The ameliorating effect of Acadian marine

plant extract against ionic liquids-induced

oxidative stress and DNA damage in
marine macroalga Ulva lactuca
Manoj Kumar, C.R.K.Reddy &
Bhavanath Jha

Journal of Applied Phycology

ISSN 0921-8971
Volume 25
Number 2
J Appl Phycol (2013) 25:369-378
DOI 10.1007/s10811-012-9871-8

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Author's personal copy

J Appl Phycol (2013) 25:369378
DOI 10.1007/s10811-012-9871-8

The ameliorating effect of Acadian marine plant extract against

ionic liquids-induced oxidative stress and DNA damage in marine
macroalga Ulva lactuca
Manoj Kumar & C. R. K. Reddy & Bhavanath Jha

Received: 12 January 2012 / Revised and accepted: 20 June 2012 / Published online: 11 September 2012
# Springer Science+Business Media B.V. 2012

Abstract Ionic liquids (ILs) are generally considered as the

green replacement for conventional volatile organic solvents. Nonetheless, their high solubility in water with proven toxic effects on aquatic biota has questioned their green
credentials. In the present study, the detoxification potential
of Acadian marine plant extract powder (AMPEP) prepared
from the brown alga Ascophyllum nodosum was investigated against the 1-alkyl-3-methylimidazolium bromide
[C12mim]Br ionic liquid-induced toxicity and oxidative
stress in marine macroalga Ulva lactuca. The IL
([C12mim]Br) at LC50 (70 M) exposure triggered the generation of reactive oxygen species (ROS) such as O2, H2O2
and OH causing membrane and DNA damage together with
inhibition of antioxidant systems in the alga. The supplementation of AMPEP (150 g mL1) to the culture medium
significantly reduced the accumulation of ROS and lipid
peroxidation together with the inhibition of lipoxygenase
(LOX) activity specially LOX-2 and LOX-3 isoforms. This
is for the first time wherein comet assay was performed to
ascertain the protective role of AMPEP against DNA damage in algal tissue grown in medium supplemented with IL
and AMPEP. The AMPEP showed protective role against
DNA damage (545 % tail DNA) when compared to those
of grown in IL alone (4570 % tail DNA). Further, specific
isomorphs of different antioxidant enzymes such as superoxide dismutase (Mn-SOD-1, ~150 kDa), ascorbate peroxidase (APX-4, ~55 kDa), glutathione peroxidase (GSH-Px2, ~55 kDa) and glutathione reductase (GR-1, ~180 kDa)
responded specifically to AMPEP supplementation. It is
evident from these findings that AMPEP could possibly be
M. Kumar : C. R. K. Reddy (*) : B. Jha
Discipline of Marine Biotechnology and Ecology,
CSIR-Central Salt and Marine Chemicals Research Institute,
Gijubhai Badheka Marg,
Bhavnagar 364002, India
e-mail: crk@csmcri.org

used for circumventing the negative effects arising from ILsinduced toxicity in marine ecosystem.
Keywords Ionic liquid . AMPEP . Marine algae .
DNA damage . Ulva lactuca . Ascophyllum nodosum

Introduction
Room temperature ionic liquids (ILs) have emerged as a
new class of solvents and are widely accepted as green
solvents and potential green replacements for conventional organic solvents due to their unique array of physicochemical properties (Pham et al. 2010). These include
excellent solvation ability for a wide range of compounds,
nonflammability, high chemical/thermal stability, favourable conductivity and negligible vapour pressure (Earle
and Seddon 2000). The low volatility of ILs limits their
release in the atmosphere and thus reduces the risk of air
pollution. Nevertheless, the higher solubility in water coupled with non-biodegradable nature of ILs is a great concern
of aquatic pollution arising from accidental spills (Romero
et al. 2008; Coleman and Gathergood 2010). Therefore, the
green credentials of these ILs have been questioned for their
use in aquatic environments (Pham et al. 2010). In view of
the growing industrial interest in ILs, concerns are rising for
their eventual release in the aquatic environment through
industrial effluents and accidental leakage, and its consequent contamination which may negatively impact the structure and function of aquatic ecosystem.
The 1-alkyl-3-methylimidazolium salts are some of the
most common ILs used in industrial application and are
reported to have poor biodegradability resulting to retain
their properties intact in water (Coleman and Gathergood
2010). Recently, there has been considerable effort to determine the toxicity of ILs on both terrestrial and aquatic

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J Appl Phycol (2013) 25:369378

organisms (Cho et al. 2008; Latala et al. 2010; Pham et al.

