamin C were measured and compared.
Administration of 20% ethanol for 16 weeks significantly increased the liver marker enzymes AST, ALT(P<0.05), reduced the pancreatic enzymatic antioxidants superoxide dismutase, lipid peroxidation, catalase, glutathione peroxidase, glutathione and vitamin C(P<0.05). Histopathological examination showed that the ethanol provoked the oxidative stress which was demonstrated as pancreatic necrosis and oedema. Simultaneous administration of n-hexane extract of E. sonchifolia (250 mg/kg body weight) protected the pancreas against the damage induced by ethanol which was confirmed by the histopathological studies and the normalization of biochemical parameters.
Thus n-hexane extract of E. sonchifolia shows a promise in therapeutic use in alcohol induced oxidative stress.

Full text

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Asian Pacific Journal of Tropical Medicine (2011)973-977
Document heading doi: 10.1016/S1995-7645(11)60229-0
Effect of Emilia sonchifolia (Linn.)DC on alcohol- induced oxidative stress
in pancreas of male albino rats
Dominic Sophia, Murugesan Gomathy, Thomas Shebin, Paramasivam Ragavendran, Chinthamony
Arulraj, Velliyur Kanniapan Gopalakrishnan*
Department of Biochemistry, Karpagam University, Coimbatore- 641 021 (T.N.), India
Contents lists available at ScienceDirect
Asian Pacific Journal of Tropical Medicine
journal homepage:www.elsevier.com/locate/apjtm
ART ICLE INFO ABSTRACT
Article history:
Received 1 August 2011
Received in revised form 11 September 2011
Accepted 15 October 2011
Available online 20 December 2011
Keywords:
Pancreas
Ethanol
Emilia sonchifolia
Antioxidants
Histopathology
*Corresponding author: Velliyur Kanniapan Gopalakrishnan, Department of
Biochemistry, Karpagam University, Coimbatore- 641 021 (T.N.), India.
Tel: 91-0422-6453777
Fax: 91-0422-2611043
E-mail: vkgopalakrishnan@gmail.com
1. Introduction
Alcoholism is a major health problem throughout the
world, regardless of racial, ethnic, and socioeconomic
factors[1]. Consumption of alcohol (ethanol) is a leading
cause of diseases and death worldwide. Ethanol exerts
profound effects on the endocrine and exocrine pancreas[2].
It has been reported that the pancreatic acinar cell can
metabolize ethanol as effectively as the liver[3]. The
pancreas can metabolize ethanol by means of oxidative
and nonoxidative pathways to generate metabolites, such
as acetaldehyde and fatty acid ethyl esters (FAEEs)[4].
The major oxidative enzyme system uses either alcohol
dehydrogenase or the cytochrome p450 system, whereas
the nonoxidative pathway uses the FAEE synthase enzyme
pathway. The metabolites generated by both oxidative and
nonoxidative pathways are injurious to both exocrine and
endocrine pancreas.
Alcohol has been linked to the causation of many human
disease states. One of the most appreciated and investigated
diseases is alcohol-induced pancreatitis[1]. Heavy alcohol
consumption is known to be a major cause of chronic
pancreatitis, which has been linked to malabsorption,
diabetes, and pancreatic cancer[5]. There is now compelling
experimental evidence in vitro and in vivo that FAEE are
toxic mediators in ethanol-induced organ injury[6].
Oxidative stress may result from exposure to a variety
of agents present in the environment. External sources of
reactive oxygen species (ROS) include radiation, UV light,
Objective: To explore the efficacy of n-hexane extract of Emilia sonchifolia (E. sonchifobia)
against ethanol induced pancreatic dysfunction in the young Wistar albino rats. Methods: The
rats were divided into four groups. Control rats in group栺received distilled water orally, group 栻
received oral administration of 20% (w/v) ethanol dissolved in drinking water, group 栿received
oral administration of 20% (w/v) ethanol in distilled water+n-hexane extract of E. sonchifolia
(250 mg/kg body weight), and group 桇 received oral administration of n-hexane extract of
E. sonchifolia (250 mg/kg body weight) alone. Liver marker enzymes aspartate aminotransferase
(AST), alanine aminotransferase (ALT), pancreatic enzymatic antioxidants superoxide dismutase,
lipid peroxidation, catalase, glutathione peroxidase, non-enzymatic antioxidants glutathione
and vitamin C were measured and compared. Results: Administration of 20% ethanol for
16 weeks significantly increased the liver marker enzymes AST, ALT(P<0.05), reduced the
pancreatic enzymatic antioxidants superoxide dismutase, lipid peroxidation, catalase, glutathione
peroxidase, glutathione and vitamin C(P<0.05). Histopathological examination showed that
the ethanol provoked the oxidative stress which was demonstrated as pancreatic necrosis and
oedema. Simultaneous administration of n-hexane extract of E. sonchifolia (250 mg/kg body
weight) protected the pancreas against the damage induced by ethanol which was confirmed by
the histopathological studies and the normalization of biochemical parameters. Conclusions:
Thus n-hexane extract of E. sonchifolia shows a promise in therapeutic use in alcohol induced
oxidative stress.

