Hepatoprotective and antioxidant effects of Azolla microphylla based gold nanoparticles against acetaminophen induced toxicity in a fresh water common carp fish (Cyprinus carpio L.)

 Please cite this paper as:
Kunjiappan S, Bhattacharjee Ch, Chowdhury R. Hepatoprotective and antioxidant effects of Azolla microphylla
based gold nanoparticles against acetaminophen induced toxicity in a fresh water common carp fish (Cyprinus
carpio L.), Nanomed J, 2015; 2(2):88-110.
Received: Oct. 19, 2014; Accepted: Feb. 5, 2015
Vol. 2, No. 2, Spring 2015, page 88-110
Online ISSN 2322-5904
http://nmj.mums.ac.ir
Original Research
Hepatoprotective and antioxidant effects of Azolla microphylla based gold
nanoparticles against acetaminophen induced toxicity in a fresh water
common carp fish (Cyprinus carpio L.)
Selvaraj Kunjiappan1
, Chiranjib Bhattacharjee1*
, Ranjana Chowdhury1
1
Department of Chemical Engineering, Jadavpur University, India
Abstract
Objective(s): Our present study sought to evaluate hepatoprotective and antioxidant effects of
methanol extract of Azolla microphylla phytochemically synthesized gold nanoparticles
(GNaP) in acetaminophen (APAP) - induced hepatotoxicity of fresh water common carp fish.
Materials and Methods: GNaP were prepared by green synthesis method using methanol
extract of Azolla microphylla. Twenty four fishes weighing 146 ± 2.5 g were used in this
experiment and these were divided into four experimental groups, each comprising 6 fishes.
Group 1 served as control. Group 2 fishes were exposed to APAP (500 mg/kg) for 24 h.
Groups 3 and 4 fishes were exposed to APAP (500 mg/kg) + GNaP (2.5 mg/kg) and GNaP
(2.5 mg/kg) for 24 h, respectively. The hepatoprotective and antioxidant potentials were
assessed by measuring liver damage, biochemical parameters, ions status, and histological
alterations.
Results: APAP exposed fish showed significant elevated levels of metabolic enzymes (LDH,
G6PDH and MDH), hepatotoxic markers (GPT, GOT and ALP), reduced hepatic glycogen,
lipids, protein, albumin, globulin, increased levels of bilirubin, creatinine, and oxidative
stress markers (TBRAS, LHP and protein carbonyl), altered the tissue enzymes (SOD, CAT,
GSH-Px and GST) non-enzyme (GSH), cellular sulfhydryl (T-SH, P-SH and NP-SH) levels,
reduced hepatic ions (Ca2+
, Na+
and K+
), and abnormal liver histology. It was observe that
GNaP has reversal effects on the levels of above mentioned parameters in APAP
hepatotoxicity.
Conclusion: Azolla microphylla phytochemically synthesized GNaP protects liver against
oxidative damage and tissue damaging enzyme activities and could be used as an effective
protector against acetaminophen-induced hepatic damage in fresh water common carp fish.
Keywords: Azolla microphylla, Acetaminophen, Antioxidant Cyprinus carpio L.,
Hepatotoxicity, Gold nanoparticles
*Corresponding author: Chiranjib Bhattacharjee,Professor, Department of Chemical Engineering, Jadavpur
University, Kolkata-700 032, India.
Tel: +91 98364 02118, Email: cbhattacharyya@chemical.jdvu.ac.in

Biological activity of Azolla microphylla-based gold nanoparticles
Nanomed J, Vol. 2, No. 1, Spring 2015 89
Introduction
The liver is one of the prime target organs,
which regulates many biological functions
such as drug metabolism, amino acid
metabolism, lipid metabolism and
glycolysis (1).
Liver injury or liver dysfunction is the
most serious ailment and are mainly
caused by excess consumption of alcohol,
high doses of acetaminophen, chemoth-
erapeutic agents, hepatic viral infection,
dantrolene sodium, valporic acid, per-
oxidised oil and isonicotinic acid
hydrazide, etc (2).
Acute and chronic liver diseases constitute
a global concern and medical treatments
for these diseases are often difficult to
handle and have limited efficiency (3).
Therefore, there has been considerable
interest in role of complementary and
alternative medicines for the treatment of
liver diseases. Developing therapeutically
effective agents from natural products may
reduce the risk of toxicity when the drug is
used clinically.
It has been reported that, among a variety
of drugs, acetaminophen (APAP or N-
acetyl-p-aminophenol or paracetamol or 4-
hydroxyacetanilide) is the most common
cause of drug-induced liver injury (4).
APAP is one of the best-selling and widely
used over-the-counter analgesic and
antipyretic drug. Although considered safe
at therapeutic doses, at higher doses (3h
following 500 mg/kg body weight),
acetaminophen produce acute liver failure
(ALF) (5-7).
When taken in therapeutic doses, greater
than 90% of acetaminophen is metabolized
to phenolic glucuronide and sulfate in the
liver by glucuronyltransferases and sulfo-
transferases and subsequently excreted in
the urine (8).
Of the remaining acetaminophen, about
2% is excreted in the urine unchanged;
approximately 5 to 10% is metabolized by
cytochrome P450, mainly the enzyme
CYP2E1 (9), to N-acetyl-p-benzoquinon-
eimine (NAPQI) (10), a highly reactive,
electrophilic molecule that causes harm by
formation of covalent bonds with other
intracellular proteins (11). This toxic
metabolites neutralized by glutathione
(GSH), which is an important cellular free
radical scavenging non-enzymes for
neutralization of toxic metabolites
produced by drugs and foreign chemicals
(12). If glutathione is not replenished,
NAPQI will begin to accumulate in the
hepatocytes (13).
NAPQI can form covalent bonds with
cysteinyl thiol groups of cellular proteins
to form protein-(cystein-S-yl)-APAP
adducts (14), which may modify the
structure and functions of the proteins
(15). This cellular functional disturbance
leads to a decrease in calcium ATPase
activities and increase in levels of
cytosolic calcium (16, 17).
Abnormal cellular calcium homeostasis
can alter the permeability of the cell,
causing the formation of blebs in the cell
membrane and loss of membrane integrity
(18). Medicinal plants have been gaining
importance of effective source of both
traditional and modern medicines to treat
or prevent human and animal diseases.
World health organization has recomme-
nded traditional plant derivatives as safe
remedies for ailments for microbial and
non-microbial origin (19).
The medicinal effects of these plants lies in
some chemical substances such as flavor-
noids, phenolic compounds, alkaloids,
anthroquinones, carbohydrates, proteins and
vitamins that produces definite therapeutic
action on the human and animal body (20).
Plant secondary metabolites (Flavonoids
and phenolic compounds) are of great
importance for the bioactivities, related to
their antioxidant activities (21) and many
enzymatic reactions, resulting in a
decrease of platelet activation and aggr-
egation, against cardiovascular diseases,
cancer chemoprevention, acute liver
failure (22) and anti-inflammatory activity
(23-25).
This suggests that flavonoid-based
pharmaceutical preparation to treat various
complications linked with human diseases.
Azolla is a genus of small aquatic fern that
is found in the temperate and tropical

