Indian Journal of Experimental Biology

Vol. 55, July 2017, pp. 469-478

Phytoextracts protect Saccharomyces cerevisiae from oxidative stress with

simultaneous enhancement in bioremediation efficacy
Shivtej P Biradar1, Asif S Tamboli1, Tejas S Patil3 Rahul V Khandare2, Sanjay P Govindwar1 & Pankaj K Pawar1*
1
Department of Biochemistry; 2Department of Biotechnology; 3Department of Zoology, Shivaji University, Kolhapur, India

Received 01 February 2017; revised 22 March 2017

Bioremediation efficacies are highly affected by abiotic stresses imparted by a verity of pollutants due to generation of
reactive oxygen species (ROS). These stressed cells can be treated using natural or synthetic antioxidants. Such an approach
could prove beneficial to bioremediation agents as the exogenously added antioxidant compounds would scavenge the
generated free radicals. This would definitely lead to increased longevity of the involved organism and carry out superior
treatments. In present study, Malachite Green (MG) was found to exert oxidative stress on Saccharomyces cerevisiae
through generation ROS. A 2 h exposure of MG though achieved 99% decolourization, the cells revealed a significant
decrease (97.8%) in colony forming units (CFU) upon further subculture. Natural antioxidants from Centella asiatica,
Phyllanthus emblica, Asperagus racemosus and Tinospora cordifolia extracts, however, restored the CFU with a loss of
only 16-33%. The MG stressed cells indicated an increase in ROS by 6.7 fold which was reduced to near normal due to
augmentation with plant extracts. MG damaged the nuclear material up to 90% and inclusion of phytoextracts protected the
cells revealing only 0-7% nuclear damage. Induction in apoptosis (92%) and necrosis (23%) in MG exposed cells was noted,
while plant extracts augmentation reduced apoptosis to 15-49% and necrosis to 10-16%. Activities of antioxidant enzymes
such as superoxide dismutase, catalase and glutathione peroxidase were significantly decreased in phyto-augmented cells
when compared to MG stressed cells. Dye degrading enzymes, namely lignin peroxidase, laccase, NADH-DCIP reductase
and MG reductase were found to show induction in activities during MG utilization. Since antioxidants from plant extracts
could protect the cells form oxidative stress, they were used to treat MG for 20 continuous decolourization cycles.
Augmentation of C. asiatica, P. emblica, A. racemosus and T. cordifolia extracts at 20th decolourization cycle revealed 75,
79, 74 and 93% superior decolorization efficacies as compared to unaugmented cells. These natural antioxidants to protect
bioremediation agents form oxidative stress, thus concluded to show enhanced treatment.

Keywords: Abiotic stress, Asperagus racemosus, Ayurvedic, Centella asiatica, Decolourization, Herbal extracts, Laccase,
Lignin peroxidase, Malachite Green, Phyllanthus emblica, Pollution, ROS scavenging, Saccharomyces cerevisiae,
Tinospora cordifolia, Vayasthapana rasayana

Bioremediation agents like bacteria, fungi and plants However, efficient system of microbes capable of
are under continuous abiotic stress for lack of nutrients, handling pollutant load containing heavy metals could
limited supply of oxygen and fluctuating pollutant not be developed. The reason underlying the
loads. This stress is the key reason behind retarded inefficiency of in situ bioremediation of heavy metals
growth, inhibited metabolic activity and ultimately the is found to be the oxidative stress generated by the
loss of viability of candidate microorganism1. Loss of target contaminant4. Similar effects of Cr (VI) on yeast
viability of remediating microbe directly affects the Candida intermedia was reported by Jamnik and
treatment efficacies. A wide array of microorganisms Raspor5.
have been shown to be stressed by the targeted Synthetic textile dye and a common antimicrobial;
contaminants, such as heavy metals, antimicrobial Malachite Green (MG) was found to have toxic effects
compounds, polychlorinated biphenyls, hydrocarbons, on cultured mammalian cells through the formation of
synthetic textile dyes, etc.2. Reduction of Cr (VI) to Cr reactive oxygen species (ROS)6. It also acts as a tumor
(III) by various microorganisms like Escherichia coli, promoter leading to malignancies in mammals7.
Shewanella oneidensis and a number of species of Toxicological implication makes it inevitable to
Pseudomonas and Bacillus spp. are reported3,4. remediate this chemical from the environment.
—————— Although, MG is a known antimicrobial, few
*Correspondence:
Phone: +91 231 2609152; Fax: +91 231 2691533 microorganisms including Saccharomyces cerevisiae
E-mail: [email protected] were proposed to degrade it8,9. Evaluation of toxicity as
470 INDIAN J EXP BIOL, JULY 2017

