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JOURNAL OF MEDICINAL FOOD

J Med Food 10 (4) 2007, 000000


Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2006.110

Antioxidant Effect of Hemidesmus indicus on Ethanol-Induced


Hepatotoxicity in Rats
Nadana Saravanan and Namasivayam Nalini
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Tamilnadu, India
ABSTRACT The antioxidant effect of the ethanolic root extract of Hemidesmus indicus, an indigenous Ayurvedic medicinal plant used in soft drinks in India, was studied in rats with ethanol-induced hepatotoxicity. Administering 20% ethanol (5
g/kg of body weight/day) for 60 days to male Wistar rats resulted in significantly decreased body weight and increased
liver/body weight ratio. The liver marker enzymes, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatae (ALP), -glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH), were elevated. In addition, the levels of
plasma, erythrocyte, and hepatic thiobarbituric acid-reactive substances (TBARS), hydroperoxides (LOOH), and conjugated
dienes (CD) were also elevated in ethanol-fed rats as compared to those of the experimental control rats. Decreased activities
of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin C, and
-tocopherol (vitamin E) were also observed in ethanol-administered as compared to control rats. Ethanolic root extract of H.
indicus was administered at a dose of 500 mg/kg of body weight/day for the last 30 days of the experiment to rats with ethanolinduced liver injury, which significantly increased body weight, significantly decreased the liver/body weight ratio, AST, ALT,
ALP, GGT, and LDH activities, and also the levels of TBARS, LOOH, and CD, significantly elevated the activities of SOD,
CAT, GPx, and GSH in plasma, erythrocytes, and liver, and also increased levels of plasma and liver vitamin C and vitamin
E at the end of the experimental period as compared to those of untreated ethanol-administered rats. Thus, our data indicate
that treatment with H. indicus extract offers protection against free radical-mediated oxidative stress in plasma, erythrocytes,
and liver of animals with ethanol-induced liver injury.
KEY WORDS:

antioxidants

ethanol

Hemidesmus indicus

INTRODUCTION

lipid peroxidation

liver

are probably the most susceptible target of free radical attack. The reaction of free radicals with the membrane lipid
components leads to lipid peroxidation. This process can
eventually cause increased membrane permeability and cell
death.7 To counteract these oxidants, cells have several
enzymatic antioxidants, including superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx),
and nonenzymatic antioxidants, including reduced glutathione (GSH), ascorbic acid, and -tocopherol, but their
levels are altered in alcoholics.
Hemidesmus indicus (Family Asclepiadaceae) is a widely
distributed medicinal plant in India. It has been used as a
traditional medicine in the treatment of biliousness, blood
diseases, diarrhea, respiratory disorders, skin diseases,
syphilis, fever, bronchitis, asthma, eye diseases, childhood
epilepsy, kidney and urinary disorders, loss of appetite,
burning sensation, and rheumatism.8 Jain and Singh9 reported that H. indicus is employed in traditional medicine
for gastric ailments. It is also used in the preparation of some
soft drinks. It mainly consists of essential oils and phytosterols, like hemidesmol, hemidesterol, and saponins. Although the protective effect of H. indicus against CCl4- and
peracetamol-induced hepatotoxicity in rats10 is known, the
effect of the extract on ethanol-induced hepatoxicity is un-

of chronic liver disthe pathogenesis of


ethanol-induced liver injury, oxidative stress plays an important role.2,3 Peroxidative damage in alcoholic patients
and enhanced lipid peroxide production in animals can be
correlated with high ethanol consumption.4 Ethanol administration has the ability to disturb the balance between the
pro- and antioxidant systems of the organism, therefore leading to oxidative stress. Generation of oxygen metabolites
such as superoxide (O2), hydrogen peroxide (H2O2), and
hydroxyl radical (OH) is believed to be important in the
pathogenesis of alcoholic liver injury.5 Increased generation
of oxygen- and ethanol-derived free radicals has been observed at the microsomal level (particularly at the ethanolinducible cytochrome P450 isoform) and with the cytosolic
xanthine and/or aldehyde oxidase, as well as through the mitochondrial respiratory chain.6 Polyunsaturated fatty acids
NE OF THE MOST COMMON CAUSES
ease is alcohol consumption.1 In

Manuscript received 23 June 2006. Revision accepted 1 December 2006.