Kunjiappan S, et al
90 Nanomed J, Vol. 2, No. 1, Spring 2015
regions of the world. Azolla microphylla is
one of the species from the genus Azolla;
it is a pteridophyte plantae belonging to
the Salvinacea family (26).
The phytochemical investigation on the
Azolla microphylla shows that tannins,
polyphenols, sugar, anthroquinone glycos-
ides and steroids are present (27).
Becerra et al. (28), Lumpkin and Plucknett
(29), Van Hove and López (30) concluded
that, Azolla is the most promising aquatic
plant for livestock feed due to its ease of
cultivation, productivity and nutritive
value. Recently this research group has
isolated and purified rutin and quercetin
from Azolla microphylla for the first time
(31). Therefore Azolla microphylla is one
of the important sources for various
antioxidants and hepatoprotective agents.
Researchers in the field of nanotechnology
are gaining new insights into its versatile
applications in the treatment of various
diseases, including hepatotoxicity.
Research into the preparation and
biological applicability of noble metal
nanoparticles with a nearly mono-
dispersed size distribution and arbitrarily
variable size and geometry has attracted
considerable research interest.
It is reported that metal nanoparticles,
especially gold nanoparticles have drawn
more attention of scientists because of
their unique nature of stability, oxidation
resistance and biocompatibility (32).
Gold nanoparticles widely applied in the
field of medicine such as chemical bio-
sensing, imaging, drug delivery and
therapeutic labeling (33).
Gold nanoparticles have a growing role in
medical biotechnology.
Production of nanoparticles can be achieved
mainly through chemical, physical, and
biological methods.
Biological methods for nanoparticles
synthesis using microorganisms, enzymes
and plants or plant extracts have been
suggested as possible ecofriendly alterna-
tives to chemical and physical methods.
However, major drawback of using
microbes such as bacteria, fungi and yeast
are the requirement of manipulation of
reaction parameters such as pH,
temperature and incubation time for the
synthesis of nanoparticles (34).
On the other hand, due to high rate of
generation of nanoparticles, plant based
synthesis is comparatively simpler and
more cost-effective (35).
Plant phytochemicals such as polyphenols,
flavonoids, triterpenes, tannins, glycosides,
vitamins, proteins and steroids are capable
of reducing the gold ions (Au3+
or Au1+
) to
neutral gold nanoparticles (Au0
) and
ensures good control over size distribution
and crystallinity of the nanoparticles (36).
The phytochemicals present in Azolla
microphylla serve a dual role as effective
reducing agents to reduce gold and also as
stabilizers to provide a robust coating on
the gold nanoparticles in a single step.
However, no reports have been found
on the protective effect of Azolla
microphylla phytochemically synthesized
gold nanoparticles on APAP-induced
hepatotoxicity.
Thus, we hypothesized that phytochemicals
rich in Azolla microphylla mediated
synthesized gold nanoparticles would
prevent APAP-linked abnormalities.
The present study examined the
hepatoprotective and antioxidant effects of
Azolla microphylla methanol extract
mediated green synthesized gold
nanoparticles in acetaminophen-induced
hepatotoxicity in common carp fish
(Cyprinus carpio L.) in terms of
hepatotoxic markers, oxidative stress,
antioxidant, hepatic ions status, glycogen,
cholesterol, protein, bilirubin, lipid levels
and H & E staining.
Materials and Methods
Materials
Acetaminophen was received as a gift
from Pharma-Fabricon pharmaceuticals Ltd,
Madurai, India.
All other chemicals and solvents were of
analytical grade and were obtained from Hi-
media laboratories Pvt. Ltd. Mumbai, India,
and Merck, Mumbai, India.
The biochemical and enzyme assay kits
were purchased from Span Diagnostics

Ltd. Surat, India and Qualigen Diagnostics
Ltd. Mumbai, India.
Plant collection and preparation of
Azolla microphylla extract
Azolla microphylla fern were received
from Vivekananda Kendra-NARDEP
(Natural Resources Development Project),
Vivekanandapuram, Kanyakumari, India.
The whole parts of the fern were washed
with tap water, rinsed with distilled water
and air-dried under shade with good
ventilation at room temperature (≈37˚C)
for a week.
The fine powder (≈60 mesh size) was
obtained from dried plant material by
using kitchen blender (Bajaj electronics
Ltd, India).
About 500g of Azolla microphylla powder
was weighed and macerated with 1000 mL
of 99.9% methanol in a 2000 mL conical
flask and kept at room temperature
for 72 h.
After 72 h, the methanol extract of Azolla
microphylla was filtered with Whatman
No: 1 filter paper.
The filtered extract was centrifuged at
10000 rpm for 10 min at 4˚C. We
discarded the precipitate that contains
debris and collected the supernatant in a
brown bottle and stored in refrigerator at
4°C until further studies.
Green chemistry approach for the
synthesis of gold nanoparticles
1 mM solution of hydrogen tetrarch-
oloroaureate (III) hydrate (Himedia
laboratories Pvt. Ltd. Mumbai) was
prepared using de-ionized water. 5 mL
(concentration 5%, v/v) of the methanolic
extract of Azolla microphylla was mixed
with 25 mL aqueous solution of
HAuCl4.3H2O (1 mM).
The mixture solution was left on constant
magnetic stirring at room temperature
(≈35°C) for 30minutes and observed for
change in color.
The bio-reduction of gold ions into gold
nanoparticles in the solution was
monitored by periodic sampling of aliquots
(1 mL) and subsequently measuring UV-
vis spectra of the solution. UV-visible
spectrophotometry analysis was carried
out by a computer controlled UV–vis
spectrophotometer (Varian Cary 50 UV-
Spec) between 400 and 800 nm possessing
a scanning speed of 400 nm/min.
Characterization of nanoparticles
Characterization of the biosynthesized
gold nanoparticles was carried out before
starting the experiment for their potent
hepatoprotective and anti-oxidative effects
in common carp fish.
Hydrogen tetracholoroaureate (III) hydrate
(aqueous) treated methanol extract of
Azolla microphylla solution was centri-
fuged at 10000 rpm for 15 min at 4˚C and
the resulted supernatant solution was
maintained at -80˚C for 24 h and then
freeze dried in a lyophilizer (Christ
Gefriertrocknungsanlagen GmbH Model
1–4) for 48 h.
Molecular shape and size of the
lyophilized gold nanoparticles was
characterized using FESEM and HRTEM
analyses.
The dose for the experimental study of the
lyophilized gold nanoparticles was
calculated as 2.5 mg/kg body weight from
previous studies (37).
Total antioxidant assay of gold
nanoparticles
The total antioxidant capacity of the
methanol extract of Azolla microphylla
mediated synthesized gold nanoparticles
was evaluated by the method of Prieto et
al. (38).
The assay is based on the reduction of Mo
(VI) to Mo (V) by various concentrations
of green gold nanoparticles (1, 2, 3, 4 and
5 mg/mL).
Lyophilized gold nanoparticles dissolved
in de-ionized water and combined with 3
mL of reagent solution (0.6 M sulfuric
acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The absorbance of
the reaction mixture was measured at 695
nm using spectro-photometer (Varian Cary
50 UV-Spec) against blank after cooling to
room temperature.

