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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 Recent studies have reported the antioxidant prop-
by quenching various free radicals and singlet molecular oxy- erties of A paniculata on in vivo and in vitro model11
gen. Andrographis paniculata (Kalmegh) is used extensively in and diabetic rats.12 A diterpene, andrographolide
the Indian traditional system of medicine as a hepatoprotec- (ANDLE), is the active constituent of A paniculata.
tive and hepatostimulative agent and has been reported to Based on our previous findings, the present article
have antioxidant effects against different hepatotoxins. The
confirms the antioxidative properties of ANDLE
present study aims to analyze antioxidant properties of an
active component, andrographolide (ANDLE), extracted
extracted from A paniculata on BHC-treated mice.
from A paniculata. This study investigates the effect of andro-
grapholide on the hepatocellular antioxidant defense system Materials and Methods
and lipid peroxidation of control mice, mice treated with
hexachlorocyclohexane (BHC) only, and andrographolide + Extraction of Active Compound
BHC. Glutathione (GSH), glutathione-s-transferase (GST), The andrographolide (a pure active compound) was
glutathione reductase (GR), glutathione peroxidase (GSH- obtained from the BV Patel Pharmaceutical Educa-
Px), γ-glutamyl transpeptidase (γγ-GTP), superoxide dismu- tional and Research Development Centre, Ahmedabad,
tase (SOD), catalase (CAT), and lipid peroxidation (LPO)
India. The andrographolide was administered orally
are studied by spectrophotometric methods. The BHC
experimental model forms an irreversible liver tumor in
in 3 doses: low (5 mg/kg body weight), medium
male mice. The activities of GSH, GR, GSH-Px, SOD, and (7 mg/kg body weight), and high (10 mg/kg body
CAT show significant (P ≤ .05) increases, while γ-GTP and weight) per animal per day for the period of 1 to
GST show significant decreases (P ≤ .05) in andrographolide- 8 months.
supplemented mice as compared with BHC-treated mice.
This study indicates that the antioxidant effect of andro- Experimental Animals
grapholide could be due to its ability to activate antioxidant Healthy, adult, pathogen-free, colony-breed male
enzymes that catalyze the reaction of oxidants and are effec- albino mice (Mus musculus) of Swiss strain, 1 month
tive in severe liver damage. old, weighing between 20 and 30 g, were used in this
study. The Indian Council of Medical Research guide-
Keywords: andrographolide; liver damage; antioxidant enzymes 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
Andrographis paniculata Nees (Acanthaceae) is a well-
Sciences, Gujarat University, Ahmedabad, India.
known medicinal plant in Ayurveda. It has been used
as a bitter and febrifuge. Recent research has thrown
Experimental Tumor Model
light on the medicinal value of the plant. The extract
Technical-grade BHC was used for inducing the car-
of whole plant has been reported to have anticancer,
cinogenic condition in the liver of male mice as per
anti-inflammatory, antiallergic, immunostimulatory,
the method of Nigam et al.13,14
antithrombotic, antiviral, hypoglycemic, and hypo-
tensive activities.1-8 Previously, we reported the anti-
NPT and UMR are at the Department of Zoology, School of
oxidant property of the whole plant extract of A Sciences, Gujarat University, Ahmedabad, India. BPP is at the Cell
paniculata on hexachlorocyclohexane (BHC)–treated Biology Division, Gujarat Cancer & Research Institute, Asarwa,
mice.9 Andrographolide, andrographiside, and neoan- Ahmedabad, India.
drographolide are active compounds of A paniculata.10 Correspondence: Upendra M. Rawal, Department of Zoology,
Gujarat University, Ahmedabad 380 009, India. E-mail: upendrarawal
DOI: 10.1177/1534735407305985 @gmail.com.