Address reprint requests to: Dr. Namasivayam Nalini, Professor, Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar-608002, Tamilnadu, India, E-mail: nalininam@yahoo.com

675

676

SARAVANAN AND NALINI

known. Because of its wide pharmacological actions and recent interest,11 the present study was undertaken to establish the hepatoprotective activity of the ethanolic root extract of H. indicus on an animal model of ethanol-induced
liver damage.

RESEARCH DESIGN AND METHODS


Materials
The chemicals used for the assay were of analytical grade.

H. indicus
The plant material (H. indicus) was obtained from a local market, identified, and authenticated by the Department
of Botany, Annamalai University. The H. indicus roots were
cleaned, shade-dried, and disintegrated. Cold aqueous
ethanolic extract was prepared according to the methodology of the Indian Pharmacopoeia12 with a Soxhlet apparatus and screened for its antihepatotoxicity property. The
ethanolic extract (yield, 9.2%) was dark brown in color and
sticky in nature and gave positive results for flavonoids, terpenoids, tannins, coumarins, and glycoside and negative results for alkaloids, anthraquinones, lactones/ester, protein/amino acids, and saponins.13

Animals

AU
1

All the animal handling and experimental procedures


were approved by the Institutional Animal Ethics Committee of Annamalai University (Registration Number
160/1999/CPCESA), and animals were cared for in accordance with the principles and guidelines of Indian National
Law on animal care and use.
Male albino Wister rats weighing 130180 g bred in the
Central Animal House, Rajah Muthiah Medical College, Annamalai University, Annamalai Nagar were used for the
study. The animals were fed ad libitum with water and normal laboratory pellet diet (Amrut Laboratory Animal Feed,
Pranav Agro Industries Ltd., Bangalore, India), consisting
of protein (22.21%), fat (3.32%), and fiber (3.11%), balanced with carbohydrates (67%), vitamins, and minerals.

Study design
The animals were divided into four groups of 10 each.
Groups 1 and 2 received normal diets and isocaloric glucose
from 27% glucose solution (1 mL contains 1.08 cal) daily
by intragastric intubation. Liver cell damage was induced in
rats of groups 3 and 4 by administering 20% ethanol (5.0
g/kg of body weight/day)14 (1 mL contains 1.08 cal) as an
aqueous solution using an intragastric tube daily for 30 days.
At the end of this period the animals were treated as follows
for the next 30 days: Group 1 animals continued to receive
standard pellet diet and isocaloric glucose from 27% glucose solution in 1% carboxy methyl cellulose (CMC) and
served as controls. Group 2 animals continued to receive

standard pellet diet and isocaloric glucose from 27% glucose solution and were administered ethanolic root extract
of H. indicus (500 mg/kg of body weight/day)10,15 in 1%
CMC by intragastric intubation every day. Group 3 animals
continued to receive standard pellet diet, 20% ethanol, and
1% CMC daily. Group 4 animals continued to receive standard pellet diet, 20% ethanol, and ethanolic extract of H. indicus (500 mg/kg of body weight/day) in 1% CMC every
day. The total duration of the experiment was 60 days.
The animals were fasted overnight and then anesthetized
with an intramuscular injection of ketamine hydrochloride
(30 mg/kg of body weight), blood samples were collected
by retro-orbital puncture, and animals were sacrificed by
cervical decapitation. Blood samples were collected in heparinized test tubes and plain tubes and then centrifuged for
the separation of plasma and serum, respectively. The erythrocytes were washed with 0.9% NaCl three times, and distilled water was added for hemolysis. The hemolysate was
then used for estimating the total hemoglobin (Hb) content.
Liver tissues were immediately homogenized and used for
various biochemical estimations.

