Antioxidant Activity of Andrographolide
Hepatoprotective Effect of Andrographolide
Against Hexachlorocyclohexane-Induced
Oxidative Injury
Neha P. Trivedi, PhD, Upendra M. Rawal, PhD, and Beena P. Patel, PhD
Many plant products are known to exert antioxidative effects
by quenching various free radicals and singlet molecular oxy-
gen. Andrographis paniculata (Kalmegh) is used extensively in
the Indian traditional system of medicine as a hepatoprotec-
tive and hepatostimulative agent and has been reported to
have antioxidant effects against different hepatotoxins. The
present study aims to analyze antioxidant properties of an
active component, andrographolide (ANDLE), extracted
from A paniculata. This study investigates the effect of andro-
grapholide on the hepatocellular antioxidant defense system
and lipid peroxidation of control mice, mice treated with
hexachlorocyclohexane (BHC) only, and andrographolide +
BHC. Glutathione (GSH), glutathione-s-transferase (GST),
glutathione reductase (GR), glutathione peroxidase (GSH-
Px), γ-glutamyl transpeptidase (γγ-GTP), superoxide dismu-
tase (SOD), catalase (CAT), and lipid peroxidation (LPO)
are studied by spectrophotometric methods. The BHC
experimental model forms an irreversible liver tumor in
male mice. The activities of GSH, GR, GSH-Px, SOD, and
CAT show significant (P ≤ .05) increases, while γ-GTP and
GST show significant decreases (P ≤ .05) in andrographolide-
supplemented mice as compared with BHC-treated mice.
This study indicates that the antioxidant effect of andro-
grapholide could be due to its ability to activate antioxidant
enzymes that catalyze the reaction of oxidants and are effec-
tive in severe liver damage.
Keywords: andrographolide; liver damage; antioxidant enzymes
Andrographis paniculata Nees (Acanthaceae) is a well-
known medicinal plant in Ayurveda. It has been used
as a bitter and febrifuge. Recent research has thrown
light on the medicinal value of the plant. The extract
of whole plant has been reported to have anticancer,
anti-inflammatory, antiallergic, immunostimulatory,
antithrombotic, antiviral, hypoglycemic, and hypo-
tensive activities.1-8 Previously, we reported the anti-
oxidant property of the whole plant extract of A
paniculata on hexachlorocyclohexane (BHC)–treated
mice.9 Andrographolide, andrographiside, and neoan-
drographolide are active compounds of A paniculata.10
DOI: 10.1177/1534735407305985
INTEGRATIVE CANCER THERAPIES 6(3); 2007 pp. 271-280
Recent studies have reported the antioxidant prop-
erties of A paniculata on in vivo and in vitro model11
and diabetic rats.12 A diterpene, andrographolide
(ANDLE), is the active constituent of A paniculata.
Based on our previous findings, the present article
confirms the antioxidative properties of ANDLE
extracted from A paniculata on BHC-treated mice.
Materials and Methods
Extraction of Active Compound
The andrographolide (a pure active compound) was
obtained from the BV Patel Pharmaceutical Educa-
tional and Research Development Centre, Ahmedabad,
India. The andrographolide was administered orally
in 3 doses: low (5 mg/kg body weight), medium
(7 mg/kg body weight), and high (10 mg/kg body
weight) per animal per day for the period of 1 to
8 months.
Experimental Animals
Healthy, adult, pathogen-free, colony-breed male
albino mice (Mus musculus) of Swiss strain, 1 month
old, weighing between 20 and 30 g, were used in this
study. The Indian Council of Medical Research guide-
lines for the care and use of laboratory animals were
followed. The research was approved by the ethical
committee of the Zoology Department, School of
Sciences, Gujarat University, Ahmedabad, India.
Experimental Tumor Model
Technical-grade BHC was used for inducing the car-
cinogenic condition in the liver of male mice as per
the method of Nigam et al.13,14
NPT and UMR are at the Department of Zoology, School of
Sciences, Gujarat University, Ahmedabad, India. BPP is at the Cell
Biology Division, Gujarat Cancer & Research Institute, Asarwa,
Ahmedabad, India.
Correspondence: Upendra M. Rawal, Department of Zoology,
Gujarat University, Ahmedabad 380 009, India. E-mail: upendrarawal
@gmail.com.
271
 Trivedi et al
Treatment Diet
The BHC-treated animals were given a basal diet
with 500 ppm BHC/kg food. This dose approxi-
mates the highest tolerable limit causing significant
mortality in test animals by Lakkad et al.15 The BHC
insecticide was weighed and dissolved in alcohol and
homogeneously mixed in the diet; the alcohol was
allowed to evaporate by drying. The dose of BHC
500 ppm/kg food is selected on the basis of previous
work on rodents.13,15
The Experimental Design
The experimental design was as follows:
• Group 1 (control): animals were given normal diet
and water ad libitum.
• Group 2 (BHC treated): animals in this group were
fed a diet containing technical-grade BHC at a dose
of 500 ppm/kg food for a period of 1 to 8 months.
• Group 3 (BHC + ANDLE supplemented): this group
of animals was given both BHC at a dose of 500 ppm/kg
food and ANDLE orally in low (5 mg/kg body
weight), medium (7 mg/kg body weight), and high
(10 mg/kg body weight) doses per animal per day for
the period of 1 to 8 months. This group was consid-
ered the supplemented group.
• Group 4 (ANDLE treated, positive control): only
ANDLE was given to this group of animals in a dose
of 7 mg/kg body weight per animal per day orally.
The various groups of animals, treatment, and
duration are shown in Table 1. At the end of each
treatment, the animals were weighed on an animal
balance and killed by cervical dislocation. The liver of
each animal was dissected out, blotted free of blood,
and weighed on a balance.
Biochemical Methods
Tissue protein. The protein content in the liver tis-
sue of mice was estimated according to the method
of Lowry et al.16
γ-Glutamyl transpeptidase. The liver γ-glutamyl trans-
peptidase (γ-GTP) activity was assayed by the method
of Tate and Meister.17 The enzyme activity was
expressed as units/min/mg protein, where 1 unit of
γ-GTP is equivalent to 1 µmol p-nitroanilide released
per minute.
Lipid peroxidation. Lipid peroxidation (LPO) in the
liver was determined by the method of Ohkawa.18 The
method is based on the formation of a red chro-
mophore that absorbs at 532 nm following the reac-
tion of thiobarbituric acid with malonyl dialdehyde
272
Table 1. Groups of Animals, Treatment, and Duration
Group Treatment and Dosage Duration, mo
1 Control 1 to 8
2 BHC treated (500 ppm/kg fed) 1 to 8
3 BHC with ANDLE (andrographolide
supplements) per day per animal orally
(a) Low dose
BHC (500 ppm/kg) + ANDLE (5 mg/kg
body weight)
(b) Medium dose
BHC (500 ppm/kg) + ANDLE (7 mg/kg
body weight)
(c) High dose
BHC (500 ppm/kg) + ANDLE (10 mg/kg
body weight) 1 to 8
4 Positive control, only ANDLE treated
(7 mg/kg body weight) per day/
animal orally 1 to 8
ANDLE = andrographolide; BHC = hexachlorocyclohexane.
(MDA) and other breakdown products of peroxidized
lipids, collectively called thiobarbituric acid reactive
substance. The results were expressed as nanomoles of
MDA/h/mg tissue weight.
Glutathione. The glutathione (GSH) levels in liver
tissue were measured using a modified Ellman
method.19 The values were expressed as µmol reduced
glutathione present/100 mg fresh tissue.
Glutathione reductase. Glutathione reductase (GR)
activity was estimated from the liver tissue by the
method of Carlberg and Mannervik.20 The activity of
GR was expressed as units/min/mg protein, where a
unit of GR activity is defined as the amount of enzyme
that catalyzes the reduction of 1 µmol GSSG per minute
Glutathione-s-transferase. The glutathione-s-trans-
ferase (GST) activity from liver tissue was assayed by
Warholm et al.21 The enzyme activity was expressed as
units/min/mg protein. One unit of GST is the amount
of the enzyme that catalyzes the formation of 1 µmol S-
2, 4-dinitrophenyl glutathione per minute at 30°C.
Glutathione peroxidase. The glutathione peroxidase
(GSH-Px) activity was assayed from liver tissue by the
modified Paglia et al method.22 The enzyme activity
was expressed as units/min/mg protein, where 1 unit
of GSH-Px is equal to 1 µmol nicotinamide adenosine
dinucleotide phosphate oxidized per minute.
Superoxide dismutase. The superoxide dismutase
(SOD) activity was assayed from liver tissue by the
method of Kakkar et al.23 The enzyme activity was
INTEGRATIVE CANCER THERAPIES 6(3); 2007
 Antioxidant Activity of Andrographolide

