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Environmental Toxicology and Pharmacology 25 (2008) 321–328

Amelioratory effect of Andrographis paniculata

Nees on liver, kidney, heart, lung and spleen
during nicotine induced oxidative stress
Sreeparna Neogy, Subhasis Das, Santanu Kar Mahapatra,
Nirjal Mandal, Somenath Roy ∗
Immunology and Microbiology Laboratory, Department of Human Physiology with Community Health,
Vidyasagar University, Midnapore 721102, West Bengal, India
Received 1 June 2007; received in revised form 21 October 2007; accepted 23 October 2007
Available online 4 November 2007

Abstract
The ameliorative properties of bioactive compound andrographolide (ANDRO), aqueous extract of Andrographis paniculata (AE-AP) and vitamin
E (vit.E) were tested against nicotine induced liver, kidney, heart, lung and spleen toxicity. A group of male Wistar rats were intraperitoneally
administered vehicle, nicotine (1 mg/kg body weight/day), nicotine + ANDRO (250 mg/kg body weight/day), nicotine + AE-AP (250 mg/kg body
weight/day) and nicotine + vit.E (50 mg/kg body weight/day) for the period of 7 days. The significantly increased levels of lipid peroxidation,
protein oxidation and the decreased antioxidant enzyme status were noted in nicotine treated group as compared to vehicle treated group. ANDRO,
AE-AP and vit.E significantly reduced the lipid peroxidation, protein oxidation and increased the antioxidant enzyme status. This indicates A.
paniculata and vit.E may act as putative protective agent against nicotine induced tissue injury and may pave a new path to develop suitable drug
therapy.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Adrographis paniculata; Andrographolide; Nicotine; Oxidative stress; Tissues; Vitamin E

1. Introduction tobacco, into two highly mutagenic nitrosamine, N -nitrosonor

Nicotine, as most biologically active chemical in tobacco
smoke, has been the subject of intense scientific scrutiny. Among
the most well characterized chemicals found in tobacco and
tobacco smoke, are polycyclic aromatic hydrocarbons (PAHs)
and the highly addictive alkaloid, nicotine and its metabolites
(Campain, 2004). To further complicate the picture, nicotine
is converted, during the production of cigarette and chewing
Abbreviations: AE-AP, aqueous extract of Andrographis paniculata;
ANDRO, andrographolide; CAT, catalase; DTNB, 5,5-dithio-bis-2-nitrobenzoic
acid; EDTA, ethylenediamine tetraacetic acid; FTIR, Fourier transform infra red
spectroscopy; GR, glutathione reductase; GSH, reduce glutathione; GSH-Px,
glutathione peroxidase; GSSG, oxidized glutathione; HPLC, high pressure liq-
uid chromatography; MDA, malondialdehyde; OFR, oxygen free radicals; PC,
protein carbonyls; SOD, superoxide dismutase; TLC, thin layer chromatography.
∗ Corresponding author. Tel.: +91 3222 275329; fax: +91 3222 275329.
E-mail address: somenathroy@hotmail.com (S. Roy).
nicotine (NNN) and 4-(methylnitrosamine)-1-(3-pyridyl)-1-
butanone (NNK) and is metabolized into cotinine. These
chemicals derivatives also exhibit a wide spectrum of biological
activity as compared to parent compound (Campain, 2004).
Nicotine has been reported to induce oxidative stress both in
vivo and in vitro (Pigeolot et al., 1990). The mechanism of gener-
ation of free radicals by nicotine is not clear. But oxidative stress
occurs when there are excess free radicals and/or low antioxi-
dant defense, and result in chemical alteration of biomolecules
causing structural and functional modification. Oxygen free rad-
icals (OFR) production has been directly linked to oxidation
of cellular macromolecules, which may induce a variety of
cellular responses through generation of secondary metabolic
reactive species (Chiarugi, 2003). Previous reports have shown
enhanced lipid peroxidation and inadequate antioxidant status
by nicotine.
Medicinal plants and their active principles have received
greater attention as anti-peroxidative agent (Lee and Park, 2002).