Kunjiappan S, et al
De-ionized water (0.3 mL) in place of gold
nanoparticles was used as the blank.
The total antioxidant activity is expressed
as the number of gram equivalents of
ascorbic acid.
Fish husbandry and maintenance
Fresh water common carp (Cyprinus
carpio L.) was obtained from commercial
hatcheries, Jadavpur, Kolkata, India. 48
healthy fish of uniform size of length (12 ±
2 cm) and weight (145.46 ± 1.24 g) were
segregated from the stock and acclimatized
to laboratory conditions for 7 days in an
aquarium (60 × 30 × 40 cm) with 50 L of
dechlorinated tap water (6-8 individuals
/aquarium).
The total mortality of fish was less than
1%. During experiment, water temperature
was 25 ± 1˚C, pH was 7.1 ± 0.25,
dissolved oxygen was 8.015 ± 0.5 mg/L,
hardness was 425 ± 3.5 mg/L CaCo3,
turbidity was 2.5NTU and total solids were
14.05 ± 0.1 mg/L.
Fishes were reared in a re-circulation
system with 12 h light-dark photoperiod
and fed with commercial diets (Tokyu
baby pellet, Floating type, Japan) twice a
day.
Feeding was withheld 1day prior to
exposure, with an average weight of 146 ±
2.5 g at the start of the experiment.
Toxicity studies
In this study, the fishes were randomly
divided into two groups consist of 6 fish in
each.
Group 1 – Control, Group 2 - GNaP
treated, Group 2 Fish were treated with
Azolla microphylla mediated synthesized
gold nanoparticles (GNaP) (2.5 mg/kg
body wt) for 48 h in static tank containing
5L of water. The control group fish were
fed with commercial baby pellet feed.
At the end of the 48 h treatment, the fish
were sacrificed by decapitation. Blood was
collected and allowed to clot at room
temperature, centrifuged at 3000 rpm at
4˚C for 20 min to obtain serum in which
analysis of total protein, cholesterol and
triglycerides were performed.
Liver was dissected immediately and fixed
with 10% buffered formalin for
histological analysis.
Experimental design
The fishes were dived into four groups
consist of 6 fish in each. Fishes were
treated as follows:
Group I Control fish (CON) were fed with
commercial baby pellet feed (crude
protein-46%, crude fat-6%, crude fibre-
5%, crude ash- 12% and spirulina nitrogen
free extract-20%) throughout the
experimental period. Group II Fish were
treated with acetaminophen (APAP) for 24
h in static tank containing 5 L of water.
Acetaminophen; 500 mg/kg was dissolved
in 100 mL water at 70˚C and cooled to
room temperature and added to the static
tank. Group III Fish were treated with
acetaminophen + Azolla microphylla
(APAP + GNaP) for 24 h in static tank
containing 5 L of water. Acetaminophen;
500 mg/kg was dissolved in 100 mL water
at 70˚C and cooled to room temperature
and combined with methanol extract of
Azolla microphylla mediated synthesized
gold nanoparticles (2.5 mg/kg body wt)
and added to the static tank. Group IV Fish
were treated with Azolla microphylla
(GNaP) (2.5 mg/kg body wt) alone for 24h
in static tank containing 5 L of water. At
the end of the experimental period, the
fishes were sacrificed by decapitation.
Blood was collected and allowed to clot at
room temperature and then centrifuged at
3000 rpm at 4˚C for 20 min to obtain
serum in which analysis of protein,
cholesterol, triglycerides and enzymes
(GPT, GOT and ALP) were carried out.
Liver was dissected immediately and
homogenized in 0.01 M Tris-HCl buffer
(pH 7.2) using Teflon homogenizer and
centrifuged at 12,000 g at 4˚C for 20 min
to obtain supernatant fractions for the
determination of metabolic enzymes and
hepatotoxic markers.

Assessment of carbohydrate metabolic
enzymes
The enzyme assays of Lactate
dehydrogenase (LDH, E.C.1.1.1.27),
Glucose-6-phosphate dehydrogenase
(G6PDH, E.C.1.1.1.49) and Malate
dehydrogenase (MDH, E.C.1.1.1.37) were
determined spectrophotometrically using
commercial kits (Span Diagnostics, Ltd.
Surat, India).
Biochemical assessment of hepatotoxicity.
Glutamate pyruvate transaminase (GPT,
E.C.2.6.1.2), Glutamate oxalate
transaminase (GOT, E.C.2.6.1.1) and
Alkaline phosphatase (ALP, E.C.3.1.3.1)
were assayed spectrophotometrically with
clinical test kits according to manufacturer’s
protocol (Span Diagnostics Ltd. Surat,
India). Glycogen content was estimated by
the method of Morales et al. (39).
Cholesterol was estimated by the method
of Natio (40). Triglycerides were estim-
ated by the method of Buccolo (41).
Protein content was estimated by the
method of Lowry et al. (42). Albumin
content was estimated by the method of
Gendler (43). Globulin content was
obtained by subtracting albumin content
from total protein. Bilirubin content was
estimated by the method of Jendrassik
(44). Creatinine was measured by the
method of Spencer (45). Levels of ions
(Ca2+
, Na+
and K+
) in the liver tissue
homogenates were estimated using Atomic
Absorption Spectrophotometer (Perkin
Elmer, Analyst spectra 200 Atomic
absorption spectroscopy) with certified
standards by AOAC method (46).
The detection limits for the assay of Ca2+
,
Na+
and K+
tend to range between 0.06,
0.01, 0.03 and 100 ± 10 mg/L, respectively.
Assessment of enzymes involved in free
radical scavenging activities
Superoxide dismutase (SOD, E.C.1.15.1.1)
was assayed by the method of Marklund
and Marklund (47). Catalase (CAT,
E.C.1.11.1.6) was assayed by the method
of Chance and Maehly (48). Glutathione
peroxidase (GSH-Px, E.C.1.11.1.9)
activities were assayed by the method of
Mohandas et al. (49). Glutathione-S-
transferase (GST, E.C.2.5.1.14) was
measured by the method of Rajasekar and
Anuradha (50). Briefly, SOD activity was
assayed by the inhibition of nicotinamide
adenine dinucleotide (reduced) phenazine
methosulphate nitrobluetetrazolium reaction
system as adapted and the results are
expressed as units (U) of SOD activity/mg
protein. CAT and GSH-Px activities were
assayed by measuring the amount of
substrate consumed (hydrogen peroxide and
glutathione, respectively) after carrying out
the reactions for a specified period of time
and the results have been expressed as units
(U) of activity/mg protein. Activity of
glutathione-s-transferase (GST) was
expressed as 1, 2-dichloro-4-nitrobenzene
µmol/mg protein/min.
Non-enzymatic glutathione (GSH) was
estimated in the liver homogenates using
dithionitrobenzoic acid (DTNB) by the
method of Ellman (51). The absorbance was
read at 412nm and the results were
expressed as µmol/mg protein/min.
Assessment of oxidative stress markers
and –SH
Lipid peroxidation of liver homogenate
was estimated by measuring the thiobarb-
ituric acid reactive substances (TBARS)
and was expressed in terms of MDA
content (nanomol/mg protein), according
to the method of Uchiyama and Mihara
(52). Lipid hydroperoxide (LHP) was
measured by the method of Ohkawa et al.
(53) and expressed as µmol/mg protein.
The level of protein carbonyl was
measured by the method of Levine et al.
(54). Total (T-SH), non-protein (NP-SH)
and protein bound (P-SH) sulfhydryl
groups were determined by the method of
Sedlak and Lindsay (55).
Histopathological studies
For liver tissue histological analysis, small
pieces of liver were immediately removed
and fixed in 10% buffered formalin. After
fixation, liver tissue was soaked with
phosphate-buffered saline and then
dehydrated through a series of various