Figure 1 Morphological observations of the mice liver in different groups. (A) Normal liver morphology of group 1 mice. (B) Liver tumor
in mice after 8 months of hexachlorocyclohexane (BHC) treatment (group 2). (C) BHC + andrographolide supplemented
(group 3) mouse liver at 6 months.

Student t test using state software. For comparison of

P < .001

20 2 or more than 2 groups at a time, ANOVA was used.

The Student t test was applied between 2 groups.
P < .001

15
P < .001

P < .01

10
Results

5 Gross Findings in the Liver

Liver tumor incidence was observed grossly in con-
0 trol and BHC-treated groups. Animals in the control
0 2 4 6 8 10
group did not show any liver lesions observed grossly
Months (Figure 1A). Similarly, animals with 2 months and
4 months of BHC exposure, respectively, did not
LW as % of BW group-I LW as % of BW group-II
show any gross nodular lesions. Only after 6 months
Figure 2 Changes in liver weight (LW) of mice during hexa-
of BHC exposure were small, multiple grayish or yel-
chlorocyclohexane (BHC)–induced hepato-carcinogen- lowish white nodules seen on the liver. Furthermore,
esis. LW as percentage of body weight (BW) in group after 8 months of BHC exposure, many large grayish
2 was compared with LW as percentage of BW in
group 1.
or yellowish white tumors up to 2 cm in diameter
(Figure 1B) with an irregular surface were found
in different liver lobes, indicating the severity of the
expressed as units/min/mg protein. One unit of the tumorous condition. The external morphological
enzyme activity is defined as the enzyme concentra- structure of the liver in animals supplemented with
tion required to inhibit the optical density at 560 nm ANDLE (group 3) for 6 months showed no severe
of chromogen production by 50% in 1 minute under morphological tumor lesion as compared to the
the specified assay conditions. group treated with BHC only (Figure 1C).