an effect of abiotic stress exerted by such chemicals, 6-sulphonic acid), superoxide dismutase assay kit
however, has been left unattended. As far as (Cat. No. 19160), catalase assay kit (Cat. No.
bioremediation using microbial cells is concerned MG CAT100), glutathione peroxidase assay kit (Cat. No.
has been reported to cause DNA damage through CGP1), 2'7'-dichlorofluorescin (H2DCF) (Cat. No.
formation of free radicals10. The antimicrobial agents D6883) and 4',6-diamidino-2-phenylindole (DAPI)
that cause formation of ROS are capable of inducing (Cat. No. D9542) were procured from Sigma Aldrich,
necrosis or programmed cell death in eukaryotic USA. Rest of the chemicals and solvents used were of
systems like yeast11. It should be borne in mind that analytical grade.
ROS production might have hampered the remediation
Organism and culture conditions
efficacy of the microbial system over a period of
Baker’s yeast (S. cerevisiae) used in present
time. Therefore, approach involving the use of
study was obtained from Korean Collection for
natural/synthetic antioxidants or free radical
Type Cultures (KCTC-7296). Culture was routinely
scavenging compounds may help in protecting the
maintained on 5% (w/v) dextrose, 1% peptone,
microbial cells used for bioremediation of toxic
1% yeast extract and 3% agar. Cells used for
compounds like MG.
present experimentation were always grown on
Ameliorative effects of apple polyphenols on ROS
slightly modified medium containing 2% dextrose,
mediated aging in yeast was reported12. A synthetic
0.1% peptone, 0.1% yeast extract (w/v). The cells
antioxidant diphenylmethyl sellenocyanate was found
required for studies were harvested from the
effective in protecting mice from MG induced
medium inoculated with 1.25× 107 Cells/mL
oxidative injuries through antioxidation and inhibition
containing inoculums at room temperature
of DNA damage13. It can, thus, be hypothesized that
(33 ± 2ºC) for 24 h by centrifugation at 7000 rpm
the stressed cells might be treated using natural or
for 7 min at 4ºC
synthetic antioxidants. This could prove beneficial to
bioremediation agents as the exogenously added Plant material and preparation of plant extracts
antioxidant compounds would scavenge the free Entire plants of Centella asiatica, Phyllanthus
radicals, and thereby lead to their increased longevity. emblica, Asperagus racemosus and Tinospora
In the present study, plants used in standard Ayurvedic cordifolia were procured from M/s Green Pharmacy,
formulation known as ‘Vayasthapana Rasayana’ Pune, India. The plant material was finely powdered
(an antiaging formulation) were selected as a source of using domestic grinder and used further for
antioxidants. Medicinal plants namely, Centella extraction. 10 g of each plant powder was added in
asiatica, Phyllanthus emblica, Asperagus racemosus 100 mL ethanol separately and kept for shaking at 150
and Tinospora cordifolia are reported to possess a rpm, 30ºC for 24 h17. The extracts were then filtered
strong in vitro free radical scavenging properties14-16. through Whatman filter paper No. 1 and centrifuged
Ethanolic extracts of these plants were used as source at 10000 rpm, 10ºC for 10 min to obtain a clear
of antioxidants supplement to the MG degrading S. supernatant. After determining the extraction yield
cerevisiae. concentration of the extract was adjusted in such a
Bioremediation processes are clearly affected if way to obtain 10 mg mL-1 working stock
remediation agents are stressed. Attempts towards concentration and was stored in amber colored bottles
reducing the abiotic stress faced by the bioremediation at 4ºC till further studies14.
agent would be highly beneficial to involved
Initialabsorbance  Observed absorbance
microorganisms leading to more efficacious treatments. Decolorization %=  100
Initialabsorbance
In this study, we investigated whether Malachite green
… (1)
(MG), a known dyestuff, exert any oxidative stress on
Saccharomyces cerevisiae, a bioremediation agent, and Cell viability studies
also studied if the phytoextracts have any ameliorative Cell of S. cerevisiae were investigated for their
effect on the stressed cells. viability as a function of exposure to MG. In this,
1 mL of culture media containing cells from the
Materials and Methods decolorization experiment were serially diluted (10-9)
Dye and chemicals in 0.85% saline solution and spread on Petri plates
MG was obtained from Hi media Laboratory, containing slightly modified culture medium
India. ABTS (2,2-azino-bis-3-ethyl benzothiazoline- mentioned above. These cultures were incubated at
 BIRADAR et al.: PHYTOEXTRACTS PROTECT BIOREMEDIATION AGENTS AGAINST OXIDATIVE STRESS 471