Kunjiappan S, et al
concentrations of alcohol in ascending
order and finally in absolute alcohol (10
min each). The tissue was then embedded
in paraffin wax. Sections of 5µm thickness
were made using a microtome.
The tissues were de-paraffinized with
xylene and treated with 100, 90 and 70%
alcohol for removing undesirable pigments
and other elements. The sections were then
stained with hematoxylin-eosin (H & E)
and observed under microscope.
Photographs of each slide were taken at
10× and 40× magnifications.
Statistical analysis
Values are expressed as mean ± standard
deviation. Data within the groups are
analyzed using one-way analysis of
variance (ANOVA) followed by Dunnett
multiple comparisons test.
Values were considered statistically
significant when p<0.05. Statistical
analyses were carried out using SPSS
statistics version 20 software.
Results
Characterization of the green synthesized
gold nanoparticles
The formation and stability of the gold
nanoparticles were confirmed by colour
change from original yellow to pink to
dark pink which is measured by UV-Vis
spectrophotometry. The dark pink colour
observed is characteristic for unique
surface plasmon resonance (SPR) of
different sizes of gold nanoparticles.
Figure 1 showed the UV-Vis absorption
spectra of biosynthesized gold
nanoparticles.
The gold surface plasmon resonance (SPR)
band (λmax 540 nm), indicates the
presence of spherical nanoparticles in the
reaction mixture.
According to the Mie theory, the small
gold nanoparticles exhibited only one
surface plasmon resonance absorption
band (56). Kinetics of the formation of
gold nanoparticles, 5% v/v methanol
extract of Azolla microphylla in 1mM
HAuCl4 solution are shown in Figure2.
Figure 3 depicts the field emission
scanning electron microscope images of
the biosynthesized gold nanoparticles.
The overall morphological shapes of the
gold nanoparticles are spherical and
rectangular at higher magnification.
In Figure 4, the HRTEM images clearly
proved the size and shape of the gold
nanoparticles as a function of concentration
of the phenolic compounds present in the
plant extract.
The shapes of the gold nanoparticles were
spherical, triangular, hexagonal and rod
shaped.
Gold nanoparticles corresponding to
HRTEM images exhibited the variation in
the particle size ranging from 3 to 20nm
with average of 8.3 nm.
Total antioxidant capacity of gold
nanoparticles
The methanol extract of Azolla
microphylla mediated biosynthesized gold
nanoparticles were subjected to screening
for their total antioxidant capacity.
The antioxidant capacity of the various
concentrations of green synthesized gold
nanoparticles was summarized in Table 1.
All the concentrations of green synthesized
gold nanoparticles were significantly
showed antioxidant activity resulted in
ranges from 48.0 ± 0.05 to 82.04 ± 0.03
mg/g ascorbic acid equivalent, while 3
mg/mL gold nanoparticles showed
efficient antioxidant capacity.
Toxicity studies
In vivo toxicity studies of the synthesized
nanoparticles were performed by exam-
ining the changes of blood serum and
histological analysis.
The fish exposed with GNaP (2.5 mg/kg
body wt) for 48 h and examined for any
changes in the morphology, behavior and
mortality.
During the experimental period all the
fishes survived without exhibiting any
abnormalities.

Figure 1. UV-vis-absorption spectra of 5% methanol extract phytochemically synthesized gold nanoparticles.
The inset image shows the 1mM HAuCl4 solution and green synthesized gold nanoparticles solution.
Figure 2. Kinetics of the formation of gold nanoparticles.
Figure3. Field emission scanning electron microscope images of green synthesized gold nanoparticles.

Kunjiappan S, et al
Figure 4. Transmission electron microscope images of green synthesized gold nanoparticles.
Table 1. Total antioxidant capacity of methanol extract of Azolla microphylla mediated synthesis of gold
nanoparticles.
Concentration of
gold nanoparticles
(mg/mL)
Total antioxidant capacity mg/g
ascorbic acid equivalent
1 48.0±0.05
2 68.2±0.02
3 82.4±0.03
4 66.1±0.02
5 64.2±0.03
Values are listed as mean± SD
The fish did not showed any symptoms of
toxicity such as fatigue, change in
orientation of color, weight loss, etc.
Comparative analysis of blood serum in
the gold treated and control fish, clearly
showed that there was no significant
alteration (Table 2).
Thus the histopatho-logical effect of the
control and gold nanoparticles treatment
was observed using light microscope. The
histological finding of the liver of the non-
toxic effect of gold nanoparticles was
showed in Figure 5.
The liver histological studies showed the
control liver with normal hepatic portal
triad and central vein (Figure 5a) and the
treatment of gold nanoparticles at a dosage
of 2.5 mg/kg body wt for 48 h did not lead
to any disruptions in the histology showed
normal hepatocytes in comparison with
control (Figure 5b).
Effect of gold nanoparticles on tissue gross
morphology
At the end of the experimental period,
observation of the gross pathology of the
liver revealed few differences between
control and experimental fish. A change in
normal appearance of liver was observed in
APAP-treated fish, (i.e.) liver appeared in
dark black color, whereas the color change
was reduced in APAP+GNaP treated fish.