Dominic Sophia et al./Asian Pacific Journal of Tropical Medicine (2011)973-977
974
chemical reagents, pollution, cigarette smoke, drugs of
abuse, and ethanol[7]. Ethanol intake causes accumulation
of ROS. There are a number of findings associated with
the ethanol induce toxicity in which oxidative stress and
proinflammatory cytokine production thought to be the
leading putative etiological factor[8]. Oxidative stress can
act through a number of important mediators to produce
cell injury and death[9]. Ethanol can lead to an increase
in the production of ROS and/or a decrease in the levels
of antioxidant defenses causing a redox imbalance and
resulting in the oxidative damage of lipids, proteins,
and DNA. A complex system of antioxidant defenses has
evolved that generally holds the oxidative attack from ROS.
On occasions, however, this balance can be perturbed,
leading to oxidative stress[10]. Therefore, oxidative stress
can contribute, at least in part, to the damage observed in
pancreas[11].
Herbal alternatives are one of the best ways to minimize
these disease conditions[12]. The protective effect of fruit and
vegetable intake can provide certain phytonutrients, such
as isothiocyanates, polyphenols and flavonoids, to act as
antioxidants to counteract the by-products of the oxidative
metabolites from alcohol[13]. Emilia sonchifolia (Family:
Asteraceae) (E. sonchifolia) commonly known as lilac tassel
flower is a traditionally used medicinal plant found in most
tropical and subtropical regions worldwide. Various parts
of the plant are used for the treatment of diseases[14]. This
weed is used in ethnomedicine against inflammation, eye
sores, convulsion, cuts, wounds, rheumatism and insect
bites[15,16]. In addition, anticonvulsant activity of aqueous
extract has previously been reported[17]. Phytochemical
studies indicated that the aerials parts of E. sonchifolia
contain alkaloids and flavonoids and terpernes[18]. The
present study is to evaluate the potentiality of E. sonchifolia
on ethanol induced oxidative stress and to assess its effect
on pancreatic antioxidant status using Wistar rats as an
experimental model.
2. Materials and methods
2.1. Plant collection
E. sonchifolia was collected from Thrissur, Kerala, India.
The plant was authenticated by Dr. G.V.S Moorthy, Botanical
survey of India, TNAU Campus, Coimbatore. The voucher
number is BSI/SRC/5/23/09-10/Tech/782. The whole fresh
plant material was washed under running tap water, air
dried and powdered.
2.2. Preparation of extract
The powder soaked in n-hexane solvent was kept in
the shaker for 48 h at room temperature. The extract was
collected and concentrated at 40 ℃ under reduced pressure
using rotary evaporator. The dried extract was stored at 4 ℃
until further use. The remaining residue was extracted again
with the fresh solvent to ensure complete extraction.
2.3. Animals
The male Wistar rats weighing between 50-55 g were
obtained from Animal house of Karpagam University,
Coimbatore. The animals were caged in well ventilated
hygienic conditions. They were divided randomly into four
groups each containing six. The study was approved by
Institutional Animal Ethical Committee constituted for the
purpose of CPCSEA, Government of India.
2.4. Experimental design
Group Ⅰ: Control rats; Group Ⅱ: Rats administered with
5 mL of 20% (w/v) ethanol; Group Ⅲ: Rats administered with
5 mL of 20% (w/v) ethanol and were treated with n-hexane
extract of E. sonchifolia (250 mg/kg body weight); Group
Ⅳ: Rats administered with n-hexane extract of Emilia
sonchifolia alone (250 mg/kg body weight).
2.5. Liver function assays
After the experimental period the animals were sacrificed
under light chloroform anesthesia. The blood was drawn
from the p-orbital venous complexes and serum separation
tubes, allowed to clot for 30 min at room temperature and
then centrifuged at 1 000 × g for 10 min and stored at
4 ℃. Serum aspartate aminotransferase (AST) and alanine
aminotransferase (ALT)[19] were estimated within 6 h of
animal sacrifice.
2.6. Lipid peroxidation and antioxidant enzyme assays
The pancreas were excised immediately, cleaned free
of extraneous material and perfused with ice cold saline
(0.9%) and stored in 10% formalin, which are used for the
antioxidant and histopathological studies respectively.
2.7. Estimation of pancreatic lipid peroxidation
Lipid peroxidation (LPO)[20] was calculated on the basis of
the molar extinction coefficient of malondialdehyde (MDA)
and expressed in terms of nanomoles of MDA/mg protein.