2010). The toxicological studies of ILs investigated so far
have demonstrated that ILs cause membrane damage and
disruption, coupled with increased generation of cellular
reactive oxygen species (ROS) manifesting the inhibition
of photosynthesis leading to oxidative stress (Yu et al.
2009). Seaweeds are one of the primary producers of the
coastal waters and thus form the first trophic level in the
food chain. These being an abundant component in coastal
waters are considered as alternative promising source of
biofuel and chemicals besides with potential to reduce
greenhouse gas emissions and global warming (APPA
2010). Thus, the accidental release of ILs into the aquatic
ecosystems may cause serious environmental risks by disturbing the food web dynamics, ecosystem productivity and
nutrient cycle.
Recently, we have evaluated the toxicity and detrimental
effects of ILs belonging to 1-alkyl-3-methylimidazolium
bromide salts with different alkyl chain lengths in the green
macroalga Ulva lactuca (Kumar et al. 2011a). The toxicity
of ILs was found to be mediated through ROS generation
that primarily induced by the activation of membrane bound
NADPH oxidase, cytochrome oxidase and lipoxygenase
enzymes which in turn lead to the severe DNA damage
and ultimately cell death. The evidence of ILs toxicity
underlines the need to investigate some remedial measures
for mitigating toxicity and adverse effects of these compounds in both terrestrial and aquatic ecosystems. In the
present study, we investigated the potential of Acadian
marine plant extract powder (AMPEP) for mitigating
ILs-induced toxicity. AMPEP is a commercially manufactured product prepared from the temperate brown alga
Ascophyllum nodosum. Recent studies have explored the
diversified functions of AMPEP which includes improved
root nodulation (Khan et al. 2008), improved plantlet regeneration in tissue culture (Hurtado et al. 2009), its function to
act as cytokinin-like elicitors (Khan et al. 2010), and its role
in enhancing daily growth rates and mitigating the occurrence of epiphytes (Loureiro et al. 2011). In this study, we
first assessed the antioxidant properties of AMPEP and
subsequently investigated its role in scavenging ROS, regulation of antioxidant system and protection from DNA
damage caused by ILs-induced toxicity in the marine macroalga U. lactuca.

autoclaved seawater to remove epiphytes and any superficial

foreign matter. The cleaned fronds were acclimatised to laboratory conditions by culturing in aerated flat bottom, Erlenmeyer flasks in PES medium (Provasoli 1968) supplemented
with GeO2 (5 mg L1) for 10 d. During the acclimatisation
period, the medium was replenished on alternate days and
maintained under cool white fluorescent tubes at an irradiance
of 50 mol photons m2 s1 with a 12:12-h light/dark cycle at
221 C.
To study the ameliorative potential of AMPEP against
ILs-induced oxidative damage, we have selected the IL 1dodecyl-3-methyimidazolium bromide ([C12mim]Br) salt
(Fig. 1) which has recently been demonstrated by our group
to induce oxidative damage in U. lactuca at LC50 value of
0.07 mM (Kumar et al. 2011a). IL [C12mim]Br was synthesised in the laboratory, as described in the literature
(Bonhote et al. 1996) with a purity of >99 % established
through 1HNMR spectroscopy by integrating the proton
signals with respect to an internal standard of tetramethyl
silane. The effective concentration of AMPEP with significant ameliorating potential against IL-induced oxidative
damage was empirically determined with cell viability assay
wherein the acclimatised thalli were cultured as mentioned
above for 4 d in the following treatments: (1) filtered natural
sea water (NSW) (control), (2) NSW+IL at concentration
0.5LC50, LC50 and 2LC50, (3) NSW+IL at concentration
0.5LC50, LC50 and 2 LC50 with AMPEP (50
250 g mL1). The TTC (2,3,5-triphenyltetrazolium chloride)-based method was employed to measure the cell
viability according to Shiu and Lee (2005). This assay
is based on the tetrazolium salt reduction to red formazon by dehydrogenase respiratory enzymes and thus
indicates the resilience of the mitochondrial component
of the cell machinery when challenged with IL-induced
stress. After treatment, the thalli (50 mg FW) were
incubated at 25 C in 3 mL NSW containing 50 mM
potassium phosphate buffer (pH 7.4) and 0.8 % (w/v)
TTC in darkness for 16 h. Thereafter, the thalli were
washed three times in NSW and the intracellular insoluble red formazon was extracted twice with 5 mL of
95 % ethanol at 80 C for 20 min. Extracts were
combined and the absorbance was determined at
530 nm. Cellular viability was calculated in percentage
while considering the absorbance of control thalli with
100 % viability.