Figure 5. Toxicity studies of green synthesized gold nanoparticles in carp fish liver. Histopathological studies
of fish liver collected from 48h treatment of gold nanoparticles, stained with hematoxylin and eosin (H and E)
showed normal morphology. (a) Control fish liver section showed normal morphology and cells arranged
around the central vein (b) Gold nanoparticles treated fish liver sections also showed normal architecture with
clear central vein.
Table 2. Blood plasma analysis revealing the nontoxic effects of green synthesized gold nanoparticles in Carp fish.
Parameters CON GNaP
Protein (mg/dL) 4.435±0.32 4.442±0.19
Cholesterol (mg/dL) 11.959±0.97 12.018±0.12
Triglyceride (mg/dL) 5.44±0.40 5.628±0.42
Values are listed as mean± SD (n=6 in each group)
CON control, GNaP green synthesized gold nanoparticles
Figure 6. Hepatoprotective effects of gold nanoparticles over Acetaminophen-induced liver damage. H and E
staining of the liver tissue in control and experimental fish (A) Normal liver architecture of control fish (B) The
APAP-treated fish showed central vein necrosis (A*), fatty changes(B*), congestion and apoptosis(C*), whereas
(C) APAP+GNaP treated fish liver showed reduced hepatocellular damage, mild degeneration and absence of
congestion. (D) GNaP-treated fish liver showed normal appearance of architecture.

Kunjiappan S, et al
GNaP treated and control fish showed
smooth and shiny, with normal appearance
of liver as that of control fish.
Effect of gold nanoparticles on liver tissue
histopathology
In the histopathological studies, liver
sections (H&E staining) of control fish
showed normal hepatic architecture
(Fig.6A), whereas that APAP treated fish
showed total loss of hepatic architecture
with intense peripheral central vein necrosis
(A*), fatty changes (B*), congestion of
sinusoid, hepatic cell hyperplasia, crowding
of the central vein, apoptosis (C*) (Fig.6B),
in case of fish treated with APAP+GNaP
(Figure 6C), these changes were reduced
(i.e. mild vacuolar degeneration, reduced
hepatocellular damage and absence of
congestion).
The above abnormalities were effectively
controlled in the GNaP (Fig. 6D)
administered fish tissues.
Effect of gold nanoparticles on
carbohydrate metabolic enzymes
The levels of carbohydrate metabolic
enzymes such as LDH, G6PDH and MDH
were significantly (p<0.05) higher in
APAP-exposed fish when compared with
the control (Figure 7), which indicates that
the effect of carbohydrate metabolic
enzymes was seen in the severity of liver
tissue damage of exposed fish rather than
the control. The elevated levels of these
enzymes were reduced by treatment of
APAP+GNaP. No significant effect on
these metabolic enzymes with GNaP-
treated fish was observed.
Figure 7. Effect of green synthesized gold nanoparticles on carbohydrate metabolic enzymes of control and
experimental fish. Values are mean± SD (n=6 in each group). #
p<0.05 as compared to CON. *
p<0.05 as
compared to APAP. †
p<0.05 as compared to APAP and CON. CON control, APAP acetaminophen, GNaP green
synthesized gold nanoparticles.
Effect of gold nanoparticles on hepato-
toxic markers
The effects of GNaP on hepatotoxic
marker enzymes such as GPT, GOT and
ALP in blood and liver tissue of control
and experimental fish are summarized in
Figure 8 a,b, respectively. Significant
elevation in the marker enzyme activities
were observed in the APAP-treated fish
when compared with the control fish.
Treatment with APAP+GNaP to intoxicate
with APAP significantly attenuated
(p<0.05) the APAP-elevated activities of
GPT, GOT and ALP. GNaP-treated
control fish did not showed any alterations
on these enzymes.

Figure 8. Effect of green synthesized gold nanoparticles on hepatotoxic markers in blood (a) and liver (b) of
control and experimental fish. Values are mean± SD (n=6 in each group). #p<0.05 as compared to CON.
*p<0.05 as compared to APAP. †
p<0.05 as compared to APAP and CON. CON control, APAP acetaminophen,
GNaP green synthesized gold nanoparticles.
Effect of gold nanoparticles on oxidative
stress markers
The effects of GNaP on oxidative stress
parameters are summarized in Table 3. The
lipid peroxidation markers such as LHP,
TBARS and protein carbonyl, were
significantly elevated (p<0.05) in the
APAP-treated fish when compared with the
control. This indicates the severity of hepatic
damage under APAP exposure, whereas in
APAP+GNaP treated fish, the levels of lipid
peroxidation (LPO) markers were
significantly depleted (p<0.05) to values
near to those in control fish. GNaP-treated
fish did not showed any changes in the
levels of LPO markers.
Table 3. Effect of green synthesized gold nanoparticles on oxidative stress markers in liver of control and experimental
fish.
Parameters CON APAP APAP+GNaP CON+GNaP
LHPd
0.4341±0.05 0.07483±0.01a
0.2687±0.18b
0.4209±0.04c
TBARSe
1.318±0.08 3.389±0.22a
1.919±0.04b
1.879±0.06c
Protein Carbonyld
2.902±0.27 4.908±0.66a
3.140±0.50b
3.005±0.46c
CON control, APAP acetaminophen, GNaP green synthesized gold nanoparticles
a
(p < 0.05) as compared to CON
b
(p < 0.05) as compared to APAP
c
(p < 0.001) as compared to CON and APAP+GNaP. Data within the groups were analyzed using one-way ANOVA followed by
Dunnett multiple comparisons test. d
µmoles/mg protein, e
nanomoles/mg protein
Effect of gold nanoparticles on enzymatic
and non-enzymatic antioxidants and thiols
The effects of GNaP on enzymatic (SOD,
CAT, GSH-Px and GST) and non-
enzymatic (GSH) antioxidants and cellular
thiols levels (T-SH, NP-SH and P-SH) in the
liver homogenates are summarized in Table
APAP-treated fish showed a decrease in the
levels of SOD, GSH-Px, GSH, T-SH, NP-
SH and P-SH and an increase in the levels of
CAT and GST, when compared with the
control fish. However, the levels of the
above enzymatic and non-enzymatic
antioxidants significantly (p<0.05) altered
upon treatment with APAP+GNaP treated
fish.
GNaP-treated control fish did not showed
any alteration in the enzymatic and non-
enzymatic antioxidants, whereas the hepatic
thiols content was increased.