Catalase. The catalase (CAT) activity from liver tis-
sue was assayed by modified Luck method.24 The
enzyme activity was expressed as units/mg protein.
One unit of CAT represents 1 µmol of H2O2 decom-
posed per minute.
Statistical Methods
The results obtained from the biochemical studies
were analyzed using analysis of variance (ANOVA) and
Liver Weight as Percentage
of Body Weight
Liver weight (LW) as a percentage of body weight
(BW) is given in Figure 2. In the BHC-exposed group
(group 2), the LW as a percentage of BW showed
a significant increase after exposure to BHC for
2 months. This increase is primarily due to a significant
increase in the liver weight. The animals exposed to
4 months and 6 months of BHC showed further

INTEGRATIVE CANCER THERAPIES 6(3); 2007 273

 Trivedi et al

Table 2. Multivariate Analysis of Antioxidant Enzymes Between Groups and Duration of Treatment

Groups (1 to 4) Duration (1 to 8 mo)

Antioxidant Enzymes F Value P Value F Value P Value

γ-Glutamyl transpeptidase 2.5847 .04336 10.77333 .00017

Lipid peroxidation 2.4948 .062 24.4034 <.0001
Glutathione 3.366 .0144 35.2292 <.0001
Glutathione reductase 5.4477 .00113 53.55774 <.0001
Glutathione peroxidase 2.669 .038352 28.90657 <.0001
Glutathione-S-transferase 2.5545 .04533 28.8918 <.0001
Superoxide dismutase 2.4986 .0492 34.7681 <.0001
Catalase 3.7959 .0081 33.39941 <.0001

Table 3. γ-Glutamyl Transpeptidase Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SE Mean ± SE P Mean ± SE P Mean ± SE P Mean ± SE P Mean ± SE P

1 44.69 ± 0.53 62.34 ± 1.46 <.01 46.31 ± 0.89 <.001 44.52 ± 0.73 <.001 44.39 ± 0.39 <.001 41.99 ± 1.06 NS
2 45.39 ± 0.69 69.03 ± 1.82 <.001 53.32 ± 0.97 <.001 52.43 ± 0.88 <.001 51.93 ± 0.79 <.001 43.29 ± 1.27 NS
3 44.72 ± 1.32 73.82 ± 1.93 <.001 59.84 ± 1.03 <.001 60.31 ± 1.13 <.001 58.14 ± 0.99 <.001 43.18 ± 1.97 NS
4 45.94 ± 1.09 82.72 ± 1.98 <.001 63.24 ± 1.44 <.001 63.11 ± 1.37 <.001 63.02 ± 1.09 <.001 45.19 ± 1.17 NS
5 42.54 ± 1.02 99.06 ± 2.08 <.001 80.97 ± 2.03 <.001 80.06 ± 1.96 <.001 79.44 ± 1.82 <.001 43.11 ± 0.97 NS
6 47.56 ± 1.43 129.26 ± 2.92 <.001 98.29 ± 2.26 <.001 95.19 ± 2.07 <.001 96.47 ± 2.17 <.001 44.89 ± 1.09 NS
7 49.03 ± 1.62 149.97 ± 3.06 <.001 115.82 ± 3.06 <.001 114.80 ± 2.97 <.001 112.92 ± 2.89 <.001 45.32 ± 1.97 NS
8 48.29 ± 1.42 199.82 ± 3.29 <.001 137.89 ± 3.29 <.001 134.82 ± 3.11 <.001 131.22 ± 3.02 <.001 46.98 ± 1.81 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit = µmol p-nitroanilide released/min/mg protein. Values
are mean ± SE of 8 animals in each group. Group 1 is compared with group 2, group 2 is compared with group 3, and group 1 is com-
pared with group 4.

increases in LW. However, there was a marked change
after 8 months of BHC exposure, with the ratio being
18.992, which is almost 8 times more than the initial
control value.
Biochemical Findings
Multivariate analysis of antioxidant enzymes between
groups and duration of treatment. γ-GTP, LPO, GSH, GR,
GSH-Px, GST, SOD, and CAT activities were com-
pared between the groups and treatment durations
using ANOVA. There were significant differences
found for each enzyme activity between the groups
and treatment duration (Table 2). Therefore, every
enzyme activation was compared between the 4 groups
using the Student t test.
γ-Glutamyl transpeptidase. γ-GTP activity in different
groups is shown in Table 3. The activity of the γ-GTP
enzyme increased in group 2 in comparison to group
1. For durations of 1 to 8 months (BHC treatment),
the difference in γ-GTP activity was highly significant
(P < .001), but after 4 months and up to 8 months of
BHC exposure, the γ-GTP activity was drastically
increased as compared with group 1 (control). A
decrease in γ-GTP activity was observed in group 3
(BHC with ANDLE supplement). The decrease in
enzyme activity was highly significant (P < .001) in
group 3 as compared with group 2, while the γ-GTP
activity in group 4 was not significant as compared
with group 1.
Lipid peroxidation.. LPO activity in different groups
is shown in Table 4. There was a highly significant
(P < .001) increase in the activity of LPO in group 2
(BHC treated) as compared with group 1. However,
the LPO activity gradually increased on BHC exposure
from 1 to 8 months in group 2. Group 3 (BHC +
ANDLE treated) showed a significant decrease (P <
.001) in enzyme activity as compared with group 2.
There were nonsignificant changes observed in group
4 as compared with group 1.