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Trivedi et al

Treatment Diet Table 1. Groups of Animals, Treatment, and Duration


The BHC-treated animals were given a basal diet Group Treatment and Dosage Duration, mo
with 500 ppm BHC/kg food. This dose approxi-
mates the highest tolerable limit causing significant 1 Control 1 to 8
2 BHC treated (500 ppm/kg fed) 1 to 8
mortality in test animals by Lakkad et al.15 The BHC
3 BHC with ANDLE (andrographolide
insecticide was weighed and dissolved in alcohol and supplements) per day per animal orally
homogeneously mixed in the diet; the alcohol was (a) Low dose
allowed to evaporate by drying. The dose of BHC BHC (500 ppm/kg) + ANDLE (5 mg/kg
500 ppm/kg food is selected on the basis of previous body weight)
(b) Medium dose
work on rodents.13,15
BHC (500 ppm/kg) + ANDLE (7 mg/kg
body weight)
The Experimental Design (c) High dose
BHC (500 ppm/kg) + ANDLE (10 mg/kg
The experimental design was as follows: body weight) 1 to 8
4 Positive control, only ANDLE treated
(7 mg/kg body weight) per day/
• Group 1 (control): animals were given normal diet
animal orally 1 to 8
and water ad libitum.
• Group 2 (BHC treated): animals in this group were ANDLE = andrographolide; BHC = hexachlorocyclohexane.
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
(MDA) and other breakdown products of peroxidized
of animals was given both BHC at a dose of 500 ppm/kg
food and ANDLE orally in low (5 mg/kg body
lipids, collectively called thiobarbituric acid reactive
weight), medium (7 mg/kg body weight), and high substance. The results were expressed as nanomoles of
(10 mg/kg body weight) doses per animal per day for MDA/h/mg tissue weight.
the period of 1 to 8 months. This group was consid-
ered the supplemented group. Glutathione. The glutathione (GSH) levels in liver
• Group 4 (ANDLE treated, positive control): only tissue were measured using a modified Ellman
ANDLE was given to this group of animals in a dose method.19 The values were expressed as µmol reduced
of 7 mg/kg body weight per animal per day orally. glutathione present/100 mg fresh tissue.

The various groups of animals, treatment, and Glutathione reductase. Glutathione reductase (GR)
duration are shown in Table 1. At the end of each activity was estimated from the liver tissue by the
treatment, the animals were weighed on an animal method of Carlberg and Mannervik.20 The activity of
balance and killed by cervical dislocation. The liver of GR was expressed as units/min/mg protein, where a
each animal was dissected out, blotted free of blood, unit of GR activity is defined as the amount of enzyme
and weighed on a balance. that catalyzes the reduction of 1 µmol GSSG per minute

Biochemical Methods Glutathione-s-transferase. The glutathione-s-trans-


Tissue protein. The protein content in the liver tis- ferase (GST) activity from liver tissue was assayed by
sue of mice was estimated according to the method Warholm et al.21 The enzyme activity was expressed as
of Lowry et al.16 units/min/mg protein. One unit of GST is the amount
of the enzyme that catalyzes the formation of 1 µmol S-
γ-Glutamyl transpeptidase. The liver γ-glutamyl trans- 2, 4-dinitrophenyl glutathione per minute at 30°C.
peptidase (γ-GTP) activity was assayed by the method
of Tate and Meister.17 The enzyme activity was Glutathione peroxidase. The glutathione peroxidase
expressed as units/min/mg protein, where 1 unit of (GSH-Px) activity was assayed from liver tissue by the
γ-GTP is equivalent to 1 µmol p-nitroanilide released modified Paglia et al method.22 The enzyme activity
per minute. was expressed as units/min/mg protein, where 1 unit
of GSH-Px is equal to 1 µmol nicotinamide adenosine
Lipid peroxidation. Lipid peroxidation (LPO) in the dinucleotide phosphate oxidized per minute.
liver was determined by the method of Ohkawa.18 The
method is based on the formation of a red chro- Superoxide dismutase. The superoxide dismutase
mophore that absorbs at 532 nm following the reac- (SOD) activity was assayed from liver tissue by the
tion of thiobarbituric acid with malonyl dialdehyde method of Kakkar et al.23 The enzyme activity was