room temperature (33 ± 2ºC) for 48 h and colony dehydration of cells19. After dehydration, cells were
forming units were counted by using colony counter. coated with gold on sputter stub surface. The stubs
were examined using JSM-6360 scanning electron
Effect of exposure of MG and augmentation of plant extracts microscope (SEM) at 30 kV (JEOL Ltd, Japan) and
on the growth of S. cerevisiae images were taken.
Growth curve of S. cerevisiae exposed to MG and Fluorescent microscopic analysis was carried out
plant extracts augmented dye along with control was on Zeiss Axio-scope A 1 trinocular phase contrast
studied in order to understand growth pattern of microscope with fluorescent attachment. The analysis
S. cerevisiae. 1 mL inoculum at the absorbance of of intracellular oxidation and nuclear chromatin
1.0 at 620 nm was inoculated in growth medium damage was studied using fluorescent H2DCF and
mentioned earlier which was fortified with MG and DAPI which were specific for intracellular oxidation
MG in combination with 1 mL of C. asiatica, and nuclear chromatin. The cells were collected
P. emblica, A. racemosus and T. cordifolia extracts during and after decolourization at 3 time intervals
independently at 1 mg mL-1. Growth of cells was (0, 1 and 2 h, respectively), washed thrice with the
monitored by measuring absorbance at 620 nm at an sterile distilled water and re-suspended in 0.1 mL of
interval of 1 h for 72 h. 50 mM phosphate buffer (pH 7.4). To this, 1 mM
ethanolic stock solution of H2DCF was added to
Determination of intracellular ROS levels, scanning electron
microscope and fluorescent microscopic analysis achieve 10 µM final concentration of the dye and
Intracellular ROS levels were determined by using incubated at 30ºC for 2 h. Cells were again
2'7'-dichlorofluorescin (H2DCF) on Fluorescence centrifuged after incubation, washed and re-
Spectrophotometer18. The dye H2DCF has chemical suspended in same buffer. The cells were observed
property to penetrate plasma membrane and react with using FITC-Spectrum Green (Chroma 3101)
oxidants (ROS) present inside the cell. Non- comprising 25 mm diameter filters, excitation filter
fluorescent H2DCF when oxidized by reactive oxygen (D480/30), beam splitter (440 DCLP) and emission
species (oxidants) shows fluorescence, which is filter (D535/40), and images were taken. For
determined spectrofluorometrically. For this analysis, observing organization of nuclear chromatin, the cells
cells were harvested during and after decolourization were harvested during and after decolorization at
at 3 time intervals (0, 1 and 2 h, respectively) by 3 time intervals (0, 1 and 2 h, respectively), washed
centrifugation in 2 mL aliquots. Harvested cells were with sterile distilled water, re-suspended in 10 µL of
washed with 2 mL of 50 mM phosphate buffer (pH 7.4) sterile distilled water and fixed by addition of
thrice and re-suspended in same buffer. The cell formaldehyde (1%). The fixed cells were stained with
suspension was pre incubated at 28ºC for 10 min. To 1 µL of 20 mg ml-1 of 4’,6-diamidino-2-phenylindole
this suspension, 1 mM ethanolic stock solution of (DAPI)19 and observed under DAPI filter (Chroma
H2DCF was added to achieve 10 µM final 3100) comprising 25 mm diameter filters, excitation
concentration of the dye and incubated at 28ºC for filter (D350/50), beam splitter (400 DCLP) and
20 min. After incubation, cell suspension was emission filter (D460/50).
centrifuged and clarified. The supernatant was Enzymatic analyses
measured for fluorescence (excitation at 488 nm S. cerevisiae cells (2 g) of control, exposed to MG
wavelength and emission at 520 nm wavelength for and cells exposed to MG augmented with C. asiatica,
each sample) using Fluorescence Spectrophotometer P. emblica, A. racemosus and T. cordifolia extracts
8300 (Agilent Technologies, USA). were homogenized in chilled 1 mL of 50 mM
The effect of MG alone and in combination with phosphate buffer (pH 7.4) using a homogenizer and
each plant extract on cell of S. cerevisiae was then sonicated with output at 60 amp, giving three
observed using scanning electron microscope (SEM). strokes each of 30 s, at 2 min intervals, at 4ºC. The
All the treated cells of S. cerevisiae were fixed using homogenate, thus obtained, was then centrifuged. The
50 mM phosphate buffer (pH 7.4) containing 2.5% supernatant was used as source of laccase, lignin
glutaraldehyde for 12 h at 4ºC. After fixation, cells peroxidase, NADH-DCIP reductase, MG reductase,
were washed with 50 mM phosphate buffer (pH 7.4) glutathione peroxidase, superoxide dismutase and
thrice and further treated with increasing catalase. Activities of lignin peroxidase, laccase,
concentration (10 to 100%) of ethanol for successive NADH-DCIP reductase and MG reductase were
472 INDIAN J EXP BIOL, JULY 2017