274 INTEGRATIVE CANCER THERAPIES 6(3); 2007

 Antioxidant Activity of Andrographolide

Table 4. Lipid Peroxidation Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SE Mean ± SE P Mean ± SE P Mean ± SE P Mean ± SE P Mean ± SE P

1 142.93 ± 2.63 163.90 ± 1.91 <.01 149.22 ± 3.06 <.01 146.14 ± 2.03 <.001 143.74 ± 1.83 <.001144.22 ± 2.11 NS
2 139.43 ± 2.34 189.32 ± 1.94 <.001 159.24 ± 2.49 <.01 151.92 ± 1.88 <.001 149.28 ± 2.17 <.001140.39 ± 3.49 NS
3 141.76 ± 2.97 189.32 ± 1.94 <.001 165.11 ± 2.17 <.01 163.17 ± 2.04 <.001 161.92 ± 1.96 <.001140.96 ± 2.11 NS
4 143.56 ± 2.76 198.47 ± 2.21 <.001 179.19 ± 1.99 <.01 174.19 ± 2.02 <.001 172.81 ± 2.31 <.001140.11 ± 2.93 NS
5 138.92 ± 1.91 229.82 ± 2.59 <.001 199.13 ± 2.58 <.001 194.15 ± 2.76 <.001 193.27 ± 2.43 <.001137.96 ± 2.87 NS
6 140.42 ± 1.07 261.14 ± 3.07 <.001 213.08 ± 2.42 <.001 209.06 ± 2.64 <.001 205.15 ± 2.83 <.001138.19 ± 2.29 NS
7 139.89 ± 1.44 277.03 ± 3.78 <.001 230.19 ± 2.91 <.001 222.14 ± 3.07 <.001 219.6 ± 2.98 <.001137.14 ± 2.17 NS
8 141.91 ± 1.09 287.11 ± 4.19 <.001 258.17 ± 3.82 <.001 252.51 ± 3.49 <.001 248.91 ± 3.38 <.001 139.1 ± 2.29 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit = nanomol MDA/h/mg tissue weight. Values are
mean ± SE of 6 to 9 observations in each group. Group 1 is compared with group 2; group 2 is compared with group 3, and group 1
is compared with group 4.

Table 5. Glutathione Level in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 7.47 ± 0.139 5.03 ± 0.049 <.001 7.94 ± 0.093 <.001 7.34 ± 0.083 <.001 8.07 ± 0.103 <.001 7.89 ± 0.103 NS
2 7.09 ± 0.183 5.18 ± 0.63 <.001 7.03 ± 0.089 <.001 7.24 ± 0.079 <.001 7.69 ± 0.098 <.001 8.89 ± 0.146 NS
3 8.03 ± 0.243 4.17 ± 0.103 <.001 7.22 ± 0.113 <.001 7.63 ± 0.143 <.001 7.49 ± 0.123 <.001 8.91 ± 0.103 NS
4 8.17 ± 0.121 3.98 ± 0.119 <.001 6.29 ± 0.133 <.001 6.81 ± 0.203 <.001 7.11 ± 0.193 <.001 9.07 ± 0.131 <.01
5 8.21 ± 0.127 3.69 ± 0.123 <.001 5.81 ± 0.187 <.001 6.09 ± 0.204 <.001 5.96 ± 0.192 <.001 9.26 ± 0.039 <.01
6 7.76 ± 0.079 3.07 ± 0.042 <.001 5.02 ± 0.039 <.001 5.28 ± 0.104 <.001 5.53 ± 0.140 <.001 9.86 ± 0.089 <.001
7 7.09 ± 0.152 2.98 ± 0.039 <.001 4.86 ± 0.032 <.001 4.92 ± 0.084 <.001 4.89 ± 0.059 <.001 10.21 ± 0.149 <.01
8 7.82 ± 0.096 2.91 ± 0.089 <.001 4.39 ± 0.084 <.001 4.53 ± 0.071 <.001 4.77 ± 0.083 <.001 9.97 ± 0.281 <.01

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (glutathione) = µmol glutathione/100 mg tissue
weight. Values are mean ± SE of 6 to 9 observations in each group. Group 1 is compared with group 2, group 2 is compared with group
3, and group 1 is compared with group 4.