Estimation of liver marker enzymes


Serum aspartate aminotransferase (AST) (EC 2.6.1.1) and
serum alanine aminotransferase (ALT) (EC 2.6.1.2) were assayed using a diagnostic kit based on the method of Reitman and Frankel.16 Serum alkaline phosphatase (ALP) (EC
3.1.2.3.1) was estimated using a diagnostic kit based on the
method of Kind and King.17 Serum -glutamyl transpeptidase (GGT) (EC 2.3.2.2) was assayed according to the
method of Rosalki and Rau.18 The activity of lactate dehydrogenase (LDH) (EC 1.1.1.27) was estimated by the
method of King.19

Lipid peroxidation and enzyme assays


Lipid peroxidation was assayed by measuring thiobarbituric
acid-reactive substances (TBARS) in the tissues by the method
of Ohkawa et al.20 and Yagi.21 The pink chromogen produced
by the reaction of malondialdehyde, a secondary product of
lipid peroxidation with thiobarbituric acid, was estimated at
532 nm. Lipid hydroperoxides (LOOH) were estimated by the
procedures of Jiang et al.22 These methods are based on the
rapid peroxide-mediated oxidation of ferrous ion (Fe2) to ferric ion (Fe3) under acidic conditions in the presence of
xylenol orange. The Fe3-xylenol orange complex was measured spectrophotometrically at 560 nm. Conjugated dienes
(CD) were estimated by the method of Recknagel and
Glende.23 Lipid peroxidation is associated with the rearrangement of double bonds in polyunsaturated fatty acids leading
to the formation of CD, which absorb light at 233 nm. The
oxidation indexes of the lipid sample were computed from the
spectrophotometic absorptions at 233 nm and 215 nm, which
reflect the diene content and the extent of peroxidation.
SOD (EC 1.15.1.1) was assayed by the method of Kakkar
et al.24 The assay was based on the 50% inhibition of the

677

H. INDICUS IN ETHANOL-INDUCED HEPATOTOXICITY

formation of NADH-phenazine methosulfate-nitro blue


tetrazolium (NBT) formazan at 520 nm. The activity of CAT
(EC 1.11.1.6) was assayed by the method of Sinha25 based
on the conversion of dichromate in acetic acid to perchromic
acid and then to chromic acetate, when heated in the presence of hydrogen peroxide. The chromic acetate formed was
measured at 620 nm. The activity of GPx (EC 1.11.1.9) was
assayed by the method of Rotruck et al.26 A known amount
of enzyme preparation was incubated with H2O2 in the presence of GSH for a specified time period. The amount of
H2O2 utilized was determined by the method of Ellman.27
The enzyme activity was expressed as mol of GSH consumed/minute/mg of protein.
GSH in the tissues was assayed by the method of Ellman.27 GSH estimation was based on the development of
yellow color when 5,5-dithiobis(2-nitrobenzoic acid) was
added to compounds containing sulfydryl groups. Ascorbic
acid was measured according to the method of Omaye et
al.28 -Tocopherol in plasma and tissues were estimated by
the method of Desai.29 Proteins were estimated by the
method of Lowry et al.30 using bovine serum albumin as the
standard.

Statistical analysis
Data were analyzed by one-way analysis of variance followed by Duncans multiple range test (DMRT) using a
commercially available statistics software package (SPSS
for Windows, version 13.0, SPSS, Chicago, IL). Results
were presented as mean  SD values. Values of P  .05
were regarded as statistically significant.

RESULTS
Table 1 shows the average weight gain, food intake, and
liver/body weight ratios of control and experimental rats during the experimental period. The average food intake and
the average weight gained were significantly reduced in
ethanol-administered rats, and the liver/body weight ratio
was significantly higher compared to that of control animals.
Rats co-administered H. indicus along with ethanol from day
31 showed significantly greater weight gain and food intake
and decreased liver/body weight ratio compared to untreated
ethanol-fed rats.

TABLE 1.

EFFECT

OF

H.