assayed spectrophotometrically in cell free extract of decolourization by measuring the absorbance at

test as well as in the control supernatant. Lignin 620 nm. This process was considered as one cycle of
peroxidase activity was calculated by monitoring the MG decolourization. Further, fresh 100 mL growth
formation of propanaldehyde at 300 nm in a reaction medium containing MG and respective plant extract
mixture of 2.5 mL containing 100 mM n-propanol, were added in previously separated cell mass and
250 mM tartaric acid, 10 mM H2O220. Laccase activity incubated as described earlier. Twenty decolorization
was determined in a reaction mixture of 2 mL cycles were performed and the decolorization
containing 10% ABTS in 0.1 M acetate buffer percentage for all the treatments was noted.
(pH 4.9), and increase in optical density was
Statistical analysis
measured at 420 nm21. NADH-DCIP reductase
All the data obtained was expressed as mean ±SD.
activity was measured in cell-free extract. DCIP
Statistical analysis was performed using one-way
reduction was monitored at 620 nm, and calculated
analysis of variance (ANOVA). P-values at α less
using an extinction coefficient 19 mM per
than 0.05 were considered to be statistically
centimeter22. The reaction mixture (5.0 mL) contained
significant..
50 mM substrate (DCIP) in 50 mM potassium
phosphate buffer (pH 7.4) and 0.1 mL enzyme. From Results and Discussion
this, 2.0 mL reaction mixture was assayed at 620 nm MG has earlier been reported to be remediated by
by addition of freshly prepared 50 mM NADH. S. cerevisiae. It should be taken into consideration
Activity of MG reductase was measured as per the that the bioremediation efficacy depends on longevity
earlier reported method of Jadhav and Govindwar9. and capacity of bioremediating organism to counter
All enzymes assayed for test sample and biotic stress. During MG remediation by S. cerevisiae, the
control sample were carried out in triplicates. yeast must be under abiotic stress. Therefore, efforts
During decolorization of MG; the cells of to prepare the remediating organisms to withstand
S. cerevisiae undergo oxidative stress and hence abiotic stress can ultimately lead to effective pollutant
expression levels of glutathione peroxidase, treatment. In this communication, toxicity of MG
superoxide dismutase and catalase were measured as a towards S. cerevisiae was evaluated by studying
function of exposure to MG alone and cells exposed various morphological, cellular and stress related
to MG augmented with C. asiatica, P. emblica, alterations. The toxic effects were hypothesized to be
A. racemosus and T. cordifolia extracts. The enzyme reduced by augmentation with the ethanolic extracts
activity was measured strictly as per the of C. asiatica, P. emblica, A. racemosus and
manufacturer’s protocol. All enzyme assays were T. cordifolia.
performed at 27ºC and were run in triplicates. The
protein content of all the samples was determined as Effect of MG exposure on S. cerevisiae cell viability with and
per Lowry’s method23. without augmentation of herbal extracts
The initial augmentation of plant extracts was not
Effect of herbal augmentation on repetitive decolorization by found to show any significant effect on dye removal
S. cerevisiae and achieved decolourization in a range of 98.9-