Glutathione. The GSH level in different groups is
shown in Table 5. There was a drastic decrease in the
level of GSH in group 2 as compared with the non-
treated group 1. The decrease in the GSH was highly
significant (P < .001) in group 2. The decreased level
of GSH was dependent on duration of BHC treat-
ment. From 4 to 8 months, the GSH level was dras-
tically decreased. The GSH level was significantly
increased in group 3 (BHC + ANDLE supplement)
when compared with group 2 (BHC treated only).
However, the GSH level increased in group 4: the level
of GSH significantly increased from 2 to 8 months in
group 4 as compared with group 1.
Glutathione reductase. GR activity in different groups
is shown in Table 6. There was a significant decrease in
GR activity in group 2 (BHC treated). The GR activity
was significantly decreased in BHC-treated animals for
a period of 8 months. From 5 to 8 months, the GR
activity showed the highest decrease (P < .001) when
compared to control animals (group 1). On the other
hand, the GR activity was significantly increased in
group 3 (BHC +ANDLE supplemented) as compared
to group 2. The GR activity in group 4 appeared to be
very close to the control value in group 1.
Glutathione peroxidase. GSH-Px activity in different
groups is shown in Table 7. GSH-Px activity was signifi-
cantly (P < .001) decreased in group 2 as compared
with group 1. The activity of GSH-Px gradually
decreased from 1 to 8 months of BHC treatment.
A highly significant (P < .001) increase was found in

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 Trivedi et al

Table 6. Glutathione Reductase Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4 (

(Control) (BHC Treated) 5 mg 7 mg 10 mg ANDLE)

Duration,
mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 36.89 ± 0.97 29.92 ± 0.73 <.001 33.11 ± 0.91 <.01 33.42 ± 0.83 <.01 34.17 ± 0.93 <.001 36.11 ± 0.79 NS
2 36.72 ± 0.79 28.64 ± 1.03 <.001 32.61 ± 0.82 <.01 33.67 ± 0.92 <.01 33.81 ± 0.88 <.001 35.93 ± 1.17 NS
3 36.14 ± 0.93 25.91 ± 1.16 <.001 29.79 ± 0.88 <.01 30.71 ± 0.92 <.001 30.93 ± 0.71 <.001 37.39 ± 0.89 NS
4 37.62 ± 1.18 23.07 ± 1.19 <.001 27.82 ± 0.93 <.01 28.03 ± 0.73 <.001 28.74 ± 0.81 <.001 38.11 ± 1.06 NS
5 35.91 ± 0.93 18.93 ± 0.97 <.001 23.11 ± 1.03 <.01 23.92 ± 0.88 <.001 24.99 ± 0.93 <.001 38.03 ± 1.73 NS
6 34.19 ± 0.92 18.14 ± 0.94 <.001 23.83 ± 0.98 <.001 23.29 ± 1.04 <.01 24.53 ± 0.98 <.001 35.74 ± 1.39 NS
7 37.12 ± 0.89 16.04 ± 0.72 <.001 20.93 ± 1.02 <.01 21.14 ± 0.98 <.001 21.92 ± 0.88 <.001 36.19 ± 0.93 NS
8 36.14 ± 0.69 14.13 ± 0.69 <.001 20.29 ± 1.11 <.001 20.89 ± 1.02 <.001 20.56 ± 0.92 <.001 36.88 ± 0.69 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (glutathione reductase) = µmol GSSG
catalyzed/min. Values are mean ± SE of 7 to 9 observations in each group. Group 1 is compared with group 2, group 2 is compared
with group 3, and group 1 is compared with group 4.

Table 7. Glutathione Peroxidase Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 229.21 ± 1.93 215.03 ± 0.96 <.001 228.04 ± 0.93 <.001 229.11 ± 0.98 <.001 229.69 ± 0.82 <.001228.14 ± 1.09 NS
2 230.17 ± 1.12 199.14 ± 1.07 <.001 214.92 ± 0.89 <.001 215.27 ± 0.97 <.001 216.13 ± 1.03 <.001231.07 ± 1.09 NS
3 232.19 ± 1.49 174.94 ± 1.04 <.001 201.66 ± 1.05 <.001 204.81 ± 1.23 <.001 203.63 ± 1.17 <.001231.14 ± 1.10 NS
4 231.66 ± 1.06 170.04 ± 1.41 <.001 197.81 ± 1.27 <.001 199.13 ± 1.07 <.001 199.94 ± 1.37 <.001232.14 ± 0.97 NS
5 232.07 ± 1.23 162.19 ± 1.73 <.001 189.23 ± 1.02 <.001 190.04 ± 1.24 <.001 190.22 ± 1.17 <.001233.03 ± 1.07 NS
6 235.12 ± 2.04 152.72 ± 1.62 <.001 179.63 ± 1.87 <.001 182.11 ± 1.29 <.001 177.32 ± 1.81 <.001230.19 ± 1.98 NS
7 241.32 ± 2.11 139.24 ± 1.83 <.001 168.14 ± 1.82 <.001 172.93 ± 1.56 <.001 170.68 ± 1.72 <.001233.38 ± 2.09 NS
8 236.64 ± 2.61 131.96 ± 2.13 <.001 153.81 ± 1.92 <.001 159.24 ± 1.63 <.001 161.22 ± 1.87 <.001231.42 ± 1.93 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (glutathione peroxidase) = µmol nicotinamide
adenosine dinucleotide phosphate oxidized/min/mg protein. Values are mean ± SE of 6 to 9 observations in each group. Group 1 is
compared with group 2, group 2 is compared with group 3, and group 1 is compared with group 4.