2.8. Antioxidant assays
The enzymatic antioxidants such as superoxide
dismutase(SOD)[21], catalase(CAT)[22], glutathione
peroxidase(GPx)[23], and the non-enzymatic antioxidants
such as reduced glutathione[24], and vitamin C[25] were
evaluated in the tissue homogenates.
2.9. Statistical analysis
The results obtained were expressed as mean暲SD. The
statistical comparisons among the groups were performed

Dominic Sophia et al./Asian Pacific Journal of Tropical Medicine (2011)973-977 975
using one way analysis (ANOVA) (SPSS 10.0) at P<0.05 level.
3. Results
3.1. Liver marker enzymes
In the present study, serum AST and ALT levels has
increased significantly in group 栻 rats after the ethanol
treatment(P < 0.05). Treatment with the n-hexane extract of
E. sonchifolia (group 栿) was found significantly effective
in the normalization of these markers when compared
to ethanol treated group 栻 rats(P < 0.05). The n-hexane
extract of E. sonchifolia alone (group 桇) did not show any
significant difference as compared to control group rats
(Table 1).
3.2. Lipid peroxidation assay
The ethanol treated group II showed a significant increase
in pancreatic LPO level (P < 0.05). Treatment of rats (group
栿) with the n-hexane extract of E. sonchifolia showed
significant protective activity against the ethanol treated
group rats(P < 0.05) (Table 2). No significant difference was
found in the MDA level between control and only n-hexane
extract of E. sonchifolia.
3.3. Pancreatic enzymatic antioxidant assays
The levels of pancreatic enzymatic antioxidants SOD, CAT,
GPx were depleted significantlyin ethanol treated group rats
as compared to control group(P < 0.05). Administration of
n-hexane extract of Emilia sonchifolia increased the levels
of enzymatic antioxidants SOD, CAT, GPx significantly as
compared to ethanol treated group. The n-hexane extract
of Emilia sonchifolia alone treated group rats exhibited no
significant changes in SOD, CAT, GPx levels as compared to
control group (Table 2).
3.4. Pancreatic non- enzymatic antioxidant assays
Table 3 shows that the ethanol treatment significantly
increased the levels of pancreatic non-enzymatic
antioxidants glutathione (GSH) and vitamin C(P < 0.05). After
the treatment of n-hexane extract of Emilia sonchifolia,
GSH and vitamin C levels were increased significantly (P <
0.05) in compare to the ethanol treated group. There was no
significant change between the n-hexane extract of Emilia
sonchifolia alone treated group and control group rats.
3.5. Histopathological analysis
Histopathological study of the pancreas in the experimental
group Ⅱ animals showed the congestion of cells and mild
Table 1
Effect of n-hexane extract of E. sonchifolia on serum biochemical parameters against oxidative stress in albino rats (IU/L) (mean依SD, n=6).
Group ALT AST
I-Control 80.75依1.07 126.56依0.47
II-Ethanol induced 140.71依0.76殼262.33依0.82殼
III-Ethanol induced+ n-hexane extract treated 92.30依0.37* 135.68依1.16*
IV-n-hexane extract alone treated 80.70依0.59 126.47依0.75
殼P < 0.05 vs. control group; *P < 0.05 vs. ethanol induced group.
Table 2
Effect of n-hexane extract of E. sonchifolia on enzymatic antioxidants against oxidative stress in albino rats(mean依SD, n=6).
Group LPO SOD CAT GPx
I-Control 1.31依0.02 6.29依0.09 15.43依0.24 7.39依0.30
II-Ethanol induced 4.03依0.10殼 3.56依0.29殼 8.57依0.33殼 4.03依0.06殼
III-Ethanol induced+ n-hexane
extract treated
1.97依0.03* 5.26依0.06* 13.44依0.32* 6.28依0.11*
IV-n-hexane extract alone
treated
1.30依0.02 6.29依0.10 15.58依0.21 7.45依0.31
殼P < 0.05 vs. control group; *P < 0.05 vs. ethanol induced group.
Units- LPO-nM/mg protein; SOD-Inhibition of 50% nitrite formation/min/mg protein; CAT-毺 moles of H2O2 consumed/mg protein; GPx-毺
moles of glutathione utilized/min/mg protein.
Table 3
Effect of n-hexane extract of E. sonchifolia on non-enzymatic antioxidants against oxidative stress in albino rats (mean依SD, n=6).
Group GSH Vitamin C
I-Control 12.46依0.24 1.41依0.06
II-Ethanol induced 7.61依0.28殼 0.66依0.06殼
III-Ethanol induced+ n-hexane extract treated 11.17依0.17* 1.17依0.04*
IV-n-hexane extract alone treated 12.35依0.26 1.46依0.07
殼P < 0.05 vs. control group; *P < 0.05 vs. ethanol induced group. Units: GSH, Vitamin C- 毺g/mg protein.