Materials and methods

Oxidative stress and histochemical localization of ROS

The vegetative thalli of U. lactuca were collected from the

intertidal region of the Veraval Coast (20 54 N, 70 22 E),
Gujarat, India. Selected, clean juvenile thalli were brought to
the laboratory in a cool pack. In order to initiate unialgal
culture, the fronds were cleaned manually with a brush in

The oxidative stress caused by [C12mim]Br IL treatment

was determined by measuring the lipid peroxidation (malondialdehyde (MDA) level) and ROS (including O2, OH and
H2O2) content in the U. lactuca. The level of lipid peroxidation in the thallus was determined as described by Kumar

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371

Protoplast isolation and comet assay

Fig. 1 General structure of 1-dodecyl-3-methylimidazolium-based

ionic liquid, [C12mim]Br

et al. (2011a). Tissue (0.5 g FW) was extracted in 2 mL of

0.5 % thiobarbituric acid prepared in 20 % trichloroacetic
acid. Extract was heated at 95 C for 30 min and then
quickly cooled on ice. After centrifugation at 10,000g
for 10 min, the absorbance of the supernatant was measured
at 532 nm. Correction of non-specific turbidity was done by
subtracting the absorbance value taken at 600 nm. The level
of lipid peroxidation was expressed as nmol of MDA
formed using an extinction coefficient of 155 mM cm1.
The O2 production rate was measured according to Liu et
al. (2010). Samples (500 mg FW) were homogenised in
4 mL of 65 mM potassium phosphate buffer (pH 7.8) and
centrifuged at 5,000g for 10 min. The incubation mixture
contained 0.9 mL of 65 mM potassium phosphate buffer
(pH 7.8), 0.1 mL of 10 mM hydroxylammonium chloride
and 1 mL of the supernatant. After incubation at 25 C for
20 min, 17 mM sulphanilic acid and 7 mM naphthylamine were added to the incubation mixture. After
reacting at 25 C for a further 20 min, the absorbance was
read at 530 nm. A standard curve, with NaNO2, was used to
calculate the production rate of O2. For the estimation of
H2O2, 250 mg fresh samples was extracted in 0.5 mL Na
acetate buffer (50 mM, pH 6.5) and incubated in reaction
media containing 50 mM Na acetate buffer, 1 mM 4aminoantipyrine, 1 mM 2,4-dichlorophenol, 50 mM MnCl2
and 0.2 mM NADH for 24 h. The oxidation of aminoantipyrine was recorded at 510 nm and the absorbance was
compared to the standard curve prepared with H2O2 in the
same reaction mixture. Determination of OH production
was performed based on the degradation of 2-deoxyribose
by OH radicals (Liu et al. 2010). The O2 and H2O2 were
visually detected in the thalli using nitroblue tetrazolium
(NBT) and 3,3-diaminobenzidine as the substrates respectively, according to Kumar et al. (2012).
Antioxidant properties of AMPEP
The antioxidant potential of AMPEP was determined by
estimating its total antioxidant activity, total phenol content,
DPPH and OH, O2, H2O2, and NO radical scavenging
activity, iron chelation and reducing power according to
Hazra et al. (2008). All the tests were performed in triplicate
in order to obtain the EC50 values using an AMPEP concentration range of 0500 g mL1.