GSH-Px in group 3 (BHC + ANDLE supplemented) as
compared with the group 2. There were no changes
observed in the activity of this enzyme in group 4 as
compared with group 1.
Glutathione-s-transferase. GST activity in different
groups is shown in Table 8. There was a gradual
increase in GST activity in group 2 as compared with
group 1. The activity of GST showed a highly signifi-
cant (P < .001) increase in group 2; during months 4
to 8 of BHC exposure, the GST activity was drastically
increased as compared with the control animals
(group 1). However, in group 3 (BHC + ANDLE sup-
plement), the GST activity showed a highly signifi-
cant (P < .001) decrease in comparison with group 2
(BHC treated). No significant changes were observed
in group 4 as compared with group 1.
Superoxide dismutase. SOD activity in different groups
is shown in Table 9. A highly significant decrease (P <
.001) in SOD activity in group 2 was observed as com-
pared with group 1. The activity of SOD in group 2
gradually decreased from 1 month to 8 months of BHC
treatment. The activity of SOD increased further in
group 3 as compared with group 2. The SOD activity
in group 4 was very close to the control animals in
group 1.
Catalase. CAT activity in different groups is shown
in Table 10. CAT activity was significantly (P < .001)
decreased in group 2 (BHC treated) mice as com-
pared with group 1 (control mice), with the decrease
being very high in 6 to 8 months of BHC treatment.
There was a highly significant increase (P < .001) in
CAT activity for 1 to 7 months in group 3 as compared

276 INTEGRATIVE CANCER THERAPIES 6(3); 2007

 Antioxidant Activity of Andrographolide

Table 8. Glutathione-S-Transferase activity in different groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration, mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 926 ± 3.19 1042 ± 3.93 <.001 952 ± 2.08 <.001 947 ± 2.27 <.001 943 ± 3.81 <.001 922 ± 3.88 NS
2 930 ± 3.77 1079 ± 4.02 <.001 997 ± 3.38 <.001 989 ± 3.57 <.001 968 ± 3.03 <.001 928 ± 2.78 NS
3 931 ± 4.03 1143 ± 4.21 <.001 1029 ± 2.96 <.001 1024 ± 3.15 <.001 1019 ± 3.67 <.001 926 ± 4.11 NS
4 927 ± 3.89 1191 ± 4.08 <.001 1043 ± 4.11 <.001 1039 ± 3.59 <.001 1034 ± 4.03 <.001 921 ± 3.59 NS
5 931 ± 4.19 1219 ± 5.49 <.001 1103 ± 3.53 <.001 1094 ± 4.74 <.001 1085 ± 4.68 <.001 925 ± 3.96 NS
6 939 ± 5.06 1329 ± 4.95 <.001 1127 ± 4.79 <.001 1111 ± 4.29 <.001 1107 ± 3.98 <.001 931 ± 4.28 NS
7 942 ± 4.87 1469 ± 4.93 <.001 1186 ± 5.07 <.001 1152 ± 6.04 <.001 1164 ± 5.49 <.001 929 ± 5.39 NS
8 930 ± 5.14 1507 ± 8.84 <.001 1198 ± 5.82 <.001 1179 ± 4.88 <.001 1180 ± 5.29 <.001 928 ± 3.93 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (glutathione-S-transferase) = µmol

S-2,4-dinitrophenyl glutathione/min. Values are mean ± SE of 7 to 9 observations in each group. Group 1 is compared with group 2,
group 2 is compared with group 3, group 1 is compared with group 4.

Table 9. Superoxide Dismutase Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 8.326 ± 0.245 5.496 ± 0.029 <.001 7.239 ± 0.067 <.001 7.492 ± 0.059 <.001 7.629 ± 0.046 <.001 8.153 ± 0.621 NS
2 7.862 ± 0.381 5.126 ± 0.06 <.001 6.927 ± 0.042 <.001 7.089 ± 0.026 <.001 7.121 ± 0.039 <.001 7.692 ± 0.247 NS
3 7.912 ± 0.135 4.518 ± 0.079 <.001 6.078 ± 0.032 <.001 6.192 ± 0.023 <.001 6.297 ± 0.029 <.001 7.625 ± 0.642 NS
4 7.341 ± 0.381 3.982 ± 0.106 <.001 5.212 ± 0.079 <.001 5.621 ± 0.026 <.001 5.829 ± 0.031 <.001 7.139 ± 0.862 NS
5 7.892 ± 0.092 3.719 ± 0.098 <.001 4.896 ± 0.018 <.01 4.982 ± 0.022 <.001 5.016 ± 0.026 <.001 7.236 ± 0.382 NS
6 7.812 ± 0.43 3.29 ± 0.086 <.001 4.519 ± 0.034 <.001 4.692 ± 0.041 <.01 4.726 ± 0.062 <.001 7.823 ± 0.231 NS
7 8.41 ± 0.62 2.784 ± 0.077 <.001 4.013 ± 0.052 <.01 4.196 ± 0.046 <.001 4.309 ± 0.054 <.001 7.831 ± 0.699 NS
8 8.260 ± 0.89 2.49 ± 0.072 <.001 3.921 ± 0.091 <.01 4.009 ± 0.061 <.02 4.017 ± 0.027 <.01 8.021 ± 0.312 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (superoxide dismutase) = unit/min/mg protein.
Values are mean ± SE of 7 to 9 observations in each group. Group 1 is compared with group 2, group 2 is compared with group 3, and
group 1 is compared with group 4.