1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.etap.2007.10.034
322 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
Andrographolide, the main active constituent of Adrographis
paniculata (A. paniculata), has excellent anti-inflammatory,
anti-bacterial and anti-viral effects. A. paniculata is an Indian
herb, well known as ‘king of bitter’. This bitter herb generally
has an affinity with heart and liver. New research has confirmed
a host of pharmacological benefits of this herb for its enor-
mous potential in far wide range of diseases. The present study
was undertaken to evaluate the amelioratory property of andro-
grapholide on tissue antioxidant status during nicotine induced
oxidative stress in male Wistar rats.
2. Materials and methods
2.1. Animals
The weight matched (120–140 g) male Wistar rats were obtained, divided
in different groups, housed in polypropylene cage and provided standard
pellet diet (Chaulia Equipment and Chemicals, Ganapatinagar, Midnapore,
India) and water ad libitum. The animals were maintained under standard
conditions of temperature (25 ± 2 ◦ C) and humidity (60 ± 5%) with an alter-
nating 12 h light/dark cycles. Animals were maintained in accordance with
the guidelines of the National Institute of Nutrition, Indian Council of Med-
ical Research, Hyderabad, India, and approved by the ethical committee of
Vidyasagar University. All the experiments were conduced with the ethi-
cal guidelines laid down by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA) constituted by the Animal
Welfare Division of Government of India on the use of animals in scientific
research.
2.2. Plant materials
A. paniculata Nees herbs were collected from the campus of IIT, Kharagpur,
West Bengal, India in July–August, 2005 and air-dried. A voucher specimen has
been deposited to the CAL herbarium, Botanical Survey of India, Howrah, India
under the accession number IIT-VU/Ap-1.
2.3. Chemicals and reagents
Standard andrographolide, epinephrine, TBA, DNPH, H2 O2 , EDTA, DTNB,
NADPH were purchased from Sigma Chemical Co., USA. Nicotine was pur-
chased from Merck, Germany. Other chemicals were procured from Merck Ltd.,
SRL Pvt. Ltd., Mumbai, India.
2.4. Treatment schedule
The animals were randomized into experimental and control groups and
divided into five groups of six animals each. Rats’ in-group ‘A’ served as control,
received 5% DMSO in physiological saline. Group ‘B’ animals received nicotine
[3-(1-methyl-2-pyrrolidinyl)pyridine, C10 H14 N2 ] 1.0 mg/kg body weight/day
(in physiological saline, pH was adjusted at 7.4 prior to injection), rats in
group ‘C’ were administered nicotine as in group ‘B’ as well as andrographolide
(250 mg/kg body weight/day) in 5% dimethyl sulfoxide (DMSO) with physio-
logical saline, rats in group ‘D’ received nicotine (1.0 mg/kg body weight/day)
as well as aqueous extract (250 mg/kg body weight/day) dissolved in 5%
DMSO with physiological saline and animals of group ‘E’ treated with nicotine
(1.0 mg/kg body weight/day) along with oral dose of vitamin E (50 mg/kg body
weight/day) in olive oil. Simultaneously, animals of group ‘A’, ‘B’, ‘C’ and ‘D’
were received olive oil orally. All the treatments were done intraperitoneally
(i.p.) except vitamin E and olive oil for the period of 7 days. The dose and
duration of nicotine were selected as per reported by many researchers (Chen
et al., 2001 and Tuncok et al., 2001) and the dose of ANDRO was chosen as
per the previous report (Madav et al., 1995). The experiment was terminated at
the end of 7 days and all animals were sacrificed by an intraperitoneal injection
of sodium pentobarbital (60–70 mg/kg body weight) (Chandran and Venugopal,
2004).
2.5. Tissue extracts preparation
After decapitation, liver, kidney, heart, lung and spleen were excised from
rats and washed with cold saline. Washed tissues were immediately immersed
in liquid nitrogen and stored at −80 ◦ C. On preparation, tissues were sliced
and homogenized in ice cold 50 mM sodium phosphate buffer (pH 7.0) con-
taining 0.1 mM ethylenediamine tetraacetic acid (EDTA) to yield 10% (w/v)
homogenate. The homogenates were then centrifuged at 1000 rpm for 10 min at
4 ◦ C. The supernatants were separated and used for enzyme assays and protein
determination (Husain et al., 2001).
2.6. Preparation of aqueous extract
The fresh aerial parts of A. paniculata was blended and extracted with dis-
tilled water (10:1). The mixture was filtered with Whatman filter paper (No. 1)
and concentrated at 38 ◦ C by a rotary evaporator, then allowed to stand at room
temperature over night. The filtration and concentration processes were repeated
to yield an aqueous solution. This solution was then centrifuged at 2000 × g for
10 min and supernatant was freeze dried to obtain the crude water extract (Zhang
and Tan, 1996).
2.7. Isolation of ANDRO by thin layer chromatography (TLC)
A. paniculata powder was homogenized and extracted with distilled water
(10:1). The mixture was filtered with Whatman filter paper (No. 1). The solution
was then centrifuged at 200 × g for 10 min and supernatant was collected. The
supernatant was mixed with ethyl acetate, yielding approximately 0.7% of ethyl
acetate fraction. Two gram of ethyl acetate fraction was dissolved in methanol
to obtain methanol extract (Zhang and Tan, 1997). The methanol extracts and
the reference andrographolide (Sigma Chemical Ltd.) was spotted, separated
on TLC plate. The plate was photographed after staining with 5% methanolic
sulphuric acid. After this, the parallel band of methanol extract matching with
the reference andrographolide was scrapped under ultra violet light (254 nm)
and then eluted out from the TLC plate in methanol. Then the methanol was
evaporated out by rotary evaporator and used for HPLC as well as biological
activity studies.
2.8. Detection of ANDRO by high performance liquid
chromatography (HPLC)
Isolated ANDRO was detected with standard andrographolide (Sigma
Chemical Ltd.) in Water HPLC at 229 nm in ␮ Bondapak C-18 column
(3.9 mm × 300 mm). The conditions are as follows:
Mobile phase: methanol:water (60:40), v/v; flow rate: 1 ml/ min; injection
volume: 20 ␮l; UV detection at 229 nm (Singha et al., 2007)
2.9. Analytical methods
2.9.1. Lipid peroxidation
The extent of lipid peroxidation was estimated as the concentration of thio-
barbituric acid reactive product malondialdehyde (MDA) by using the method of
Ohkawa et al. (1979). One hundred microliters of tissue homogenate was added
to 100 ␮l of double-distilled water and 50 ␮l of 8.1% sodium dodecyl sulfate
(SDS) and incubated at room temperature for 10 min. Three hundred seventy-
five microliters of 20% acetic acid (pH 3.5), along with 375 ␮l of thiobarbituric
acid (0.6%), was added to the tissue solution and placed in a boiling water
bath for 60 min. After incubation, 250 ␮l of double-distilled water and 1.25 ml
of 15:1 butanol–pyridine solution were added to the mixture and centrifuged
for 5 min at 2000 × g. The resulting supernatant was removed and measured at
532 nm with the use of the Hitachi U-2000 spectrophotometer. Malondialde-
hyde concentrations were determined by using 1,1,3,3-tetraethoxypropane as
standard.
2.9.2. Protein carbonyl
Protein carbonyl (PC) levels were measured according to method described
by Reznick and Packer (1994); based on spectrophotometric detection of the
 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328 323