Following IL and AMPEP treatments, protoplasts were isolated according to Kumar et al. (2011a) and suspended in
saline phosphate buffer at pH 7.4 and kept on ice at 4 C.
The protoplast yields were estimated by counting the cells
using a haemocytometer under an inverted microscope.
For comet assay the protoplast solution (50 L) containing approximately 1103 protoplasts was mixed with 50 L
of 1 % low-melting temperature agarose (LMPA) dissolved
in phosphate-buffered saline. From this mixture, an aliquot
of 80 L was layered on to a base slide, which was precoated with 1 % agarose, and then covered with a coverslip.
When the agarose gel solidified, the coverslip was gently
slide off and another agarose layer (90 L, 0.5 % LMPA)
was layered while covering it with a new coverslip and then
left for 10 min on a chilled metal plate to solidify the
agarose layer. Following this, the coverslip was removed
and the slides were submerged in alkali lysis solution
(2.5 MNaCl, 10 mM Trizma and 100 mM EDTA) at pH>
13 overnight at 4 C. Thereafter, the glass slide was incubated in fresh, cold electrophoresis buffer (i.e. 0.3 M NaOH
and 1 mM EDTA at pH 13) in a horizontal electrophoresis
tank (Model POWER PAC 200, Bio-Rad Co., USA) for
30 min at room temperature to allow for DNA unwinding.
Electrophoresis was performed for 10 min at 25 V and
300 mA in a chamber cooled in an ice bath. After electrophoresis, the glass slides were neutralised in 0.4 M TrisHCl
(pH 7.5) buffer, washed twice in distilled water and left
overnight for drying at room temperature. The slides were
stained following the silver staining method of Nadin et al.
(2011). Stained slides were examined using an Olympus
Microscope model-BX60 fitted with an Olympus-DP72
camera. The classification of comet category and their tail
measurement was carried out according to Garcia et al.
(2007).
Determination of the enzymatic antioxidants
Extracts for determining the activities of antioxidant
enzymes namely superoxide dismutase (SOD), ascorbate
peroxidase (APX), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) and lipoxygenase (LOX) were
prepared under ice-cooled conditions in the extraction buffer
at a proportion of 1:2 (w/v). For SOD, thalli were homogenised in buffer [0.05 M potassium phosphate buffer (PPB,
pH 7.8), 1 % (w/v) polyvinylpyrrolidone (PVP), 0.1 mM
Na2EDTA and 1 % (v/v) Triton X-100], for APX [0.1 M
PPB (pH 6.8), 1 mM Na2EDTA, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.5 mM L-AsA (freshly prepared)], for GR [0.1 M PPB (pH 6.8), 3 mM Na2EDTA, 1 %
(w/v) PVP and 1 % (v/v) Triton X-100] and for GSH-Px
[100 mM Tris HCl (pH 7.9), 1 % ascorbate and 1 % (w/v)

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Table 1 Effect of ionic liquid [C12mim]Br exposure on cell viability (in %) in U. lactuca in various treatments, viz. control, 0.5LC50, LC50 and 2
LC50, with and without Acadian marine plant extract powder (AMPEP)
ILs concentration

Control
0.5 LC50
LC50
2xLC50

Without AMPEP

100 a
73.292.6b
48.802.8 c
10.331.7 d

With AMPEP (g mL-1)

50

100

96.771.9 a
71.443.5 b
52.965.7 c
14.051.1 d

97.873.6
77.823.8
62.852.8
14.722.4

a
b
c
c

150

200

250

102.223.4a
81.242.7 b
83.183.6 b
16.450.97 c

98.985.6 a
82.904.6 b
81.335.1 b
16.732.2 c

97.044.6
83.834.2
82.975.3
16.270.6

a
b
b
c

Different lower case letters in a columns indicate significant difference at p<0.01 analysed by one way ANOVA (meanSD, n03)

PVP]. The homogenates were centrifuged at 12,000g for

15 min at 4 C, and supernatants were assayed for respective
enzyme activity according to Kumar et al. (2010a, 2011b).
Extracts for LOX activity were prepared in extraction buffer
containing [50 mM PPB (pH 7.5), 0.2 mM CaCl2, 1 mM
GSH, 1 mM PMSF, 0.3 mM DDT, 0.2 mM EDTA, 1 % PVP
and 0.1 % Triton X-100]. The activity of antioxidant
enzymes and LOX was determined according to Kumar et
al. (2010a); their isoenzyme analysis and molecular mass
were determined with 10 or 12 % non-denaturating polyacrylamide gels using their specific activity staining procedures (Kumar et al. 2011b).
Statistical analysis
Results are expressed as the mean of three replicates with
standard deviation. Statistical analysis was performed by
one way analysis of variance (ANOVA). Significant differences among the mean values were determined by least
significant difference at p<0.05 or p<0.01.

Fig. 2 Effect of Acadian

marine plant extract powder
(AMPEP, 150 g mL1) on
detoxification of reactive
oxygen species (O2, OH and
H2O2) and prevention from
damage of membrane (MDA
level) in U. lactuca exposed to
ionic liquid [C12mim]Br
concentrations of LC50
(70 M), 2LC50 (140 M).
Different lowercase letters on
similar shade columns indicate
significant difference at p<0.01
analysed by one way ANOVA
(meanSD, n03) (Kumar et al.
2011a)

Results
Cell viability
Cell viability of U. lactuca was influenced significantly by
IL exposure and decreased with the increase of IL concentration (Table 1). However, the supplementation of AMPEP
to the cultures during IL exposure (LC50) resulted in a
considerable linear increase in the cell viability. Application
of AMPEP (150 g mL1) led to an increase in cell viability
of U. lactuca to 83.18 % (LC50) when compared to 48.80 %
during IL exposure (LC50). A further increase in the concentration of AMPEP however did not result in
corresponding increase in cell viability. Nevertheless,
AMPEP supplementation (at all the studied concentration)
was inefficient in protecting the algal cells from IL-induced
toxicity (2LC50) and exhibited minimum cell viability of
10.3316.73 % when compared to control. Based on these
findings, the potential of AMPEP to detoxifying the ROS,
protection from DNA damage and the effects on the