with group 2. Furthermore, in the eighth month in
group 3 (BHC + ANDLE supplement), the CAT activ-
ity was significantly increased (P < .02) as compared
with group 2. In group 4, the CAT activity showed no
significant changes as compared with group 1.
Discussion
Many plant products are known to exert antioxidative
effects by quenching various free radicals and singlet
oxygen, as mentioned by Aruna and Sivaramakrishnan.25
In the present study, while studying effects of ANDLE on
liver tumors, an attempt has also been made to deter-
mine the antioxidative effect of ANDLE. This plant
extract activates antioxidant enzymes that catalyze the
reaction of oxidants in BHC-treated mice.
The liver is the first organ in which investigations
of dose-response relationships in chemical carcino-
genesis were carried out systematically.26 The deter-
mination of the induction and the total dose
required for the production of liver tumors by differ-
ent hepatocarcinogens led to the well-known concept
of Druckrey27 that the primary carcinogenic effects of
all individual doses act additively to form the final
tumor. In the present study, the incidence of tumors
was directly correlated with the total dose level of
BHC, as indicated by the duration of exposure. No

INTEGRATIVE CANCER THERAPIES 6(3); 2007 277

 Trivedi et al

Table 10. Catalase Activity in Different Groups

Group 3 (BHC + ANDLE)

Group 1 Group 2 Group 4

(Control) (BHC Treated) 5 mg 7 mg 10 mg (ANDLE)

Duration,
mo Mean ± SD Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P Mean ± SD P

1 68.65 ± 0.36 53.09 ± 0.32 <.001 68.89 ± 0.39 <.001 68.73 ± 0.51 <.001 69.14 ± 0.43 <.001 67.69 ± 0.87 NS
2 69.96 ± 0.76 48.19 ± 0.69 <.001 61.77 ± 0.63 <.001 63.89 ± 0.63 <.001 64.09 ± 0.98 <.001 70.14 ± 0.69 NS
3 70.79 ± 0.74 42.89 ± 0.47 <.001 57.41 ± 0.69 <.001 59.17 ± 0.47 <.001 61.69 ± 0.82 <.001 71.63 ± 0.81 NS
4 68.79 ± 0.69 38.19 ± 0.62 <.001 53.11 ± 58 <.001 54.93 ± 0.81 <.001 56.07 ± 0.49 <.001 70.09 ± 1.03 NS
5 70.25 ± 0.49 32.79 ± 0.87 <.001 46.11 ± 0.93 <.001 47.72 ± 0.89 <.001 49.23 ± 0.88 <.001 72.06 ± 0.98 NS
6 71.09 ± 0.81 29.17 ± 1.06 <.001 40.06 ± 0.39 <.001 41.29 ± 0.76 <.001 43.09 ± 0.69 <.001 73.44 ± 1.29 NS
7 69.97 ± 0.26 24.86 ± 0.81 <.001 33.81 ± 0.72 <.001 35.14 ± 0.66 <.001 37.59 ± 0.83 <.001 70.96 ± 0.87 NS
8 67.82 ± 0.52 20.92 ± 0.89 <.001 27.96 ± 0.61 <.01 29.11 ± 0.67 <.001 31.96 ± 0.81 <.001 71.06 ± 0.98 NS

ANDLE = andrographolide; BHC = hexachlorocyclohexane; NS = nonsignificant. Unit (catalase) = unit/min/mg protein. Values are mean
± SE of 6 to 9 observations in each group. Group 1 is compared with group 2, group 2 is compared with group 3, and group1 is com-
pared with group 4.