INDICUS ON

BODY WEIGHT

AND

In Table 2, it can be seen that administration of ethanol


produced severe liver damage as indicated by marked increases in AST, ALT, ALP, GGT, and LDH activities. However, with H. indicus co-administration there was a normalization of these values.
Tables 3, 4, and 5 show the levels of TBARS, LOOH, and
CD of control and experimental animals. Lipid peroxidation
levels indicated by TBARS, LOOH, and CD were significantly
higher in plasma, erythrocytes, and liver of ethanol-administered animals compared to those of normal control rats.
TBARS, LOOH, and CD levels were significantly lower in
the plasma, erythrocytes, and liver cells of ethanol-administered animals treated with H. indicus extract.
Tables 6, 7 and 8 show the activities of SOD, CAT, and
GPx in the plasma, erythrocytes, and liver of control and
experimental animals. SOD, CAT, and GPx activities in the
plasma, erythrocytes, and liver of ethanol-administered rats
(group 3) were significantly lower than the control rats
(group 1), but were significantly higher, near control values, when H. indicus extract was co-administered with
ethanol.
As shown in Tables 6, 7, and 8 the concentration of GSH
was significantly lower in plasma, erythrocytes, and liver
cells of rats receiving ethanol (group 3) as compared to control rats (group 1). Treatment with H. indicus extract of
ethanol-administered rats (group 4) resulted in significantly
higher GSH levels as compared with those receiving ethanol
administration alone (group 3).
Table 9 shows the levels of vitamin C and vitamin E in
plasma and liver of control and experimental animals. Vitamin C and E levels in plasma and liver of rats receiving
ethanol supplementation were significantly lower than in the
control rats. Ethanol-treated rats given H. indicus extract had
significantly higher vitamin C and E levels as compared with
those animals receiving ethanol supplementation alone.

DISCUSSION
Chronic ethanol feeding caused significantly less weight
gain in rats compared to untreated controls. Wastage of energy during ethanol metabolism by the microsomal ethanol
oxidizing system (MEOS) (CYP2E1) can be one of the
causes for the decreased weight gain with ethanol supplementation.31 Other causes are mitochondrial insufficiency in

LIVER/BODY WEIGHT

OF

CONTROL

AND

ETHANOL-ADMINISTERED RATS

Body weight (g)


Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

Day 1

Day 60

Net gain (g)

Average food intake (g)

Liver weight 100/body weight

145.11  6.34
142.04  6.84
140.54  7.33
144.39  6.20

226.07  10.93
224.46  12.33
152.61  6.53
198.94  3.46

80.97 
82.42  5.51c
12.07  0.96a
54.56  5.67b

9.18 
9.27  0.25c
7.55  0.26a
8.71  0.22b

2.77  0.14a
2.81  0.23a
5.19  0.38c
3.18  0.07b

4.61c

0.25c

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

678

SARAVANAN AND NALINI


TABLE 2.

EFFECT

OF

H.

INDICUS ON

HEPATIC MARKER ENZYMES

OF

CONTROL

AND

ETHANOL-ADMINISTERED RATS

Level (IU/L)
Group

AST
75.66 
80.69  2.50a
127.91  7.13c
97.56  4.49b
3.39a

Control
H. indicus
Ethanol
Ethanol  H. indicus

ALT

ALP

GGT

LDH

26.89 
21.41  0.56a
58.11  1.85c
27.81  2.16b

102.77 
111.80  14.09a
158.39  5.43b
108.64  23.15a

2.27 
2.03  0.46a
7.33  1.23c
3.11  0.06b

120.17  3.08a
130.02  3.34b
332.95  8.55d
159.58  4.10c

1.28b

22.11a

0.66a

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

TABLE 3.

EFFECT

OF

Group

H.

INDICUS ON

PLASMA LIPID PEROXIDATIVE MARKERS

CONTROL

OF

AND

ETHANOL-ADMINISTERED RATS

TBARS (nmol/mg of protein)

LOOH ( 10
5 mmol/mg of protein)

CD (nmol/mg of protein)

1.56  0.03a
1.46  0.04a
3.13  0.25c
1.77  0.08b

9.57  0.62b
7.77  0.51a
16.27  0.74c
10.00  0.78b

0.67  0.02b
0.56  0.02a
1.39  0.06d
0.75  0.03c

Control
H. indicus
Ethanol
Ethanol  H. indicus

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

TABLE 4. EFFECT OF H. INDICUS ON ERYTHROCYTE LIPID PEROXIDATIVE


MARKERS OF CONTROL AND ETHANOL-ADMINISTERED RATS
Level (nmol/mg of Hb)
Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

TBARS

LOOH

CD

1.52  0.06b
1.26  0.03a
3.08  0.23c
1.60  0.07b

6.87  0.11a
6.51  0.18a
13.25  1.02c
8.53  0.21b

6.07  0.10b
5.25  0.19a
10.73  0.96d
6.63  0.22c

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at
P  .05 (DMRT).