Role of augmentation with C. asiatica, P. emblica, 99.8%. However, the cell viability assay after MG
A. racemosus and T. cordifolia extracts in increasing decolorization revealed that the S. cerevisiae cells
the decolourization efficacy of S. cerevisiae was were highly affected by the presence of dye. Exposure
investigated. For this, 2 g cells of S. cerevisiae were to MG was observed to reduce the CFU of
collected by centrifugation at 7000 rpm for 7 min S. cerevisiae by 97.8%. Whereas, independent
under cold conditions and were transferred to a augmentation with natural antioxidants in the form
100 mL broth containing MG (100 mg L-1) alone and of C. asiatica, P. emblica, A. racemosus and
another three sets containing MG augmented with T. cordifolia extracts decreased the CFU by 32.9,
plant extracts of C. asiatica, P. emblica, A. racemosus 25.3, 23.1 and 16.5%, respectively, when compared to
and T. cordifolia (1 mg mL-1), respectively and kept at control cells (Fig. 1). These results clearly indicate
room temperature (33±2ºC) with shaking at 150 rpm. that the cell viability of bioremediation agent was
The decolourization of the MG was measured after retained to significant levels upon augmentation of the
every 2 h and cells were separated by centrifugation. herbal extracts. T. cordifolia was found to be superior
The removed media was used for the study of among the tested plants as far as cell viability was
 BIRADAR et al.: PHYTOEXTRACTS PROTECT BIOREMEDIATION AGENTS AGAINST OXIDATIVE STRESS 473

concerned. Textile dyes like Auramine, Methylene Effect of MG exposure and herbal augmentation on
Blue, Gentian Violet, Phenol Red and Dichloran have S. cerevisiae cells in terms of ROS accumulation, nuclear
organization, apoptosis, necrosis and cellular morphology
been shown to decrease colony diameter and CFU in
The oxidative stress gives more ROS accumulation
Deuteromycetes and Zygomycetes24.
which can be detected with ROS specific fluorescent
dye19. Accumulation of ROS in S. cerevisiae due to
MG stress with and without augmentation of
C. asiatica, P. emblica, A. racemosus and
T. cordifolia extract was visualized by using H2DCF
dye. A 95% of cells exposed to MG showed
accumulation of ROS when compared to control cells
(2%) cells. On the other hand, augmentation of
C. asiatica, P. emblica, A. racemosus and
T. cordifolia extracts unveiled only 8, 15, 12 and 13%
cells with accumulation of ROS, respectively
(Fig. 2A). Reduced ROS accumulation after herbal
augmentation can be attributed to natural antioxidant
present in these extracts. Nuclear damage is
considered as the key reason behind reduced/lost cell
viability. Therefore, MG exposed cells and the herbal
Fig. 1 — Decolorization of MG by S. cerevisiae and effect of extract augmented cells were further tested for nuclear
phytoextract augmentation on cell viability. [Each observation
expressed as means ± Standard Deviation for six independent
damage. In control set, only 20% of the cells were
experiments. (n=6)] observed to show nuclear damage. On the other hand

Fig. 2 — Effect of exposure of MG alone and in combination with plant extracts on (A) accumulation of ROS; (B) nuclear damage;
C(a) apoptosis; C(b) necrosis; and (D) cell morphology.
474 INDIAN J EXP BIOL, JULY 2017