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

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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 (P < .001), but after 4 months and up to 8 months of
after 8 months of BHC exposure, with the ratio being BHC exposure, the γ-GTP activity was drastically
18.992, which is almost 8 times more than the initial increased as compared with group 1 (control). A
control value. decrease in γ-GTP activity was observed in group 3
(BHC with ANDLE supplement). The decrease in
Biochemical Findings enzyme activity was highly significant (P < .001) in
Multivariate analysis of antioxidant enzymes between group 3 as compared with group 2, while the γ-GTP
groups and duration of treatment. γ-GTP, LPO, GSH, GR, activity in group 4 was not significant as compared
GSH-Px, GST, SOD, and CAT activities were com- with group 1.
pared between the groups and treatment durations
using ANOVA. There were significant differences
found for each enzyme activity between the groups Lipid peroxidation.. LPO activity in different groups
and treatment duration (Table 2). Therefore, every is shown in Table 4. There was a highly significant
enzyme activation was compared between the 4 groups (P < .001) increase in the activity of LPO in group 2
using the Student t test. (BHC treated) as compared with group 1. However,
the LPO activity gradually increased on BHC exposure
γ-Glutamyl transpeptidase. γ-GTP activity in different from 1 to 8 months in group 2. Group 3 (BHC +
groups is shown in Table 3. The activity of the γ-GTP ANDLE treated) showed a significant decrease (P <
enzyme increased in group 2 in comparison to group .001) in enzyme activity as compared with group 2.
1. For durations of 1 to 8 months (BHC treatment), There were nonsignificant changes observed in group
the difference in γ-GTP activity was highly significant 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 GR activity in group 2 (BHC treated). The GR activity
shown in Table 5. There was a drastic decrease in the was significantly decreased in BHC-treated animals for
level of GSH in group 2 as compared with the non- a period of 8 months. From 5 to 8 months, the GR
treated group 1. The decrease in the GSH was highly activity showed the highest decrease (P < .001) when
significant (P < .001) in group 2. The decreased level compared to control animals (group 1). On the other
of GSH was dependent on duration of BHC treat- hand, the GR activity was significantly increased in
ment. From 4 to 8 months, the GSH level was dras- group 3 (BHC +ANDLE supplemented) as compared
tically decreased. The GSH level was significantly to group 2. The GR activity in group 4 appeared to be
increased in group 3 (BHC + ANDLE supplement) very close to the control value in group 1.
when compared with group 2 (BHC treated only).
However, the GSH level increased in group 4: the level Glutathione peroxidase. GSH-Px activity in different
of GSH significantly increased from 2 to 8 months in groups is shown in Table 7. GSH-Px activity was signifi-
group 4 as compared with group 1. cantly (P < .001) decreased in group 2 as compared
with group 1. The activity of GSH-Px gradually
Glutathione reductase. GR activity in different groups decreased from 1 to 8 months of BHC treatment.
is shown in Table 6. There was a significant decrease in A highly significant (P < .001) increase was found in