TABLE 5.

EFFECT OF H. INDICUS ON HEPATIC LIPID PEROXIDATIVE MARKERS


CONTROL AND ETHANOL-ADMINISTERED RATS

OF

Level (mmol/mg of tissue)


Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

TBARS

LOOH

CD

0.73 
0.65  0.02a
1.92  0.06d
0.82  0.04c

64.96 
59.30  1.67a
90.47  3.25c
62.78  2.28ab

52.32  1.13b
43.73  1.78a
86.88  3.22c
59.90  2.26b

0.05b

4.13b

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at P 
.05 (DMRT).

679

H. INDICUS IN ETHANOL-INDUCED HEPATOTOXICITY


TABLE 6.

EFFECT

OF

H. INDICUS ON ACTIVITIES OF PLASMA SOD, CAT, GPX,


NORMAL AND ETHANOL-ADMINISTERED RATS

AND

GSH

OF

Activity (U/mg of protein)


Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

SOD

CAT

GPx

GSH (mmol/mg of protein)

2.60  0.06c
2.54  0.07c
1.59  0.08a
2.29  0.05b

3.54  0.32c
4.17  0.12d
1.76  0.03a
2.95  0.07b

22.70  1.43c
25.85  0.79d
13.15  0.49a
21.08  0.51b

35.22  0.61c
37.93  0.76d
15.06  0.71a
29.83  0.51b

Data are mean  SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for
50% inhibition of NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute,
respectively.
Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

TABLE 7.

EFFECT

OF

H.

INDICUS ON

ACTIVITIES

OF

ERYTHROCYTE SOD, CAT, GPX,

AND

GSH

OF

NORMAL

AND

ETHANOL-ADMINISTERED RATS

Activity (U/mg of Hb)


Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

SOD

CAT

GPx

GSH (mmol/mg of Hb)

2.03  0.06b
2.89  0.09c
1.29  0.37a
2.02  0.06b

154.21  2.56c
164.48  4.91d
96.75  2.89a
145.55  4.35b

14.37  0.42c
16.18  0.43d
7.55  0.20a
12.46  0.24b

3.61  0.19b
4.36  0.30c
1.52  0.14a
3.46  0.27b

Data are mean  SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for 50% inhibition of
NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute, respectively.
Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

TABLE 8.

EFFECT

OF

H.

INDICUS ON

ACTIVITIES

OF

HEPATIC SOD, CAT, GPX,

AND

GSH

OF

NORMAL

AND

ETHANOL-ADMINISTERED RATS

Activity (U/mg of protein)


Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

SOD

CAT

GPx

GSH (mmol/mg of protein)

5.75  0.15b
5.89  0.12b
2.07  0.19a
5.89  0.16b

75.31  1.72c
77.17  1.25c
52.34  2.67a
67.55  1.97b

13.22  1.06b
15.02  1.20c
6.00  0.48a
12.50  1.00b

17.64  0.37c
18.50  0.34d
10.02  0.39a
15.15  0.32b

Data are mean  SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for 50% inhibition of
NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute, respectively.
Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

TABLE 9.

EFFECT

OF

H.

INDICUS ON

NORMAL

LEVELS OF PLASMA AND HEPATIC VITAMIN C


AND ETHANOL-ADMINISTERED RATS

Plasma (mg/dL)
Group
Control
H. indicus
Ethanol
Ethanol  H. indicus

AND

VITAMIN E

OF

Liver (mg/100 g of tissue)

Vitamin C

Vitamin E

Vitamin C

Vitamin E

2.06  0.04c
2.31  0.08d
1.25  0.15
1.76  0.04b

2.68  0.17c
2.84  0.11d
1.28  0.17
2.16  0.05b

0.84  0.10b
1.02  0.11c
0.51  0.49a
0.82  0.73b

5.46  0.16c
5.96  0.35d
3.39  0.11a
5.10  0.22b

Data are mean  SD values for 10 rats in each group.