Kunjiappan S, et al
Table 4. Effect of green synthesized gold nanoparticles on enzymatic and non-enzymatic free radical scavenging
antioxidants and thiols in liver of control and experimental fish.
Parameters CON APAP APAP+GNaP CON+ GNaP
SODd
4.560±0.30 2.070±0.15a
3.880±0.25b
4.301±0.26c
CATe
7.408±0.20 27.879±0.54a
11.414±0.24b
7.901±0.22c
GSH-Pxf
1.045±0.07 0.4102±0.23a
0.7102±0.05b
0.9185±0.05c
GSTg
0.3432±0.02 1.526±0.05a
0.4313±0.02b
0.3965±0.02c
Non-enzymatic free radical scavenging antioxidants
GSHd
0.6128±0.012 0.3326±0.01a
0.4734±0.01b
0.6687±0.012c
Thiols
NP-SHd
0.8810±0.028 0.4260±0.012a
0.8062±0.023b
0.8363±0.025c
T-SHd
2.542±0.059 1.362±0.021a
2.360±0.059b
2.395±0.056c
P-SHd
1.661±0.036 0.9360±0.012a
1.554±0.037b
1.559±0.035c
a
b
c
(p < 0.05) as compared to CON and APAP+GNaP. Data within the groups were analyzed using one-way ANOVA followed by
Dunnett multiple comparisons test.
d
µmoles/min/mg protein
e
µmoles of H2O2 consumed/min/mg protein
Effect of gold nanoparticles on blood
plasma and hepatic proteins, bilirubin,
glycogen and lipids
Table 5 gives the details of the serum
protein, lipids (triglyceride, cholesterol) of
control and experimental fish. The levels
of protein, triglyceride and cholesterol
were significantly (p<0.05) reduced in the
APAP-treated fish compared to those of
the control fish. Whereas, the metabolic
alterations were restored to near-control
(p<0.05) in the APAP+GNaP treated fish.
GNaP treated control fish did not showed
any alterations in the protein and lipid
levels. Table 6 showed the activities of the
liver homogenate in protein, albumin,
globulins, bilirubin, glycogen, triglyceride,
cholesterol and creatinine of control and
experimental fish.
The levels of protein, albumin, globulins,
glycogen, triglyceride and cholesterol were
significantly (p<0.05) reduced and
increased the value of bilirubin and
creatinine in the APAP-treated fish
compared to those of the control fish.
Whereas, the metabolic alterations of these
parameters were significantly restored to
near-control (p<0.05) in the APAP+GNaP
treated fish.
any alterations in the proteins, creatinine
and lipid levels.
Table 5. Effect of green synthesized gold nanoparticles on blood plasma protein and lipid levels of control and
experimental fish.
Proteind
4.357±0.33 1.507±0.12a
2.846±0.17b
4.404±0.18c
Cholesterold
12.363±0.97 8.983±0.28a
10.539±0.60b
12.312±0.61c
Triglycerided
5.144±0.40 2.499±0.12a
3.543±0.36b
4.968±0.21c
a
b
c
(p < 0.05) as compared to CON and APAP+GNaP. Data within the groups were analyzed using one-way ANOVA followed
by Dunnett multiple comparisons test
d
mg/dL

Table 6. Effect of green synthesized gold nanoparticles on hepatic proteins, glycogen, bilirubin and lipids in
liver of control and experimental fish.
Proteind
1.858±0.018 1.443±0.013a
1.585±0.014b
1.760±0.018c
Albumind
0.08027±0.001 0.02496±0.009a
0.03524±0.009b
0.08370±0.009c
Globulind
1.778±0.018 1.419±0.013a
1.550±0.014b
1.617±0.018c
Glycogend
1.664±0.023 0.8699±0.012a
1.373±0.018b
1.552±0.021c
Bilirubind
0.03748±0.003 0.06695±0.008a
0.05828±0.002b
0.04070±0.001c
Cholesterold
9.039±0.43 7.103±0.27a
8.401±0.35b
9.073±0.34c
Triglycerided
4.531±0.29 0.2774±0.08a
1.635±0.14b
4.110±0.22c
Creatinined
0.435±0.08 0.983±0.11a
0.548±0.07b
0.431±0.078c
a
b
c
(p < 0.05) as compared to CON and APAP+GNaP. Data within the groups were analyzed using one-way ANOVA followed
by Dunnett multiple comparisons test
d
mg/g tissue
Effect of gold nanoparticles and Azolla
microphylla on hepatic ions
The effects of GNaP on hepatic ions such
as Na+
, K+
and Ca2+
in the liver
homogenate of control and experimental
fish are summarized in Figure 9. The
levels of hepatic ions were significantly
(p<0.05) reduced in the APAP-treated fish
compared to those of the control fish.
Whereas, the altered hepatic ions were
significantly elevated to near-control
(p<0.05) in the APAP+GNaP treated fish.
any alterations in the hepatic ions.
Figure 9. Effect of green synthesized gold nanoparticles on the hepatic ions status of control and experimental
fish. Values are mean± SD (n=6 in each group). #
p<0.05 as compared to CON. *
p<0.05 as compared to APAP.
†
p<0.05 as compared to APAP and CON. CON control, APAP acetaminophen, GNaP green synthesized gold
nanoparticles.

Kunjiappan S, et al
Discussion
Nanoparticles have drawn more attention
in biology and medicine because they can
be used as carriers for delivering small
molecules such as drugs, proteins and
genes (57). Most commonly studied metal
nanoparticles include gold, silver, titanium
oxide and iron nanoparticles (58). Among
these, gold being inert and relatively less
cytotoxic is extensively used for various
applications including drug and gene
delivery (59-61). Biosynthesis of gold
nanoparticles with the help of medicinal
plants has come into the limelight in
nanobiotechnology due to the growing
need to develop environmental friendly
benign technologies (62). No evidence
indicates that green synthesized gold
nanoparticles causes toxicity at the
clinical, histological, cellular and
molecular levels (63).
In this study, we have reported total
antioxidant capacity of various
concentrations of green synthesized gold
nanoparticles. From the analysis, it was
evident that antioxidant capacity increases
with increase in concentration up to certain
level (3 mg/mL) and beyond this
concentration saturation is observed. The
results are in agreement with the content of
flavonoids found in the surface of the gold
nanoparticles.
Previous studies have demonstrated that,
Cassia fistula aqueous extract and
phytochemically synthesized gold nano-
particles as progression of hypoglycemic
treatment for diabetic mellitus in mice
(62). A Bacillus licheniformis biomass
has also been reported for its antioxidant
as well as antidiabetic effect against
streptozocin-induced diabetes in mice (37).
However, studies on possible beneficial
effects of plant phytochemicals mediated
synthesized gold nanoparticles against
drug-induced toxicity.
The present study evaluated the effect of
green synthesized gold nanoparticles on
APAP-induced hepatotoxicity and liver
alterations in a fresh water fish (Cyprinus
carpio L.). In the current work, no
mortality, alteration in behavior of
experimental fish, changes in swimming
behavior, breathing and orientation or
color pattern were not observed during the
experimental period. The appearance of
control fish liver showed smooth and
shiny, with normal, whereas APAP-treated
fish liver appeared in dark black colour,
which is confirmed the liver toxicity of
APAP.
GNaP treated fish showed normal
appearance of liver as that of control fish.
Examination of pathological changes in
the livers of both control and experimental
fish revealed the highest hepatoprotective
activity of GNaP. Our finding was similar
to those of Wonkchalee et al. (64), who
treated hepatotoxic Syrian hamster with
Thunbergia laurifolia Linn.
The observed hepatoprotective activity
might be due to its several flavonoids on
the surface of the gold nanoparticles and
Azolla microphylla extract.
Acetaminophen-induced hepatic failure is
the second leading cause of liver
transplantation and accounts for
considerable levels of morbidity and
mortality (65).
Hepatotoxicity of acetaminophen has been
attributed to the formation of reactive
metabolite (N-acetyl-p-benzoquinone-
imine) by the actions of hepatic CYP450
enzymes (66). However, an overdose of
APAP causes depletion of cellular
glutathione level in the liver due to toxic
NAPQI, directly react with glutathione
(67).
Depletion of glutathione may have two
adverse effects. First, it reduces the
inactivation of the reactive metabolite and
tends to increase its covalent binding to
proteins (68, 69).
Second, it may therefore aggravate the
toxic effects of the reactive metabolite (70,
71).
In the present study, significantly elevated
levels of carbohydrate metabolic enzymes
such as LDH, G6PDH and MDH in the
liver homogenates of APAP-treated fish