reaction of 2,4-dinitrophenylhydrazine with protein carbonyl to form protein

hydrazones. Briefly, after precipitation of protein with an equal volume of 1%
trichloroacetic acid (TCA), the pellet was resuspended in 10 mM DNPH in 2N
HCl or with 2N HCl as control blank. Next after the washing procedure with
1:1 ethanol/ethylacetate, the final palette was dissolved in 6 M Guanidine. The
carbonyl group was determined from the absorbance at 370 nm. The result was
expressed as micromoles of carbonyl groups per milligram of protein with molar
extinction coefficient of 22,000 M−1 cm−1 .

2.9.3. Superoxide dismutase

Superoxide dismutase activity was determined at room temperature accord-
ing to the method of Misra and Fridovich (1972). Ten microliters of tissue
homogenate was added to 970 ␮l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodium
carbonate buffer. Twenty microliters of 30 mM epinephrine (dissolved in 0.05%
acetic acid) was added to the mixture to start the reaction. Superoxide dismutase
activity was measured at 480 nm for 4 min on a Hitachi U-2000 spectrophotome-
ter. Activity was expressed as the amount of enzyme that inhibits the oxidation
of epinephrine by 50%, which is equal to 1 U/mg of protein.

2.9.4. Catalase
Catalase activity was determined at room temperature by using a slightly
modified version of Aebi (1984). Ten microliters of ethanol was added to 100 ␮l
of tissue homogenate. The tissue mixture was then placed in an ice bath for
30 min and tubes were brought at room temperature, followed by the addition of
10 ␮l of Triton X-100 RS. Ten microliters of the tissue homogenate was added
to a cuvette containing 240 ␮l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodium Fig. 1. TLC of methanol extract (T) and standard andrographlide (S) after
phosphate buffer, and 250 ml of 0.066 M H2 O2 (dissolved in sodium phosphate derivatisation with 5% methanolic sulphuric acid at visible.
buffer) was added to start the reaction. Catalase activity was measured at 240 nm
for 1 min with the use of the Hitachi U-2000 spectrophotometer. The molar was expressed in terms of nanomole NADPH consumed/min per milligram of
extinction coefficient of 43.6 M cm−1 was used to determine CAT activity. One protein.
unit of activity is equal to the millimoles of H2 O2 degraded per minute per
milligram of protein.
2.10. Statistical analysis
2.9.5. Reduced glutathione
The data were expressed as mean ± S.E.M. Comparisons of the means of
Reduced glutathione estimation was performed by the method of Grifith
control, nicotine, nicotine with ANDRO, nicotine with AE-AP and nicotine with
(1980). The required amount of tissue homogenate was mixed with 12% sul-
vit.E group were made by two-way ANOVA with multiple comparison t-test,
fosalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitated
P < 0.05 as a limit of significance.
proteins. 0.1 ml of protein free supernatant, 0.7 ml of 0.3 mM NADPH, 0.1 ml
of 6 mM DTNB and 0.48 units of glutathione reductase were combined and the
absorbance of 5-thio-2-nitrobenzoic acid (TNB) was read at 412 nm. A standard 3. Results
curve was obtained with standard reduced glutathione. The level of GSH was
expressed as microgram per mg protein. 3.1. Isolation of ANDRO
2.9.6. Oxidized glutathione
Isolation of ANDRO was carried out by TLC (Fig. 1). The
Oxidized glutathione estimation was performed by the method of Grifith
(1980). The required amount of tissue homogenate was mixed with 12% sul- band parallel to reference was eluted out and used for HPLC
fosalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitated analysis and supplemented in rats.
proteins. 0.1 ml of protein free supernatant incubated in room temperature with
0.005 ml of 2 M 2-venyl pyridine for 1 h. Following incubation, 0.4 ml of 0.5 mM 3.2. Detection of ANDRO by HPLC
NADPH, 0.1 ml 6 M DTNB and 0.48 unit of glutathione reductase were added
and measured at 412 nm. A standard curve was obtained with standard oxi-
dized glutathione. The level of GSSG was expressed as microgram per mg The detection of andrographolide was done with stan-
protein. dard andrographolide (Sigma Chemical Co., USA) and the
peck of both isolated compound and standard andrographolide
2.9.7. Redox ratio (GSH/GSSG) matches, hence it confirmed the presence of andrographolide
Redox ratio was determined for all the five groups by taking the ratio of (Fig. 2).
reduced glutathione/oxidized glutathione.