80% cells were found with nuclear damage in the MG superior growth even upon MG exposure. In presence
exposed S. cerevisiae. Presence of C. asiatica, of extract, the lag phase was found to lasts for 5-8 h
P. emblica, A. racemosus and T. cordifolia however followed by the log phase of growth which lasted for
could significantly arrest the nuclear damage and 10-35 h. Among the tested plants, T. cordifolia extract
showed only 30, 28, 32 and 35% cells with damage, augmented culture could attend the stationary phase
respectively (Fig. 2B). Accumulation of ROS of growth at 28 h and lasted up to 72 h even in
followed by DNA damage was shown to be the real presence of MG. Similar protective effect was
cause for apoptosis in S. cerevisiae25. Reduced observed with augmentation of P. emblica and
nuclear damage in the augmented cells can, thus, be A. racemosus extracts. The C. asiatica extract,
attributed to the strong radical scavenging potential of however, assisted a long log phase of around 40 h
these plants which is reported earlier by Mukherjee et followed by the stationary phase (Fig. 3). The
al.14. MG exposed cells showed apoptosis in 92% and restoration of normal growth curve patterns is an
necrosis in 23% cells, while C. asiatica, P. emblica, indication of protected cells after inclusion of plant
A. racemosus and T. cordifolia extracts augmentation extracts rich in natural oxidants, in presence of the
reduced apoptosis to 28, 25, 15 and 49% [Fig. 2 C(a)] ROS generating agent.
and necrosis to 15, 13, 10 and 16% [Fig. 2C(b)]. Intracellular ROS level, antioxidant and dye degradation
Imaging by SEM can be effectively used to study enzyme activity analyses during MG removal
changes in cellular morphology during stress Measuring intracellular reactive oxygen species
conditions. MG exposed cells and the herbal extract (ROS) was imperative as accumulation of ROS
augmented cells showed morphological changes as triggers apoptotic cell death in S. cerevisiae25. The
compare to control cells. MG exposed cells shown MG exposed and herbal extract augmented cell
damaged cell surface but in herbal extract augmented revealed dissimilar accumulation when observed
cells shown relatively less damaged cell surface as under fluorescent microscope. In the present study,
compare to MG exposed cells. It proves that the herbal augmented cells stained with H2DCF showed
herbal extracts protect the cell surface of S. cerevisiae significantly reduced fluorescence meaning that the
from damage (Fig. 2D). ROS were notably scavenged. Presence of MG gave a
fluorescence of 17.529 au which was 6.6 fold more
Effect of natural antioxidant from herbal extracts on growth when compared to control (2.749 au). Augmentation
of S. cerevisiae after MG exposure of C. asiatica, P. emblica, A. racemosus and
Generation of ROS in wide range of organisms T. cordifolia extracts, however, showed significant
including the yeast like S. cerevisiae is known as the reduction in fluorescence i.e. 3.81, 4.73, 3.98 and 3.63
key modulator of apoptosis26. In the light of the fact au, respectively. These results revealed that the
that MG causes ROS production and exerts stress on natural antioxidants scavenged the ROS generated
the cells, it can be concluded that the toxicity of MG after MG exposure. The fluorescence of augmented
is by virtue of its ability to generate oxidative stress27. samples as clearly seen are comparable with the
Thus, it must be adversely affecting the growth of control cells [Fig. 4A(a)]. Exposure of MG to
S. cerevisiae and therefore, the natural antioxidant C. albicans was found to show a high rate of necrosis
should alter these effects. Induction of apoptosis due in flow cytometry analysis28. The accumulation of
to MG stress may lead to drastically hampered
quorum resulting in loss of viable cells. MG at a
concentration of 100 mg L-1 was found to extend the
lag phase up to 40 h prior to initiation of log phase
which lasted up to 50 h of culture. On contrary,
normal lag phase lasts only for 4-6 h and log phase
lasted around 20 h. The delay in initiation of log
phase in presence of MG must be because of its
toxicity and antimicrobial action. Most of the actively
dividing cells must have been destroyed in the lag Fig. 3 — Changes in growth pattern of S. cerevisiae as a function
of exposure to MG alone and in combination with C. asiatica,
phase itself.
P. emblica, A. racemosus and T. cordifolia. [Each observation
Augmentation of herbal extracts of C. asiatica, expressed as means ± Standard Deviation for six independent
P. emblica, A. racemosus and T. cordifolia gave much experiments. (n=6)]
 BIRADAR et al.: PHYTOEXTRACTS PROTECT BIOREMEDIATION AGENTS AGAINST OXIDATIVE STRESS 475