revealed that increased permeability of the
hepatocytes and cellular leakage. An
increase in the levels of such enzymes
reflects the liver damage and related
oxidative stress (72, 73). Lactate
dehydrogenase catalyzes the reversible
oxidation of pyruvate to lactate in the
terminal step of glycolysis. It is also
involved in gluconeogenesis in tissues in
which lactate is converted to glycogen
through gluconeogenesis (74). An
increased LDH activity in fresh water fish
by Arsenite treatment was reported.
G6PDH activity was moderately increased
in paranchymal and inflammatory cells.
Inflammatory and epithelial cells are
responsible for ROS (reactive oxygen
species) generation. Moreover G6PDH is
responsible for increased metabolic
activity of liver by glucose oxidation
(oxidation of G-6-P) via hexose
monophosphate (HMP) shunt which is
essential for synthesis of fat. HMP shunt
pathway is the major source of NADPH,
which maintains the reductive
environment for all biosynthetic processes
using NADPH as a co-factor (75).
Increased level of MDH in APAP induced
fish indicated the change in the metabolic
pathways of energy generation and glucose
synthesis by gluconeogenesis (76).
Generally liver damage reflects
disturbances of cellular metabolism, which
lead to characteristic changes in cellular
and liver enzymes (77, 78).
Acetaminophen-induced hepatic damage
was accompanied by significant elevation
of both blood and hepatic tissue GPT,
GOT and ALP about 60%. The increased
levels of GPT, GOT and ALP, soluble
enzymes in cytoplasm of liver cells may be
a result of liver injury lead to increased
permeability of cell membranes (74). GPT
and GOT are two mitochondrial enzymes,
which plays an important role in the
conversion of amino acids to keto acids.
These results are in agreement with those
reported earlier by Senthilkumar et al.
(79). Treatment with green synthesized
gold nanoparticles, 2.5 mg/kg body
weight, significantly restored elevated
levels of both blood plasma and liver
tissue GPT, GOT and ALP.
Lipid peroxidation is an important
parameter of oxidative stress and usually
was reflected by increased levels of MDA
and TBARS, a lipid peroxidation end
product of lipid hydroperoxide (LHP) (50).
The increased levels of TBARS, LHP in
the liver tissue of the fresh water fish
indicated the tissue lipid peroxidation and
oxidative stress exerted by APAP. This
could be due to APAP-mediated gene-
ration of ROS and increased peroxidation.
On the other hand, excess of ROS
generation initiates protein-oxidation,
forming protein carbonyls and advanced
oxidation protein product (AOPP), which
have been described as reliable markers
for protein damage (80, 81). Our result is
supported by other researchers, who have
observed that oral exposure of APAP (500
mg/kg) in a fresh water fish (Pangasius
sutchi) for 24h duration induces liver
damage and alters the antioxidants.
Abraham (82) suggests that, paracetamol-
induced renal damage was accompanied
by an increase in lipid peroxidation on
rats. Vengerovskii et al. (83) reported,
paracetamol induced LPO processes in the
liver, along with calcium ions and pro-
inflammatory cytokines, damage the
barrier and matrix functions of hepatocyte
membranes. Lipid peroxidation in liver is
induced mainly by hepatotoxic chemicals
including paracetamol and alcohol either
directly or indirectly (84).
A novel finding in the present study is that,
green synthesized gold nanoparticles (2.5
mg/kg) when administered in a fresh water
fish helps in the prevention of hepatic
damage (completely) caused by paracet-
amol.
The biochemical changes and alterations in
enzyme activities induced by oxidative
stress on liver function was also explored.
The present results showed that, high dose
of APAP, deplete the hepatic levels of
proteins, albumins, globulins, glycogen,
triglycerides, cholesterol as well as elevate

Kunjiappan S, et al
the levels of bilirubin and creatinine. Since
there is a close relationship between the
rate of protein synthesis in the liver tissue
and total protein concentrations in the
plasma. The depleted level of total protein
(composed of albumin and globulin) in
plasma reflects the decrease of protein
synthesis in liver tissue. Administration of
green synthesized gold nanoparticles and
showed significant effect on both blood
and liver tissue protein, albumin and
globulin levels. The rise of globulin level
in the experimental fish may improve the
immune system mediated immune
responses (85).
Experimentally determined elevated levels
of liver bilirubin and creatinine indicate
the severe hepatic damage and renal
impairment. It also confirms the hepat-
otoxic nature of APAP. Bilirubin is
considered as an index for the assessment
of hepatic function and abnormality
indicates hepatobiliary disease and severe
disturbance of hepatocellular architecture
(86). An increase in creatinine indicates
the pathological changes in hepaticbiliary
flow (87).
The main function of the liver is to store
energy in the form of glycogen or lipids
(cholesterol or triglycerides). High dose of
APAP reduces the levels of glycogen,
cholesterol and triglycerides in the carp
fish, indicates liver injury. In our study,
green synthesized gold nanoparticles
+APAP were restored the depleted levels
of glycogen and lipids near-control fish.
Simultaneously Azolla microphylla
particles were also maintained the levels of
glycogen and lipids significantly higher
than the APAP exposed fish.
In the present study, we found that the
activities of antioxidant enzymes such as
SOD, CAT, GSH-Px and GST and non-
enzymatic GSH were significantly altered
by higher doses of APAP. The altered
levels of these enzymes were maintained
by Azolla microphylla phytochemically
synthesized gold nanoparticles. Similarly,
APAP+GNaP treated fish restored the
levels near to control. SOD, CAT, GSH-Px
are known to be inactivated by H2O2, O2*-
and *OH respectively. SOD and CAT are
the major antioxidant defense components
(88). CAT primarily catalyzes the
conversion of H2O2 to H2O. Increased CAT
activity in the liver of APAP treated fish
revealed the free radical induced damage
of hepatic cells. Excessive generation of
free radicals may result in alterations in the
biological activity of macromolecules. It
has been proved that, CAT is so efficient
that it cannot be saturated by H2O2 at any
concentrations (89). SOD has been
reported as one of the most important
enzymes in the enzymatic defense system.
It scavenges the superoxide anion to form
H2O2, thus diminishing the toxic effect
caused by this radical. SOD is necessary
because superoxide reacts with sensitive
and critical cellular targets. It reacts the
NO radical and makes toxic peroxynitrite
(ONOO-
) and also SOD catalyze the
dismutation of superoxide (O2*-
) radicals
to oxygen and H2O2 (90). In Azolla
microphylla phytochemically synthesized
gold nanoparticles, a significant decline in
hepatic SOD activity was observed and
thus reduces the reactive free-radical
induced oxidative damage in liver.
H2O2 is normally detoxified in cells by
either CAT or GSH-Px. In GSH-Px
catalyzes the reduction of hydrogen and
organic H2O2, utilizing reduced glutathione
(GSH) as cofactor, hence having an
important role in protecting cells from
lipid peroxidation. GSH is readily oxidized
to glutathione disulfide (GSSG) by the
GSH-Px reaction. GSSG can be reduced
by NADPH-dependent reaction catalyzed
by glutathione reductase (91, 92). NADPH
is an essential cofactor for the generation
of GSH. The reduced tissue GSH-Px
activity in carp fish under APAP treatment
indicates the damage of GSH-Px protein.
The observed reduction in GSH-Px
activity could be due to the structural
alteration of GSH-Px protein by APAP or
its toxic metabolites (NAPQI). Addit-