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373

Fig. 3 Histochemical localization of reactive oxygen species generation: a O2 and b H2O2 in U. lactuca following exposure to the ionic
liquid [C12mim]Br for 4 d. Reactive radicals were detected with nitroblue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) staining,

respectively. Characters (ad) inside figures represent the various treatments, viz. control, LC50, 2LC50, LC50 +AMPEP, respectively. The
AMPEP was used at a concentration of 150 g mL1 in the culture
medium (Kumar et al. 2011a)

antioxidant system of the alga were studied using the concentration of LC50 [C12mim]Br together with AMPEP at
150 g mL1.

oxidative damage to lipid membranes accompanied with

significant accumulation of ROS in alga by 1.255.25-fold
higher than the control. However, the exogenous application
of AMPEP at concentration 150 g mL1 in the media
supplemented with [C12mim]Br IL at LC50 and 2LC50
concentration was found to be highly efficient in detoxifying the ROS by nearly 6070 % (Fig. 2), at least in case of
LC50 exposure, but was incompetent to rescue the plants
cultured in lethal concentration of IL (i.e. 2LC50). Histochemical staining (Fig. 3) used for in situ localization of
O2 and H2O2 radicals apparent the distribution of reduced
NBT-dependent dense blue formazone and H2O2-dependent
brown precipitate, respectively, all over the tissue in plants
exposed to LC50 and 2LC50 concentrations of [C12mim]Br
IL with intense blue formazone and brown precipitate at
extreme lethal dose of IL. Further, Fig. 3 confirmed
AMPEP-mediated complete alleviation of O2 and H2O2
accumulation in plants exposed to LC50 concentration of
[C12mim]Br salt.

Generation, histochemical localization of ROS

Algal exposure to IL at concentrations LC50 and 2LC50
results in induction of oxidative stress (Fig. 2) with a considerable increase in membrane lipid peroxidation (MDA
level) estimated to be 2.8- and 4.2-fold higher, respectively,
over the control (3.08 nmol g1 FW). In addition, there is

Table 2 Scavenging of reactive oxygen species, iron chelation, total

phenol and total antioxidant potential of Acadian marine plant extract
powder (AMPEP)
Activity

Superoxide anion ( O2) scavenging

Hydroxyl radical (OH) scavenging
Hydrogen peroxide (H2O2) scavenging
Nitric oxide radical (NO) scavenging
DPPH radical scavenging
Iron chelation
Total phenolic content (mg PGE g1 extract)
Total antioxidant (mg AAE g1 extract)

IC50 (g mL-1)
MeanSD, n03
57.962.65
127.237.84
177.787.89
67.654.59
85.014.15
93.981.90
62.065.94
1.550.12

AAE ascorbic acid equivalent, PGE phloroglucinol equivalent

Antioxidant properties of AMPEP

The results of the antioxidant properties of AMPEP and its
ability to scavenge reactive oxygen radicals are presented in
Table 2. AMPEP had a total antioxidant activity and phenolic content with values of 1.550.12 mg ascorbic acid
equivalent g1 extract and 62.065.94 mg phloroglucinol
equivalent g1 extract, respectively. The EC50 values for
scavenging free radicals were 57.962.65, 127.237.84

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Fig. 4 Reducing power of

AMPEP compared with
commercial synthetic
antioxidant butylated
hydroxytoluene (BHT).
Different lowercase letters on
similar shade columns indicate
significant difference at p<0.01
analysed by one way ANOVA
(meanSD, n03)

and 67.654.59 g mL1 for superoxide, hydroxyl and

nitric oxide, respectively. The EC50 for hydrogen peroxide
scavenging was 177.787.89 g mL1. The extract was
found to be a potent scavenger of DPPH radicals and iron
chelator with EC50 values of 85.01 4.15 and 93.98
1.90 g mL1. The reducing power of the extract was found
to be almost similar to that of standard butylated hydroxytoluene (BHT) and increased with increasing amounts of the
extracts (Fig. 4).

70 % of DNA in tail) and 4 (very high damage, >70 % of

DNA in tail) increased significantly (p <0.01) in plants
treated with LC50 concentrations (Table 3). Nevertheless,
supplementing the culture media with AMPEP (150 g mL-1)
inhibited the IL (LC50)-induced DNA damage to an appreciable amount whereby majority of the comets were
rated in 0 (i.e. no damage, 05 % of DNA in tail), 1 (525 % of
DNA in tail) and 2 (2545 % of DNA in tail) categories, as
compared to the control with comets rating in categories 0 and
1 only.