Fig. 4 — Effect of exposure of MG and herbal intervention on A(a) intracellular ROS generation, and induction in the activities of A(b)
glutathion peroxidase, A(c) superoxide dismutase and A(d) catalase; B(a) Laccase, B(b) Lignin peroxidase, B(c) NADP-DCIP Reductase
and B(d) MG Reductase in S. cerevisiae. [Each observation expressed as means ± Standard Deviation for six independent experiments.
(n=6). Values were significantly different from that of Malachite green group at P <0·05]
ROS is believed to be the key factor for this damage. reductions in activities in augmented cells.
Accumulation of ROS resulted in oxidation of the S. cerevisiae cells due to MG stress showed
vital biomolecules like carbohydrates, proteins, lipids inductions in CAT, SOD and GPx activities by 100,
and DNA ultimately resulting in abnormal cellular 166 and 248% after a 2 h exposure which was clearly
membrane, reproductive functions and lethality29. indicating generation of ROS. On the contrary,
Depletion of ROS leading to hypoxia was proposed to activities of CAT, SOD and GPx after C. asiatica,
prevent apoptosis in S. cerevisiae25. P. emblica, A. racemosus and T. cordifolia extract
The activities of CAT, SOD and GPx enzymes augmentation showed inductions in (i) in CAT by 42,
tested after exposure to MG and independent 50, 33 and 1.69% [Fig. 4A(b)]; (ii) in SOD by 17, 12,
augmentations of plant extracts revealed noteworthy 16 and 0.4% [Fig. 4A(c)]; and (iii) in GPx by 33, 71,
476 INDIAN J EXP BIOL, JULY 2017

57 and 53% [Fig. 4A(d)], respectively when degradation capacity of the cells after repeated dye
compared to the respective controls. The presence of exposure. Accumulation of products after biocatalysis
natural antioxidants of plant origin must have has been shown to be toxic to the cells inhibiting there
scavenged the ROS minimizing their exposure to cell growth and in some cases death32. The extract
organelles which is evident from reduced induction of unaugmented set showed a 59% reduction in
antioxidant enzyme activities. These plants have performance after 20th cycle when compared to the
already been reported to possess in vitro ROS 1st cycle of decolourization. The herbal augmentation
scavenging activity and our findings are in agreement was found to reveal much superior decolourization
with the previous report by Mukherjee et al.14. performances. Evaluation of all setups at 20th cycle of
Well known textile dye degrading enzymes like decolourization revealed that sets augmented with
laccase, LiP, NADH-DCIP reductase and MG C. asiatica, P. emblica, A. racemosus and
reductase checked before and after 2 h of dye T. cordifolia showed 75, 79, 74 and 93% superior
exposure revealed their involvement to varying decolourization efficacies, respectively when
extents when compared to their controls (0 h). The compared to unaugmented set (Fig. 5).
yeast cells exposed to MG alone and supplied Stress tolerance ability imparted by the natural
independently with extracts of C. asiatica, P. emblica, antioxidants in herbal extracts to MG remediating
A. racemosus and T. cordifolia revealed an induction cells thus resulted in enhanced performance.
in activities of laccase, LiP, NADH-DCIP reductase T. cordifolia extract augmentation to cells was
and MG reductase by 250, 155, 164, 109, and 117% found to be the most effective among the tested
[Fig. 4B(a)]; 180, 85, 48, 53 and 84% [Fig. 4B(b)]; plants. Chemically synthesized diphenylmethyl
951, 505, 771, 700 and 400% [Fig. 4B(c)]; and 900, selenocyanate was found to show protection in mice
1900, 1400, 2150 and 1383% [Fig. 4B(d)], against MG stress13. Polyphenols from apple has
respectively. MG reductase among the tested enzymes showed an increase in life span of S. cerevisiae which
was observed to be modulated most significantly due was drastically reduced in presence of H2O212. The
to its specificity towards this particular dye after protection of bioremediating cells from abiotic stress
herbal augmentation. Other enzymes were also becomes inevitable if efficient bioreactors have to be
induced but to lesser extents when compared to cells designed and constructed. Approaches like herbal
exposed purely to MG. Induction in the activity of extract supplementation ultimately could be helpful in
MG reductase in S. cerevisiae has also been reported increasing efficiency of bioreactor system utilized for
by Jadhav and Govindwar9. Aquatic hyphomycetes remediation of toxic chemicals and further can be
like Varicosporium elodeae and Heliscus submerses
after an abiotic stress by Cu and Zn reviled
enhancement in SOD and CAT activities30.
Aspergillus niger due to presence to H2O2 in the
media were also found to show induction in the
actives of CAT, SOD and GPx31.