ionally, APAP-mediated GSH depletion
could also be one of the reasons for the
down regulated activity of GSH-Px in the
liver tissue of APAP-treated fish.
Glutathione-S-transferases (GST) are an
inducible phase II detoxification enzymes
that catalyze the conjugation of glutathione
with reactive metabolites formed during
phase I of metabolism (93). Induction of
GST synthesis is a protective mechanism
that occurs in response to APAP exposure.
Our finding is supported by Sun et al. (94)
who explained the decline in GSH level at
lower concentrations and inductions of
GST activity were observed in Carassius
auratus, exposed to pyrene. Glutathione
(GSH) provides a first line of defense and
scavenges free radical oxygen species.
GSH is a tripeptide (Cys-Gly-Glu), non-
enzymatic biological antioxidant, present
in liver. GSH plays an important role in
protecting the liver against APAP-induced
hepatotoxicity, because NAPQI is
detoxified by conjugation with GSH.
During overdose of APAP, protein
sulfhydryls groups (P-SH, T-SH, and NP-
SH) in liver tissue are significantly
reduced. Protein sulfhydryls groups are
important targets of oxidation stress,
where protein oxidation can acts as a
cellular redox switch to modulate protein
function, particularly those involving cell
death (95). The depletion of non-protein
thiols and protein thiols in the liver, about
50-85% of which is APAP toxic byproduct
NAPQI, as well as in enhanced lipid
peroxidation. Lipid peroxidation resulting
from oxidative stress contributes to the
initiation and progress of liver damage.
ROS can cause the S-hydroxylation of
protein sulfhydryls to slightly oxidized
state, sulfenic acid, which is reversible.
Sulfenic acid can be further oxidized to
sulfinic acid and sulfonic acid, which are
irreversible modifications. The changes or
loss of protein sulfhydryl groups may
cause the mitochondrial dysfunction that is
observed at high dose of APAP.
The ability of the Azolla microphylla
phyto-chemically synthesized gold
nanoparticles reduces the toxic byproduct
NAPQI, by regeneration of glutathione
and also maintains the protein sulfhydryls
groups in the liver.
The APAP –treated fish liver tissue
showed reduced Na+
, K+
and Ca2+
ions
when compared with control fish, indicates
that APAP-treated fish experienced ionic
disturbances.
In fresh water fish, membrane bound
ATPase (Mg2+
and Na+
/K+
-ATPase) play
a significant role in ionic regulation of
cellular components and maintenance of
tissue osmolority (96) against concen-
tration gradients and across membranes
(97).
Na+
and K+
are the principal cations of
intracellular fluid and play an important
role in the maintenance of acid-base
balance. The decreased levels of Na+
and
K+
observed in the present investigation in
APAP-treated fish liver tissue may be
attributed to the pathophysiology of
hepatic damage and the underlying disease
process (98). The calcium concentration
gradient between the inside the cell (10-7
M) and the extra cellular fluid (10-3
M) is
maintained by an active membrane-
associated calcium and magnesium
effluxing adenosine triphosphate (ATPase)
enzyme system which is important
potential target for toxicants. Chemically
induced hepatotoxicity may lead to the
disruption of calcium homeostasis (99,
100). Disruption of calcium homeostasis
may result in the activation of
mitochondrial metabolism, ATP synthesis
and damage of microfilaments used to
support cell structure. Similarly,
APAP+GNaP treated fish showed hepatic
ions level near-control, which indicates the
membrane-stabilizing action against APAP
toxicity.
Histopathological examination of APAP-
treated fish liver showed total loss of
architecture.
The abnormalities were effectively
controlled in the Azolla microphylla
particles treated fish.

Kunjiappan S, et al
Conclusions
Thus, the present study concluded that
phytochemically synthesized gold
nanoparticles protects liver against
oxidative damage and tissue damaging
enzyme activities and could be used as an
effective protector against acetaminophen-
induced hepatic damage in fresh water
common carp fish. Reduction of gold ions
by methanol extract of Azolla microphylla
resulted in the formation of stable and
biocompatible nanoparticles. The flavono-
ids present in Azolla microphylla served
dual role as effective reducing agents to
reduce gold and also as stabilizers to
provide a robust coating on the gold
nanoparticles in a single step. The
hepatoprotective and antioxidant action of
GNaP was probably accomplished via
elevating cellular antioxidative capacity,
protecting GSH depletion, inhibiting lipid
peroxidation and enhancing protein
synthesis. The gold nanoparticles have
been proven for their non-toxic and
protective effects over the cells. The
observed hepatoprotective and antioxidant
effects of GNaP might be due to its several
flavonoids on the surface of the gold
nanoparticles. Thus Azolla microphylla
particles may be used as a safe, cheap,
effective alternative chemopreventive and
protective agent in the management of
liver diseases. Further studies are,
however, required to investigate the
detailed molecular mechanisms of the
protective effect of flavonoids based gold
nanoparticles against toxicant-induced
liver injury in fish and other animals.
Acknowledgments
This work was supported and sponsored
by Department of Chemical Engineering,
Jadavpur University, Kolkata. The authors
also thank M/S. Renuka Clinical
Laboratory, Namakkal India, for valuable
support in performing histopathological
analysis throughout the research. The
authors also wish to acknowledge DST
unit on Nanoscience, Indian Institute of
Technology, Madras for HRTEM meas-
urements. They also thank Dr. S. Muru-
gesan, Assistant Professor, Department of
Pharmacy, Birla Institute of Technology
and Science, Pilani, Rajasthan, India, for
timely help and support.
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Hepatoprotective and antioxidant effects of Azolla microphylla based gold nanoparticles against acetaminophen induced toxicity in a fresh water common carp fish (Cyprinus carpio L.)