DNA damage
Enzymatic antioxidants
Comet assay was used as a sensitive biological marker for
measuring DNA damage in cells exposed to oxidative stress
representing the disproportion between free radical production and functions of the antioxidant system. Results
obtained from comet assay clearly evident the IL-induced
DNA damage in Ulva. Following exposure to IL, the number of comets belonging to category 3 (high damage, 45

Table 3 Effect of ionic liquid [C12mim]Br exposure on DNA damage

in U. lactuca assessed by COMET assay in various treatments, viz.
control, LC50 and LC50 +AMPEP
Comet
category

0
1
2
3
4

% DNA
in tail

05
525
2545
4570
>70

Tail length
(M)

Number of comets
Control

LC50

LC50 +
AMPEP

4.28.4
8.524.8
25.038.7
39.055.8

48
9
3

10
4
3
37

14
24
21
1

56.096.3

The AMPEP was used at a concentration of 150 g mL1 in the

culture medium. A total of 60 comets were examined for each treatment with two replicates (Kumar et al. 2011a)

Algal exposure to IL (LC50) decreased the enzyme activities

of SOD, APX and GSH-Px by 1.41.65-fold (p<0.01) over
the control activities with 87.45, 0.44 and 2.63 U mg1
protein, respectively (Fig. 5a). In contrast, the activity GR
was quite stable to the IL toxicity and was comparable to
that of control activity with 0.46 U mg1 protein. The application of AMPEP to the culture media containing IL (LC50)
reverted the toxic effects of IL by elevating the antioxidant
enzyme activities ranging from 1.38- to 3.12-fold over the
control values. Interestingly, IL treatment enhanced the LOX
activity by 3.0-fold in plants, while it reduced dramatically
with AMPEP application from 2.46 U mg1 protein (LC50) to
1.08 U mg1 protein, which was comparable to the control
activity.
The apparent higher activity of SOD observed in LC50 +
AMPEP treatments were solely attributed to Mn-SOD (SOD1,~150 kDa) and Fe-SOD (SOD-2, ~120 kDa) confirmed by
using H2O2/KCN as inhibitors. In the control, one Fe-SOD
and two Zn-SOD (SOD-3, ~20 kDa and SOD-4, ~35 kDa)
were observed (Fig. 5a). However, exposure of IL (LC50)
resulted in partial or complete inhibition of Fe and Zn-SOD
isoforms. The enzyme APX showed differential regulation in
response to IL (LC50) with induction of APX-1 (~125 kDa)
and APX-5 (~45 kDa), partial inhibition of APX-3 (~65 kDa)

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Fig. 5 Effect of ionic liquid [C12mim]Br concentration on a activity of

antioxidative enzymes (in U mg1 protein) and b their isoenzyme
analysis in U. lactuca in various treatments, viz. control, LC50 and
LC 50 + AMPEP. The AMPEP was used at a concentration of

150 g mL1 in the culture media (Kumar et al. 2011a). Different

lowercase letters on columns indicate significant difference in enzyme
activities of a specific enzyme at p<0.01 analysed by one way ANOVA
(meanSD, n03)

and complete inhibition of APX-2 (~100 kDa). The presence

of AMPEP resulted in an induction of APX-4 (~55 kDa)
together with the increased intensity of APX-3. However,
the control plants exhibited only two APX isoforms (i.e.
APX-2 and APX-3). The activity gel of GSH-Px showed only
two isoforms GSH-Px-1 (~80 kDa) and GSH-Px-2 (~55 kDa)
in control; however, the intensity of these forms in IL (LC50)exposed plants was diminished and increased sharply in the
presence of AMPEP (Fig. 5b). Two forms of GR (GR-1,
~180 kDa; GR-2, ~135 kDa) were visualised in a 10 %
activity staining gel in AMPEP-treated plants, while only a
single isoform (GR-1) has been observed in control. The
exposure of IL (LC50) markedly inhibited the activity of
GR-1 with the induction of GR-2 isoform. Further, IL
(LC50) treatment result in induction of two new isoforms of
LOX (i.e. LOX-2 and LOX-3) with molecular weights ~80
and ~55 kDa, respectively, in addition to LOX-1 (~125 kDa)
recorded in the control. Nevertheless, AMPEP application
significantly reduced the LOX activity with the inhibition of
LOX-2 and LOX-3 isoforms.