Dominic Sophia et al./Asian Pacific Journal of Tropical Medicine (2011)973-977
976
lymphocytic infiltration. Treatment with n-hexane extract
of E. sonchifolia restored the damaged tissue to its normalcy
(Figure 1).
Figure 1. Histopathology of pancreas.
A - Pancreas section of the normal rats (group 栺) showing the normal
exocrine acini and endocrine islets;
B - Pancreas section of the ethanol-induced rats (group 栻) showing
the congestion of cells and mild lymphocytic infiltration;
C - Pancreas section of the ethanol-induced rats treated with the
n-hexane extract of E. soncifolia (group 栿) showing the absence of
congestion and cellularity- reversal of toxic changes;
D - Pancreas section of the n-hexane extract of E. soncifolia alone
(group 桇) treated rats showing the normal exocrine acini and
endocrine islets.
4. Discussion
Liver damage is a common problem due to alcohol
consumption and ethanol may be responsible for the
increment of AST and ALT, important marker enzyme of the
liver function test[12]. Various experimental and clinical
studies suggest that ethanol produces pancreatic injury
in which oxidative stress may play a crucial role in the
pathogenesis. Ethanol intake causes accumulation of ROS,
like superoxide, hydroxyl radical, and hydrogen peroxide[26]
or a decrease in the levels of antioxidant defenses causing
a redox imbalance and resulting in the oxidative damage
of lipids, proteins, and DNA. These reactive moieties cause
lipid peroxidation. Ethanol treatment has been reported
to activate ERK1/2, increase COX-2 immunoreactivity,
enhance activation of transcription factors like NF-毷B or
AP-1, and induce rapid lipid peroxidation[4,27]. Alcohol
toxicity is caused not only directly by ethanol but also by its
metabolic products, including the ROS produced during its
biotransformation.
In agreement with the previous reports, the present
study also revealed that the administration of ethanol
significantly increased the lipid peroxidation, reduced the
antioxidant levels and provoked damage to the organs.
Biological systems show different mechanisms to protect
themselves from the damaging effects caused by activated
species. These mechanisms include free radical scavenging,
termination of chain reactions and activities of antioxidant
enzymes such as SOD, CAT and GPx[28].
SOD, a metalloprotein, is involved in the antioxidant
defense mechanism as the first enzyme by lowering the
amount of O2
-. CAT is a hemoprotein, localized in the
peroxisomes and induces the decomposition of H2O2 to
water and oxygen[29]. GPx reduces hydrogen peroxide and
other organic peroxides at the expense of GSH, which is
in turn oxidized to form glutathione disulfide (GSSG). GSH
is regenerated by GR with the consumption of NADPH[30].
A number of research studies have shown evidence that
acute ethanol administration decreases hepatic GSH
content[31,32]. GSH is the major antioxidative tripeptide in the
cell and plays pivotal role in the detoxification of toxicants,
metabolism of nutrients and regulation of various pathways
to maintain cellular homeostasis[33]. Different enzymatic and
non enzymatic reactions could be involved in GSH mediated
scavenging of free radicals and other oxygen species. It
has been observed that acute and chronic intake of ethanol
causes GSH depletion both in time as well as dose dependent
fashion[34,35]. Vitamin C may effectively protect against the
deleterious effects of ethanol-induced abnormalities. It may
also interact with ethanol extracellularly and hence alleviate
the overall teratogenic effect of ethanol[36].
Several studies have shown that the activity of antioxidant
enzymes is significantly reduced in the animals when
exposed to ethanol. All these biochemical alterations
are likely to be responsible for ethanol-induced multi-
organ damages including brain, liver, esophagus,
urinary bladder and pancreas as well as stomach and the
persistence or perpetuation of these damages are related
to the carcinogenesis of each organ. Previous studies in
the rat enteral ethanol model have also shown that alcohol
increases free radical formation and oxidative stress in
pancreas[37,38-41]. Several in vivo studies have indicated that
antioxidant treatment by using herbs can prevent or reduce
growth retardation and/or the occurrence of malformations
in the organs upon ethanol exposure[12,42,43]. Similarly, in
the present study treatment with the n-hexane extract of
E. sonchifolia significantly ameliorated the damage caused
by ethanol by preventing the elevation of serum AST and
ALT and restored its antioxidant levels which may be
due to its antioxidant capacity. This is confirmed by the
histopathological studies also. The congestion of cells and
mild lymphocytic infiltration in the pancreas of the ethanol
induced rats were restored to its normalcy when treated
with n-hexane extract of E. sonchifolia. Thus the n-hexane
extract of E. sonchifolia can be effectively used as the
therapeutic drug for the management of alcohol-induced
oxidative stress.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
We, the authors are thankful to our Chancellor, Advisor,
Vice Chancellor, and Registrar of Karpagam University for
providing facilities and encouragement.
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