Effect of augmentation with natural antioxidants from plants

on restoration of bioremediation efficacy of S. cerevisiae over
repeated exposure of MG
It has earlier been reported that the yeast cells
could remove toxic compound like MG9. In the
present work, the effect of repeated exposure of MG
on same biomass was evaluated and the dye removal
efficacy of S. cerevisiae was observed to be
significantly reduced. This may be due to the toxic
effect of MG which was evident from earlier data that
indicated reduced CFU, increased intracellular ROS, Fig. 5 — Effect of augmentation with natural antioxidants from
greater nuclear damage and enhanced induction of plants on restoration of bioremediation efficacy of S. cerevisiae
over repeated exposure of MG for 20 bioremediation cycles.
enzymes involved in antioxidant defence. These [Each observation expressed as means ± Standard Deviation for
factors must have contributed in the reduced dye six independent experiments. (n=6)]
 BIRADAR et al.: PHYTOEXTRACTS PROTECT BIOREMEDIATION AGENTS AGAINST OXIDATIVE STRESS 477

advocated to develop sustainable, ecofriendly and cost embryo (SHE) cells in culture by Malachite Green: an agent of
effective technology for bioremediation. environmental importance. Indian J Exp Biol, 37 (1999) 904.
8 Safarik I, Ptackova L & Safarikova M, Adsorption of dyes on
magnetically labeled baker’s yeast cells. Eur Cells Mater, 3
Conclusion (2002) 52.
Oxidative stress generated by MG was observed to 9 Jadhav JP & Govindwar SP, Biotransformation of Malachite
be a retarding factor for the overall growth and Green by Saccharomyces cerevisiae MTCC 463. Yeast, 23
(2006) 315.
proliferation of S. cerevisiae while performing 10 Sudova E, Machova J, Svobodova Z & Vesely T, Negative
bioremediation. This ultimately resulted in decreased effects of Malachite Green and possibilities of its
treatment performance of the cell mass. The replacement in the treatment of fish eggs and fish: A review.
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showed ameliorative effects on stressed cells by 11 Eisenberg T, Carmona-Gutierrez D, Büttner S, Tavernarakis
N & Madeo F, Necrosis in yeast. Apoptosis, 15 (2010) 257.
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restored the growth pattern, reduced the intracellular Med Cell Longev, (2012) 1.
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Bhattacharya S, Diphenylmethyl selenocyanate attenuates
damage and altered the expression of antioxidant and Malachite Green induced oxidative injury through
dye degradation enzymes. The protective effect antioxidation and inhibition of DNA damage in mice. Indian
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Acknowledgement 16 Anita, Dubey MK, Khati A & Chauhan RS,
Authors are thankful to University Grants Immunostimulatory and growth promoting potential of
Commission (UGC), New Delhi for funding under Tinospora cordifolia (Thunb.) Miers on fingerlings of Amur
Special Assistance Programme (SAP-DRS II, Grant carp. Indian J Exp Biol, 54 (2016) 659.
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