Discussion
The earlier investigations aimed at studying the toxicity of
ILS have indicated that the contamination of marine ecosystem with ILs cause a serious threat to the survival of seaweeds (Kumar et al. 2011a). The present findings revealed
significant changes in biochemical processes in U. lactuca
following exposure to IL (LC50). In general, IL exposure
resulted in reduced cell viability, inhibition of enzymatic
antioxidant defence system, increase in biomarkers level
(ROS and MDA level) and DNA damage. These findings
suggest that ionic liquids may have negative impact on the
structure and function of aquatic ecosystem by accumulating in higher trophic levels through the food chain. Further,
ILs contamination may alter population demographic rates,
species interaction within communities and biogeochemical
processes (Bernot et al. 2005). Our results are in agreement
with the previous findings of Yu et al. (2009) who reported
the production of ROS coupled with the disturbance in
antioxidant system of Daphnia magna exposed to 1-alkyl-

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methyl-methylimidazolium bromide ionic liquid [C8min]Br

at 0.95 g mL1 for 48 h.
Apparently, the presence of AMPEP in the culture medium efficiently detoxified the ROS generated in the algae
when exposed to [C12mim]Br at LC50 concentration for 4 d.
The application of AMPEP alone and/or in combination
with plant growth regulators have recently been demonstrated the induction of callus in the red alga, Kappaphycus
(Hurtado et al. 2009). Furthermore, the present study for
the first time reports the antioxidant potential of AMPEP
having higher concentration of phenolic compounds, excellent reducing power and considerably higher antioxidant
and free radical scavenging activities which were comparable to known commercial antioxidants such as BHT and
BHA. The present study demonstrated significant alteration
in various biochemical processes (decrease in ROS, MDA
level and LOX activity) in U. lactuca following the exposure to [C12mim]Br in the presence of AMPEP. It has been
evident from this study that the extract of Ascophyllum (at
150 g mL1) has acted as a promising natural source of
antioxidants eventually prevented the Ulva from the oxidative assault following the IL exposure, at least for LC50
value of [C12mim]Br. A significant decrease in LOX activity
with the inhibition of LOX-2 and LOX-3 isomorphs in
cultures supplemented with AMPEP and [C12mim]Br LC50
resulted in less ROS generation via enzymatic pathways
involving LOX. This enzyme is known to generate singlet
oxygen and superoxide anions while oxidising the n-3 and
n-6 PUFAs of membrane lipids (Kumar et al. 2012). Therefore, the presence of AMPEP would have prevented the
oxidation of PUFAs (n-3 and n-6) in plants exposed to
[C12mim]Br LC50 +AMPEP and thus maintained the rigidity
of cell membrane. Recently, the application of Ascophyllum
extract has been shown to inhibit the activities of LOX and
peroxidases in greenhouse cucumbers subjected to fungal
pathogens (Jayaraj et al. 2011). The volatile aldehyde-like
MDA is a suitable marker to measure the membrane lipid
peroxidations and thus the oxidative stress in a cell. The
algal thalli exposed to IL for 4 days at LC50 and 2LC50
exhibited a considerable rise in MDA level over control and
was positively correlated with increased activity of LOX. A
possible reason for increase MDA level following IL exposure could be attributed to the incorporation of alkyl chain of
[C12, 14 min]Br in to the polar head group of phospholipid
bilayer, which in turn led to the disruption of membrane
bound proteins and lipids (Couling et al. 2006). The presence of longer alkyl chain lengths in ILs have been shown to
allied with more lipophilic nature (Ranke et al. 2007), which
may facilitate their quick entry in to the cell while exerting
the severe oxidative stress, coupled with the generation of
ROS. The reduced accumulation of MDA in algal thalli
cultured in presence of AMPEP suggests its role in minimising the lipid peroxidation caused by ILs. Recently,