2.9.8. Glutathione peroxidase (GSH-Px)

The GSH-Px activity was measured by the method of Paglia and Valentine
(1967). The reaction mixture contained 50 mM potassium phosphate buffer (pH
7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reduc-
tase and 1 mM reduced glutathione. The sample, after its addition, was allowed
to equilibrate for 5 min at 25 ◦ C. The reaction was initiated by adding 0.1 ml
of 2.5 mM H2 O2 . Absorbance at 340 nm was recorded for 5 min. Values were
expressed as nanomoles of NADPH oxidized to NADP by using the extinc-
tion coefficient of 6.2 3 103 M−1 cm−1 at 340 nm. The activity of GSH-Px
3.3. Lipid peroxidation
MDA levels were significantly (P < 0.05) increased in liver,
kidney, heart, lungs and spleen by 30.21%, 22.42%, 121.43%,
176.92% and 40.40%, respectively, as compared to the control
group.
In liver, supplementation with ANDRO, AE-AP and vit.E
showed significant (P < 0.05) diminution of MDA content by
324 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
Fig. 2. Detection of andrographolide by HPLC. Black: Isolated andro-
grapholide. Blue: Standard andrographolide (Sigma Chemical Co.). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the web version of the article.)
13.45%, 16.42% and 17.14%, respectively, as compared to nico-
tine treated group.
In kidney, significantly (P < 0.05) decreased level of MDA
was seen after supplementation with ANDRO, AE-AP and vit.E
by 10.78%, 13.29% and 15.21%, respectively. ANDRO did not
show any significant change.
In heart, supplementation with AE-AP and vit.E showed sig-
nificant (P < 0.05) diminution in MDA levels by 19.75% and
22.18%, respectively, as compared to nicotine treated group.
ANDRO did not response against nicotine toxicity in heart.
In lungs, MDA level showed a significant (P < 0.05) diminu-
tion by 27.89% on ANDRO supplementation to nicotine treated
animals. Supplementation with AE-AP and vit.E to nicotine
treated group, significantly (P < 0.05) decreased the MDA level
by 44.79% and 44.51%, respectively.
In spleen, supplementation with ANDRO, AE-AP and vit.E
decreased MDA levels by 52.36%, 45.28% and 48.58%, respec-
tively, in comparison to nicotine treated animals (Fig. 3).
3.4. Protein oxidation
Protein carbonyl levels were significantly (P < 0.05) elevated
by 69.09%, 130.17%, 55.94%, 73.80% and 68.55%, respectively
in all the treated tissues (liver, kidney, heart, lung and spleen) in
comparison to control.
Fig. 3. MDA concentration in liver, kidney, heart, lung and spleen. Values are
expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05
compared to nicotine.
Supplementation with ANDRO, AE-AP and vit.E signifi-
cantly (P < 0.05) decreases the liver PC content by 23.66%,
26.52% and 32.97%, respectively, as compared to the nicotine
treated group.
Renal PC content was significantly decreased by 21.72%,
26.22% and 43.07%, respectively in ANDRO, AE-AP and vit.E
supplementation as compared to control animals.
In heart, supplementation with AE-AP and vit.E showed
significant (P < 0.05) fall in PC level by 27.80% and 31.83%,
respectively, as compared to nicotine treated group. ANDRO did
not show any significant alteration of PC level in heart against
nicotine toxicity.
In lung, PC content was significantly (P < 0.05) decreased
by 25.57%, 36.52% and 39.26% on ANDRO, AE-AP and vit.E
administration to nicotine treated animal.
In spleen, all three supplementation (ANDRO, AE-AP and
vit.E) significantly (P < 0.05) decreased PC level by 23.50%,
32.83% and 33.58%, respectively as compared to nicotine
treated group and the decreased was significant in relation to
control (Fig. 4).
3.5. Superoxide dismutase activity
The SOD activity of liver was significantly (P < 0.05) reduced
by 57.67% due to nicotine treatment in relation to control. Sig-
nificant (P < 0.05) variation in SOD activity by 43.05% and
33.33%, respectively, was seen on supplementation with AE-AP
and vit.E to nicotine treated group. But ANDRO did not increase
SOD activity significantly as compared to nicotine treated group.
Similarly, in kidney SOD activity was declined significantly
(P < 0.05) by 56.41% due to nicotine administration. Supple-
mentation with ANDRO, AE-AP and vit.E to nicotine treated
groups showed, 27.05%, 42.35% and 55.29% increase of SOD
activity significantly (P < 0.05), respectively.
The SOD activity of heart was significantly (P < 0.05)
reduced by 46.66% due to nicotine toxicity as compared to
Fig. 4. PC concentration in liver, kidney, heart, lung and spleen. Values are
expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05