liver tumors developed in animals after 2 months of
BHC exposure. However, tumors appeared after
BHC treatment for longer durations of time, and the
maximum tumor development occurred after 8
months of BHC treatment. The result of the present
study indicates a dose-dependent relationship for the
incidence of tumors, as suggested by Druckrey27 and
McGee et al.28
Because chlorinated insecticides are metabolized in
the liver, the increase in LW as a percentage of BW
observed in these experiments in group 2 (BHC-treated
group) may be an indicator of the overall effect of BHC
on the liver. The increase in LW as a percentage of BW
after exposure of various hepatotoxic chemicals includ-
ing organochlorine insecticides has been reported.29
Toxic chemical effects were associated with an increase
in liver weight, observed within the initial few weeks of
exposure, indicating that the liver is involved at an early
stage of exposure. These changes were more pro-
nounced with a longer duration of exposure, and sub-
stantial cellular damage took place when the duration
of BHC exposure was longer than 6 months. In the
period of 6 to 8 months, the LW as a percentage of BW
was at a maximum. The change was very significant after
6 months of exposure. Eltze et al30 reported that liver
undergoing neoplastic changes increased more
markedly in size and weight. On the basis of these sug-
gestions, BHC exposure as a liver tumor model was
investigated, and effects of ANDLE were observed.
γ-GTP, formerly known as γ-glutamyl transferase,
is the most sensitive indicator of liver disease and is a
useful marker in patients with liver metastases.31 A
number of drugs and chemicals are known to increase
γ-GTP activity by the induction of microsomal enzymes,
such as barbiturates, antidepressants, anticonvulsants,
and contraceptives.32 The present study showed signifi-
cantly increased activity of γ-GTP after BHC exposure.
During 4 months to 8 months of BHC exposure, the γ-
GTP activity indicated a many-fold increase. Tumors
were observed after BHC exposure for 6 to 8 months.
However, even by that time, a significant elevation of γ-
GTP activities was observed as compared with control
mice. Such a multifold increase in γ-GTP activity in
chemically induced rat hepatoma has been reported
earlier.33 After chronic treatment and until the develop-
ment of hepatocellular carcinoma, a 20- to 60-fold
increase in liver γ-GTP was reported, which is compara-
ble to the activity measured in fetal rat liver.34 Earlier
work in immunohistochemical and enzyme histochem-
ical studies on γ-GTP in rat liver has been used during
3-me-DAB hepatocarcinogenesis.35 Pompella et al36
showed the decreased levels of protein thiols in GGT-
positive nodules induced by chemical carcinogenesis in
the rat liver.
These findings correlate with the results of this
study, showing elevated levels of γ-GTP in liver
tumors induced by BHC. The hepatoprotective prop-
erty of silymarin has also been related to the inhibi-
tion of γ-GTP, and the decrease of γ-GTP activity was
reported.37
The GSH/GST detoxification system is an impor-
tant part of the cellular defense against a large array of
injurious agents. Reduced GSH, a tripeptide, is essen-
tial to maintain structural and functional integrity of
cells. The maintenance of GSH levels depends on the
activities of various enzymes, such as GR and GST.
Since GR affects the reduction of GSH, the level of this
enzyme is also of importance in detoxification of per-
oxides.38 An effective defense against oxidative dam-
age is the GSH cycle, which includes oxidation of GSH

278 INTEGRATIVE CANCER THERAPIES 6(3); 2007


to GSSG during detoxification of peroxidation by
GSH-Px and GST and further reduction of GSSG to
GSH by GR.
LPO reflects the interaction between molecular
oxygen and polyunsaturated fatty acids, and it results
in the oxidative deterioration of the latter. The nat-
ural targets for such reactions are the biological
membranes; therefore, oxidative damage to these
membranes results in an impairment of cellular and
subcellular functions, changes in permeability, and
morphological alterations. Free radicals disrupt the
equilibrium of biological systems by damaging their
constituent molecules, leading eventually to cell death.
The inhibitory effect of ANDLE on LPO is related to
membrane-stabilizing activity.
SOD is the principle protective enzyme that dis-
mutes the reactive oxygen radical to hydrogen per-
oxide and oxygen. Hydrogen peroxide produced
can then be decomposed enzymatically by CAT or by
GSH-PX. GSH-PX not only decomposes hydrogen
peroxide but is also capable of interacting with LPO.
In the present study, marked increases in SOD, CAT,
and GSH-PX activities in ANDLE-supplemented ani-
mals showed the adaptive nature of the system
against damaging effects of superoxide radicals in
liver as brought about by severe hepatic damage by
BHC. Decreased activities of GSH, GR, CAT, SOD,
and GSH-PX in BHC-treated mice may increase their
susceptibility to oxidative injury. However, the over-
expression of the antioxidant molecule ANDLE is
indicative of its ability to reactivate hepatocellular
antioxidant defense in the liver. Koul and Kapil10
reported the effects of diterpenes from A paniculata
on antioxidant defense systems and LPO. They
showed the adaptive nature of the system against
hepatotoxicity from CCl4. Campos et al39 and
Chander et al40 have reported that hepatoprotective
plant principles also possess antioxidative activity.
A recent report by Sheeja and Kuttan41 showed that
cyclophosphamide-induced urothelial toxicity in mice
via reactive oxygen species production and treatment
with ANDLE resulted in elevated levels of GSH in the
liver and bladder of cyclophosphamide-treated mice.
Nitric oxide is a highly reactive free radical associated
with the severity of cyclophosphamide-induced blad-
der injury.41 Administration of ANDLE has been
shown to reduce the level of nitric oxide metabolite
and protect mice from cyclophosphamide-induced
urinary bladder injury.41
The results of the present study suggest that the
antioxidative effect of ANDLE may be due to its abil-
ity to activate antioxidant enzymes that catalyze the
reduction of oxidants and that its use for recovery of
hepatic damage may be beneficial.
INTEGRATIVE CANCER THERAPIES 6(3); 2007
Antioxidant Activity of Andrographolide
Acknowledgment
The authors are thankful to the Indian Council of
Medical Research for financial support.
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