J Appl Phycol (2013) 25:369378

Queguineur et al. (2012) also demonstrated the protective

role of Ascophyllum extract with reduced accumulation of
ROS and MDA level in HepG2 cells exposed to tert-butyl
hydroperoxide.
The ILs-induced oxidative stress caused a differential
regulation of antioxidant defence system in U. lactuca.
Similar conditions were also observed in D. magna following exposure to methylimidazolium ILs (Yu et al. 2009).
Macroalgae have evolved with antioxidant defence mechanisms to cope with the oxidative damage caused by ROS
and have been reported that higher antioxidant enzyme
activities are associated with higher stress tolerance in various algae (Kumar et al. 2010a, b, 2011b). In the present
study, the higher activities of SOD particularly Fe- and ZnSOD isoforms in LC50 +AMPEP coincided with the higher
activities of other antioxidant enzymes. The higher SOD
activity suggest a rapid breakdown of O2 radicals to keep
their levels below the toxic limits at the site of their generation and was subsequently followed by the action of APX,
GR and GSH-Px. The increased antioxidant enzyme activities with the supplementation of AMPEP indicate its potential to regulate the enzymatic antioxidant system which
eventually scavenge O2 and H2O2 and allowed algae to
survive under oxidative stress condition caused by IL exposure. Recently, Kumar et al. (2010a, 2011b) also demonstrated the enhanced SOD activity specifically attributed to
Mn-SOD in U. lactuca (Cd exposure) and Gracilaria corticata (desiccation stress) confirming its potential to scavenge
O2 more efficiently. The rescuing action of AMPEP in
plants treated with LC50 +AMPEP may be attributed to (1)
direct scavenging of ROS, (2) induction of higher Fe- and
Zn-SOD activities and (3) efficient chelation of Fe which
release from the Fe-sulphur centres of various enzymes by
O2 attack. These processes may collectively prevented the
induction of oxidative stress mediated by the cascading
events of Fe and O2 reactions via Fenton chemistry. On
the contrary, a significant decrease in SOD and APX activity
in plants exposed to LC50 concentrations suggested the
enhanced production of ROS to a level that besieged the
antioxidant system, leading to cell and tissue damage. In the
present study, APX activity increased to 1.6-fold together
with the induction of APX isoforms, APX-3 and APX-4
during LC50 +AMPEP exposure suggests their greater ability for H2O2 detoxification. On the contrary, the inhibition
of APX-2 reveals its sensitivity to H2O2. The higher activity
of APX compared to other enzymes (i.e. GPX and GR)
involved in H2O2 detoxification could be attributed to its
high availability throughout the cell and higher substrate
affinity in the presence of AsA as a reductant (Willekens
et al. 1995). In the present study, a sizeable increase in GR
and GPX activity following LC50 +AMPEP treatment invariably indicated the potential of AMPEP in enhancing the
antioxidant capacity of U. lactuca under IL-induced

Author's personal copy

J Appl Phycol (2013) 25:369378

oxidative stress. Recent studies also investigated the

AMPEP potentials in elicit activation of natural defences
against pathogen attack in Kappaphycua alvarezii which
eventually ameliorated the negative impact of oxidative
bursts caused by elevated production of H2O2 (Loureiro et
al. 2011). The potentials of Ascophyllum extract in enhancing the endogenous antioxidant activity and phenolic content of Spinacia oleracea subjected to oxidative and thermal
stress have also been examined (Fan et al. 2011).
Comet assay, otherwise called as single cell gel electrophoresis, is a rapid and sensitive microscopic method for the
detection of DNA damage in an individual cell and has increasingly been employed in molecular epidemiological studies. In this study, for the first time, we have evaluated the
potential of AMPEP in protecting the cells of U. lactuca from
DNA damage mediated through ROS triggered by IL exposure. The comets observed in the cells treated with [C12mim]Br
(LC50) showed greater DNA damage with significant amounts
of tail DNA (most of comets belong to category 3) as compared to the control. However, the relative occurrence of
comets belonging to categories 0, 1 and 2 in the thalli treated
with LC50 +AMPEP was comparable to control and thereby
indicated that AMPEP can protect the cell from IL-induced
DNA damage. There are few reports highlighting the functions
of AMPEP as an elicitor of plant growth regulation (Hurtado et
al. 2009), suppression of disease induced by fungal pathogen
in cucumber (Jayaraj et al. 2011) and elicitation of a cytokininlike activity in Arabidopsis thaliana (Khan et al. 2010). The
present study describes a newer finding wherein AMPEP
found to protect the DNA from damage arising from ROS
accumulation as a function of ILs exposure. Further, AMPEP
has also been shown to contain some antioxidants, including
phenolics with strong Fe chelating activity and direct ROS
scavenging functions to attenuate the oxidative stress induced
by high temperature (Fan et al. 2011). The strong inhibition
activities on formation of free radicals and DNA damage in
thalli treated with LC50 +AMPEP could possible due to induction of enzymatic antioxidants as well as presence of high
amounts of phlorotannins, a major phenolic substance in
AMPEP itself (Holdt and Kraan 2010).
Acknowledgments The first author (MK) gratefully acknowledges
the Council of Scientific and Industrial Research, New Delhi, India for
Senior Research Fellowship. We are especially grateful to Dr Alan T.
Critchley (Acadian Seaplants Limited, Canada) for generously providing the Acadian marine plant extract powder and offering valuable
suggestions on the first draft of manuscript.

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