Values not sharing a common superscript letter within each column differ significantly at P  .05 (DMRT).

680

SARAVANAN AND NALINI

fatty acid oxidation secondary to chronic ethanol consumption and acetaldehyde toxicity.32 Significant improvement
in body weight gain was observed with H. indicus treatment,
which may be due to the inhibitory effect of H. indicus on
CYP2E1 in liver microsomes. The ratio between liver
weight and total body weight was significantly lower in
ethanol-fed rats supplemented with H. indicus extract compared to that of the unsupplemented ethanol-fed rats, which
may be because the extract increases the elimination of
ethanol directly from the intestines without absorption or
perhaps because H. indicus consumption prevents fat accumulation in the liver.
Liver damage after ethanol ingestion is a well-known phenomenon, and the obvious sign of hepatic injury is the leakage of cellular enzymes into plasma.33 The increased levels
of serum enzymes such as AST, ALT, ALP, GGT, and LDH
observed in ethanol-administered rats may indicate increased permeability, damage, and/or necrosis of hepatocytes.34 H. indicus extract supplementation showed a
marked hepatoprotective effect that is consistent with the results of previous researchers,10 and it is supported by the reversal of changes produced by ethanol. Moreover, the observed decrease in the activities of these enzymes shows that
H. indicus, to a certain extent, preserves the structural integrity of the liver from the toxic effects of ethanol.
Oxidation of polyunsaturated fatty acids (lipid peroxidation) of membranes is a common process in living organisms, since they are the target of oxygen-derived free radicals produced during mitochondrial electron transport.35
Increased lipid peroxidation associated with chronic ethanol
administration has often been used as an indicator of oxidative stress in both animal models and human clinical trials. Excess lipid peroxidation, as measured by formation of
TBARS, LOOH, and/or CD, has been found in most studies.36 In agreement with these findings, ethanol-administered rats showed increased blood and tissue levels of lipid
peroxidation markers such as TBARS, LOOH, and CD. The
increased peroxidation can result in changes in cellular metabolism of the hepatic and extrahepatic tissues. Products of
lipid peroxidation formed in the primary site reaching the
other organs and tissues via the bloodstream provoke lipid
peroxidation there and consequently cause cellular and tissue damage.37 Increased accumulation of lipid peroxidation
products in cells can result in cellular dehydration, whole
cell deformity, and cell death.38
Lipid peroxidation is an important cause of alcoholic liver
disease.36 Free radical generation and lipid peroxidation
products play a pivotal role in the mechanism by which
ethanol may exerts its toxic effects on the liver and other
extrahepatic tissues.36 The H. indicus co-administered rats
showed significantly lower levels of these lipid peroxidative
markers compared to ethanol-fed rats. H. indicus also exhibited its potent antioxidant activity by decreasing lipid peroxidation in control rats administered H. indicus extract
compared to normal controls. In this context, Mary et al.39
have also reported that H. indicus has an antioxidant property using in vitro studies. Decreased lipid peroxidation with