compared to nicotine.
 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
the control group. While administration of ANDRO to nico-
tine treated rats showed no change in SOD activity, there was a
significant (P < 0.05) increase of the latter on AE-AP and vit.E
supplementation. Thus SOD activity of the heart, which was pre-
viously lowered by nicotine treatment, was increased by 46.78%
and 76.78%, respectively on AE-AP and vit.E administration.
Similarly, in lungs SOD activity was significantly (P < 0.05)
reduced by 51.00% due to nicotine treatment as compared to
control group. Though, supplementation with ANDRO produces
no significant variation in SOD activity, where as AE-AP and
vit.E supplementation significantly (P < 0.05) increase the activ-
ity by 65.16% and 79.64%, respectively, as compared to the
nicotine treated group. Thus a fall in SOD activity was partly
compensated by AE-AP and vit.E administration.
The SOD activity of spleen was significantly (P < 0.05)
reduced by nicotine administration in relation to the control
group. The reduction was as much as 53.07%. ANDRO, AE-AP
and vit.E administration to nicotine treated groups significantly
(P < 0.05) increases the SOD activity by 74.69%, 78.22% and
83.82%, respectively (Fig. 5).
3.6. Catalase activity
In liver CAT activity was decreased by 50.93% due to nicotine
administration. A significant rise (P < 0.05) in CAT activity was
observed on supplementation with ANDRO, AE-AP and vit.E
by 25.41%, 35.49% and 63.16%, respectively, as compared to
nicotine treated group.
There was a significant fall (P < 0.05) in kidney CAT activ-
ity by 38.19% on nicotine administration. ANDRO, AE-AP and
vit.E supplementation to nicotine treated groups showed a sig-
nificant rise (P < 0.05) in CAT activity by 51.48%, 63.75% and
45.83%, respectively.
There was a significant (P < 0.05) reduce in CAT activity of
heart due to nicotine toxicity by 24.84%. While CAT activity
of heart was significantly (P < 0.05) elevated by ANDRO, AE-
AP and vit.E supplementation by 36.44%, 59.32%, 48.31%,
respectively to nicotine treated group.
Fig. 5. SOD activity in liver, kidney, heart, lung and spleen. Values are expressed
as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared
to nicotine.
325
Fig. 6. CAT activity in liver, kidney, heart, lung and spleen. Values are expressed
as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared
to nicotine.
The CAT activity of lungs was significantly (P < 0.05)
reduced by 49.25% due to nicotine treatment in relation to con-
trol. Significant (P < 0.05) deviation by 46.95% and 48.75% in
catalase activity of lungs was seen on supplementation with
AE-AP and vit.E to nicotine treated group. But ANDRO did
not increase CAT activity significantly as compared to nicotine
treated group.
Similarly, in spleen catalase activity was declined signif-
icantly (P < 0.05) by 51.64% due to nicotine administration.
Supplementation with ANDRO, AE-AP and vit.E to nicotine
treated groups showed, 40.50%, 60.45% and 69.49% increase
of CAT activity, respectively (Fig. 6).
3.7. GSH concentration
In liver GSH content was decreased by 62.61% due to nicotine
administration. A significant rise in GSH content was observed
on supplementation with ANDRO, AE-AP and vit.E by 63.52%,
89.49% and 143.97%, respectively, as compared to nicotine
treated group.
There was a fall in kidney GSH content by 29.78% on nicotine
administration. AE-AP and vit.E supplementation to nicotine
treated groups showed a significant rise in GSH content by
28.20% and 35.22%, respectively.
The GSH content of heart was significantly (P < 0.05)
reduced by 43.09% due to nicotine toxicity as compared to
the control group. While administration of ANDRO to nicotine
treated rats showed no change in GSH content, there was a sig-
nificant (P < 0.05) raise on AE-AP and vit.E supplementation.
Thus GSH content of the heart, which was lowered by nicotine
treatment, was increased by 21.47% and 32.98%, respectively
on AE-AP and vit.E supplementation.
Similarly, in lungs GSH content was significantly (P < 0.05)
reduced by 56.43% due to nicotine toxicity as compared to
control group. Supplementation with ANDRO, AE-AP and
vit.E significantly (P < 0.05) increase by 31.88%, 35.16% and
49.45%, respectively, as compared to the nicotine treated group.
The GSH content of spleen was significantly (P < 0.05)
reduced by nicotine administration in relation to the control
group. The reduction was as much as 47.38%. ANDRO, AE-AP
326 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328