INTEGRATIVE CANCER THERAPIES 6(3); 2007 275


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 Superoxide dismutase. SOD activity in different groups
compared with the group 2. There were no changes is shown in Table 9. A highly significant decrease (P <
observed in the activity of this enzyme in group 4 as .001) in SOD activity in group 2 was observed as com-
compared with group 1. pared with group 1. The activity of SOD in group 2
gradually decreased from 1 month to 8 months of BHC
Glutathione-s-transferase. GST activity in different treatment. The activity of SOD increased further in
groups is shown in Table 8. There was a gradual group 3 as compared with group 2. The SOD activity
increase in GST activity in group 2 as compared with in group 4 was very close to the control animals in
group 1. The activity of GST showed a highly signifi- group 1.
cant (P < .001) increase in group 2; during months 4
to 8 of BHC exposure, the GST activity was drastically Catalase. CAT activity in different groups is shown
increased as compared with the control animals in Table 10. CAT activity was significantly (P < .001)
(group 1). However, in group 3 (BHC + ANDLE sup- decreased in group 2 (BHC treated) mice as com-
plement), the GST activity showed a highly signifi- pared with group 1 (control mice), with the decrease
cant (P < .001) decrease in comparison with group 2 being very high in 6 to 8 months of BHC treatment.
(BHC treated). No significant changes were observed There was a highly significant increase (P < .001) in
in group 4 as compared with group 1. 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 extract activates antioxidant enzymes that catalyze the
group 3 (BHC + ANDLE supplement), the CAT activ- reaction of oxidants in BHC-treated mice.
ity was significantly increased (P < .02) as compared The liver is the first organ in which investigations
with group 2. In group 4, the CAT activity showed no of dose-response relationships in chemical carcino-
significant changes as compared with group 1. genesis were carried out systematically.26 The deter-
mination of the induction and the total dose
Discussion required for the production of liver tumors by differ-
Many plant products are known to exert antioxidative ent hepatocarcinogens led to the well-known concept
effects by quenching various free radicals and singlet of Druckrey27 that the primary carcinogenic effects of
oxygen, as mentioned by Aruna and Sivaramakrishnan.25 all individual doses act additively to form the final
In the present study, while studying effects of ANDLE on tumor. In the present study, the incidence of tumors
liver tumors, an attempt has also been made to deter- was directly correlated with the total dose level of
mine the antioxidative effect of ANDLE. This plant 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 and contraceptives.32 The present study showed signifi-
BHC exposure. However, tumors appeared after cantly increased activity of γ-GTP after BHC exposure.
BHC treatment for longer durations of time, and the During 4 months to 8 months of BHC exposure, the γ-
maximum tumor development occurred after 8 GTP activity indicated a many-fold increase. Tumors
months of BHC treatment. The result of the present were observed after BHC exposure for 6 to 8 months.
study indicates a dose-dependent relationship for the However, even by that time, a significant elevation of γ-
incidence of tumors, as suggested by Druckrey27 and GTP activities was observed as compared with control
McGee et al.28 mice. Such a multifold increase in γ-GTP activity in
Because chlorinated insecticides are metabolized in chemically induced rat hepatoma has been reported
the liver, the increase in LW as a percentage of BW earlier.33 After chronic treatment and until the develop-
observed in these experiments in group 2 (BHC-treated ment of hepatocellular carcinoma, a 20- to 60-fold
group) may be an indicator of the overall effect of BHC increase in liver γ-GTP was reported, which is compara-
on the liver. The increase in LW as a percentage of BW ble to the activity measured in fetal rat liver.34 Earlier
after exposure of various hepatotoxic chemicals includ- work in immunohistochemical and enzyme histochem-
ing organochlorine insecticides has been reported.29 ical studies on γ-GTP in rat liver has been used during
Toxic chemical effects were associated with an increase 3-me-DAB hepatocarcinogenesis.35 Pompella et al36
in liver weight, observed within the initial few weeks of showed the decreased levels of protein thiols in GGT-
exposure, indicating that the liver is involved at an early positive nodules induced by chemical carcinogenesis in
stage of exposure. These changes were more pro- the rat liver.
nounced with a longer duration of exposure, and sub- These findings correlate with the results of this
stantial cellular damage took place when the duration study, showing elevated levels of γ-GTP in liver
of BHC exposure was longer than 6 months. In the tumors induced by BHC. The hepatoprotective prop-
period of 6 to 8 months, the LW as a percentage of BW erty of silymarin has also been related to the inhibi-
was at a maximum. The change was very significant after tion of γ-GTP, and the decrease of γ-GTP activity was
6 months of exposure. Eltze et al30 reported that liver reported.37
undergoing neoplastic changes increased more The GSH/GST detoxification system is an impor-
markedly in size and weight. On the basis of these sug- tant part of the cellular defense against a large array of
gestions, BHC exposure as a liver tumor model was injurious agents. Reduced GSH, a tripeptide, is essen-
investigated, and effects of ANDLE were observed. tial to maintain structural and functional integrity of
γ-GTP, formerly known as γ-glutamyl transferase, cells. The maintenance of GSH levels depends on the
is the most sensitive indicator of liver disease and is a activities of various enzymes, such as GR and GST.
useful marker in patients with liver metastases.31 A Since GR affects the reduction of GSH, the level of this
number of drugs and chemicals are known to increase enzyme is also of importance in detoxification of per-
γ-GTP activity by the induction of microsomal enzymes, oxides.38 An effective defense against oxidative dam-
such as barbiturates, antidepressants, anticonvulsants, age is the GSH cycle, which includes oxidation of GSH

278 INTEGRATIVE CANCER THERAPIES 6(3); 2007


Antioxidant Activity of Andrographolide

to GSSG during detoxification of peroxidation by Acknowledgment


GSH-Px and GST and further reduction of GSSG to The authors are thankful to the Indian Council of
GSH by GR. Medical Research for financial support.
LPO reflects the interaction between molecular
oxygen and polyunsaturated fatty acids, and it results
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280 INTEGRATIVE CANCER THERAPIES 6(3); 2007