H. indicus extract administration suggests a decreased impact of reactive oxygen species (ROS) on lipid membranes,
and therefore increased protection against ethanol-induced
liver injury. Thus the inhibition of lipid peroxidation by H.
indicus may be one of the mechanisms by which H. indicus
exerts its protection against ethanol-mediated tissue injury.
Some studies have shown that treatment with silymarin protects the liver, probably through decreasing lipid peroxidation.40 H. indicus may have a similar mode of action.
Free radical scavenging enzymes such as SOD, CAT, and
GPx are the first line of defense against oxidative injury.
SOD scavenges excess superoxide anions and converts
them to H2O2. The primary role of CAT is to scavenge
H2O2 that has been generated by free radicals or by SOD
and convert it to water. GPx works in tandem with CAT to
scavenge excess H2O2 as well as other free radicals in response to oxidative stress. The equilibrium between these
enzymes is important for the effective removal of oxidative
stress in intracellular organelles. This antioxidant defense
system is significantly altered by ethanol administration.
Our results show decreased activities of SOD, CAT, and
GPx activities in chronic ethanol-administered rats. It has
been reported that ethanol impairs the antioxidant system
of the tissues in proportion to the amount of ethanol ingestion.41 Biphasic fluxes of these enzyme activities are
common; an increase or decrease may relate to the presence of excess ROS. The decreased activities of enzymatic
antioxidants observed in ethanol-administered rat erythrocytes and tissues may be a consequence of irreversible inactivation of enzyme proteins from increased free radical
production resulting from ethanol metabolism.42 Lowered
activities of these enzymes will result in the accumulation
of highly reactive free radicals, leading to deleterious effects such as loss of cell membrane integrity and membrane
function.43 There was a significant increase in the activities of these enzymes with H. indicus extract co-administration. H. indicus is reported to scavenge superoxide radicals and hydrogen peroxide.39 Because of these properties,
it was expected that H. indicus might decrease the workload of enzymatic antioxidants and reduce the free radicalmediated inactivation of enzyme proteins and thereby maintaining the activities of enzymatic antioxidants. Thus H.
indicus has the ability to increase the activity of the endogenous antioxidant enzymes.
The second line of defense consists of the nonenzymatic
scavengers, such as GSH, ascorbic acid, and -tocopherol,
which scavenge residual free radicals escaping decomposition by the antioxidant enzymes. Moreover, enzymatic antioxidants are inactivated by the excessive levels of free radicals, and hence the presence of nonenzymatic antioxidants
is presumably essential for the removal of these radicals.44
Glutathione is a major non-protein thiol in living organisms
that plays a central role in co-ordinating the antioxidant defense process in our body. Glutathione reacts directly with
ROS and electrophilic metabolites, protects essential thiol
groups from oxidation, and serves as a substrate for several
enzymes, including GPx.

H. INDICUS IN ETHANOL-INDUCED HEPATOTOXICITY

We observed lower level of plasma, erythrocyte, and hepatic GSH in ethanol-fed rats, a consequence of increased
utilization to counter the increased oxidative stress. The
shortage of NADPH (due to the increased oxidation of
ethanol by the MEOS, which uses NADPH as a cofactor)
suppresses the reduction of oxidized glutathione by glutathione reductase and subsequently decreases glutathione
content. Generation of large quantities of acetaldehyde during ethanol metabolism ultimately will deplete cellular GSH
pools by forming S-conjugation with the
SH group. Perturbation in the GSH redox status not only can impair cell
defense against toxic compounds, but also result in increased
oxidative stress and oxidative injury.45
H. indicus co-administered rats exhibited significantly
improved GSH levels compared to ethanol-fed rats. The results obtained suggest that the maintenance of GSH by H.
indicus was mainly due to inactivation of ROS via its radical scavenging effects, sparing antioxidant enzymes such as
SOD46 and CAT. Restoration of GSH levels has been shown
to inhibit ethanol toxicity.47,48 Therefore, it was presumed
that the effects of H. indicus might be related to a normalization mechanism by maintaining adequate levels of GSH
for detoxification of xenobiotics.
Antioxidant systems other than GSH may also play a role
in preventing lipid peroxidation under experimental and clinical conditions. -Tocopherol and ascorbic acid are naturally
occurring free radical scavengers.49 Both ascorbic acid and tocopherol are known to be decreased in liver diseases, particularly in alcoholics.50 Under these conditions, thiol compounds,
such as GSH, might be involved in regenerating -tocopherol
from its radical form.51 The observed decrease in the levels of
-tocopherol and ascorbic acid may be due to their increased
utilization for scavenging ethanol- and/or oxygen-derived radicals. We have observed near normal levels of these antioxidants with H. indicus supplementation. The decrease in lipid
peroxidation with H. indicus treatment can be correlated with
the elevated levels of antioxidants. The ability of H. indicus to
enhance the levels of antioxidants along with its anti-lipid peroxidative activity suggest that this extract might be potentially
useful in countering free radical-mediated injuries involved in
the development of liver damage caused by alcohol abuse.
This study provides convincing evidence that H. indicus
treatment can effectively protect against oxidative injury in
alcoholics.

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NADANA SARAVANAN
AU1
One word in affiliation. Why different here?