Fig. 7. GSH concentration in liver, kidney, heart, lung and spleen. Values are Fig. 8. GSSG concentration in liver, kidney, heart, lung and spleen. Values are
expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05
compared to nicotine. compared to nicotine.

and vit.E administration to nicotine treated groups significantly 3.10. GSH-Px activity
(P < 0.05) increases the GSH content by 31.15%, 39.64% and
65.04%, respectively (Fig. 7). Due to nicotine administration, GSH-Px activity was
decreased in liver by 52.55%. A significant rise (P < 0.05)
in GSH-Px activity was observed on supplementation with
3.8. GSSG concentration
ANDRO, AE-AP and vit.E by 45.06%, 63.44% and 91.98%,
respectively, as compared to nicotine treated animals.
The GSSG level of liver was significantly (P < 0.05) reduced
There was a significant decrease (P < 0.05) in kidney GSH-Px
by 53.16% due to nicotine treatment in comparison to con-
activity in kidney by 50.02% on nicotine administration. Sup-
trol. Significant (P < 0.05) variation in GSSG level was seen on
plementation with ANDRO, AE-AP and vit.E to nicotine treated
supplementation with ANDRO, AE-AP and vit.E by 60.55%,
groups showed a significant rise (P < 0.05) in GSH-Px activity
66.97% and 69.72%, respectively, to nicotine treated group.
by 36.76%, 59.1% and 78.72%, respectively.
In kidney, GSSG level was declined significantly (P < 0.05)
As a result of nicotine toxicity, there was a significant
by 51.61% due to nicotine administration. Supplementation with
(P < 0.05) reduce in GSH-Px activity in heart by 42.68%. On the
ANDRO, AE-AP and vit.E to nicotine treated groups showed,
other hand GSH-Px activity of heart was significantly (P < 0.05)
50.30%, 73.36% and 104.24% rise of GSSG level, respectively.
elevated by the supplementation of ANDRO, AE-AP and vit.E
There was a fall in GSSG level of heart by 48.54%, on nicotine
by 29.83%, 41.93%, 59.67%, respectively as compared to nico-
administration. A treatment with AE-AP and vit.E significantly
tine treated group.
(P < 0.05) increased the GSSG level by 46.23% and 38.67%
The GSH-Px activity of lungs was significantly (P < 0.05)
as compared to nicotine treated group. While administration of
reduced by 48.19% due to nicotine treatment in relation to con-
ANDRO showed no significant variations.
trol. Significant (P < 0.05) deviation by 62.59%, 72.38% and
Similarly, in lungs GSSG level was decreased by 78.88% due
to nicotine administration. A significant rise in GSSG level of
lungs was observed on supplementation with ANDRO, AE-AP
and vit.E by 261.76%, 323.53% and 282.35%, respectively, as
compared to nicotine treated group.
There was a fall in spleen GSSG level by 53.43% on nicotine
administration. ANDRO, AE-AP and vit.E supplementation to
nicotine treated groups showed a significant rise in GSSG level
by 91.80%, 83.61% and 90.16%, respectively (Fig. 8).

3.9. Redox ratio (GSH/GSSG ratio)

As a result of nicotine toxicity, a significant increase

(P < 0.05) in the redox ratio was observed in nicotine treated
group as compared to control animals. The increased was
observed in all the tissues studied. The supplemented groups
showed the significantly decreased (P < 0.05) the redox ratio in
comparison to nicotine treated animals (Fig. 9).
Fig. 9. GSH/GSSG ratio in liver, kidney, heart, lung and spleen. Values are
expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05
compared to nicotine.
 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
Fig. 10. GSH-Px activities in liver, kidney, heart, lung and spleen. Values are
expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05
compared to nicotine.
80.10% in GSH-Px activity of lungs was seen on supplementa-
tion with ANDRO, AE-AP and vit.E in comparison to nicotine
treated animals.
In spleen, GSH-Px activity was declined significantly
(P < 0.05) by 42.0% due to nicotine administration. Supplemen-
tation with ANDRO, AE-AP and vit.E to nicotine treated groups
showed, 32.78%, 51.61% and 56.02% increase of GSH-Px activ-
ity, respectively (Fig. 10).
4. Discussion
In the present study, the protective effect of A. paniculata
Nees has been carried out against nicotine-induced toxicity in
liver, kidney, heart, lung and spleen. For this, we have chosen
major bioactive metabolite andrographolide (ANDRO), crude
aqueous extract (AE-AP) and vit.E as a positive standard.
ANDRO was isolated and characterized by TLC and HPLC.
It was confirmed that presence of ANDRO in the isolated com-
pound from A. paniculata Nees.
Nicotine, a pharmacologically active ingredient in tobacco, is
generally regarded to a primary risk factor in the development of
cardiovascular disorders, myocardial infraction, stroke, kidney
cancer, pulmonary diseases and certain immunological dysfunc-
tion (Jung et al., 2001). This highly addictive alkaloid has been
reported to induce oxidative stress both in vivo and in vitro
(Suleyman et al., 2002). The mechanisms of free radicals gener-
ation by nicotine are not clear. However, it has been reported that
nicotine disrupts the mitochondrial respiratory chain leading to
an increase generation of superoxide anions and hydrogen per-
oxide (Yildiz et al., 1998). Previous studies have suggested that,
superoxide anion and hydrogen peroxide are the main source of
nicotine induced free radicals depleting the cellular antioxidants
(Wetscher et al., 1995).
In this study, significant elevation of malondialdehyde
(MDA) and protein carbonyls contents were observed in nicotine
induced hepatocytes, myocytes, spleenocytes, renal and lung
tissues. Lipid peroxidation is known to disturb the integrity
of cellular membranes, leading to the leakage of cytoplasmic
327
enzymes (Bagchi et al., 1995). Enhanced lipid peroxidation asso-
ciated with antioxidant depletion in different tissues, may yield
a range of toxic aldehydes that are capable of damaging mem-
brane proteins (Husain et al., 2001; Hedley and Chows, 1992).
Recent report by our laboratory suggested that, increased lipid
peroxidation and decrease antioxidant enzyme status can be an
indicator of disease progression of oral cavity cancer patients
(Das et al., 2007). PC formation has been proposed to be an
earlier marker of protein oxidation (Reznick and Packer, 1994).
Oxidative modification of proteins may lead to the structural
alteration and functional inactivation of many enzyme proteins
(Davies, 1988). Thus the nicotine induced oxidatively modi-
fied proteins is due to either excessive oxidation of proteins or
decreased capacity to cleanup oxidatively damaged proteins. In
the present investigation, significantly increased MDA and PC
levels were either partially or completely returned to the con-
trol levels, were may be due to the free radicals scavenging
properties of the herbal product and vit.E. In our recent report,
we have demonstrated that ANDRO is having hepato-renal pro-
tective activity by reducing the lipid peroxidation level against
ethanol induced toxicity in mice (Singha et al., 2007). The con-
stant exposure of the tissue to the herbal supplementation for the
mentioned schedule may have been able to detoxify the nicotine
toxicity by modulating the extent of lipid peroxidation and pro-
tein oxidation. These modulatory mechanisms may either be a
mutual biomolecular interaction among the reactive radicals or
by interaction with herbal bioactive molecules.
Antioxidant enzymes are considered to be a primary defense
that prevents biological macromolecules from oxidative dam-
age. Aerobic cells contain various amounts of two main
antioxidant enzymes: superoxide dismutase (SOD) and catalase
(CAT). SODs rapidly dismutate superoxide anion (O2 −• ) to less
dangerous H2 O2 , which is further degraded by CAT and glu-
tathione peroxidase (GSH-Px) to water and oxygen (Wetscher
et al., 1995). The results of the present study showed a significant
fall in SOD activities, in the nicotine treated groups. SOD, dis-
mutate O2 −• and the same in turn is a potent inhibitor of CAT
(Ashakumari and Vijayammal, 1996). The depletion in SOD
activity was may be due to dispose off the free radicals, produced
due to nicotine toxicity. Beside this, on nicotine administration,
H2 O2 produced by dismutation of superoxide anion, may have
been efficiently converted to O2 by CAT and the enzyme activ-
ities showed a marked reduction. The depletion of antioxidant
enzyme activity was may be due to inactivation of the enzyme
proteins by nicotine-induced ROS generation, depletion of the
enzyme substrates, and/or down-regulation of transcription and
translation processes. Glutathione is an important cellular reduc-
tant, which offers protections against free radicals, peroxide and
toxic compounds. It is reformed from GSSG by donation of
hydrogen from NADPH, the reaction being catalyzed by glu-
tathione rductase (GR) (Meister, 1994 and John, 2003). In this
study, a significant fall in GSH level and GSH-Px activity was
observed in nicotine treated animals, may be due to enhanced
free radical production (as evidence by increase lipid peroxida-
tion and protein oxidation) and apart from catalase, GSH-Px also
involved in the removal of H2 O2 . H2 O2 generated due to nico-
tine toxicity, engage more GSH, which thereby get converted
328 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
to GSSG in presence of GSH-Px. Hence, the GSH, and GSSG
level decreases on nicotine administration. The toxic effects of
nicotine may be prevented during ANDRO, AE-AP and vit.E
exposure, since it restores redox ratio.
In this scientific investigation, supplementation with
ANDRO, AE-AP and vit.E were able to compensate the antiox-
idant enzyme status, more or less near to the control levels. One
possible reason behind this findings is may be the anti-oxidative
properties of the supplements. The extracts of A. paniculata
increase the glutathione level in all tissues. Increased glutathione
level may have efficiently scavenged ROS, increased the antiox-
idant enzyme status and prevent the oxidative damage in all
supplemented groups. Vitamins are the most important micronu-
trients that are known to modulate the defense mechanisms. In
the present study, A. paniculata products (ANDRO and AE-
AP) and vit.E treatment to the nicotine treated groups, showed
an increase in the antioxidant enzyme status. This lipid soluble
vitamin may have effectively reduced the free radicals produc-
tion by scavenging the generated OFRs and thus obstruct the
peroxidation chain reactions. As a well-acquainted fact, vit.E
acts as a cytosolic antioxidant in the lipid domains. The synthe-
sis of glutathione is regulated by oxidants, antioxidants, growth
factors (MacNee and Rahman, 2001). In the nicotine treated
groups, ANDRO, AE-AP and vit.E may have been able to elevate
the glutathione level, which thereby disposes the free radicals,
generated by nicotine toxicity and the chain reaction of ROS
is prevented. So, a raise in hepatic, renal, cardiac, lung and
spleenic antioxidant levels indicate the involvement of A. panic-
ulata products and vit.E in antioxidant defense against nicotine
induced oxidative stress mediated tissue injury.
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