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Antioxidative and radioprotective activities of semiquinoneglucoside derivative (SQGD) isolated from Bacillus sp. INM-1
Raj Kumar • Deen Dayal Bansal • Dev Dutt Patel •
Saurabh Mishra • Yana Karamalakova • A. Zheleva •
Vessilina Gadjeva • Rakesh Kumar Sharma
Received: 23 July 2010 / Accepted: 15 November 2010 / Published online: 30 November 2010
� Springer Science+Business Media, LLC. 2010
Abstract A semiquinone glucoside derivative (SQGD)
was isolated from a radioresistant bacterium Bacillus sp.
INM-1 and its antioxidant and radioprotective activities
were evaluated using in vitro assays. Natural stable free
radical properties of SQGD in solid as well as in solution
form were estimated using Electron Paramagnetic Reso-
nance (EPR) spectrometry. Results of the study were dem-
onstrated high reducing power (1.267 ± 0.03356 Uabs) and
nitric oxide radicals scavenging activity (34.684 ± 2.132%)
of SQGD. Maximum lipid peroxidation inhibitory activity
of SQGD was found to be 74.09 ± 0.08% at 500 lg/ml
concentration. Similarly, significant (39.54%; P \ 0.05)
protection to the liposomal artificial membrane against
gamma radiation was observed by SQGD in terms of neu-
tralization of gamma radiation-induced TBARS radicals
in vitro. OH- radicals scavenging efficacy of SQGD was
estimated in terms of % inhibition in deoxy D-ribose deg-
radation by non-site-specific and site-specific assay. The
maximum (54.01 ± 1.01%) inhibition of deoxy D-ribose
degradation was observed in non-site-specific manner,
whereas, site-specific inhibition was observed to be
46.36 ± 0.5% at the same concentration (250 lg/ml) of
SQGD. EPR spectroscopic analysis of the SQGD indicated
*80% reduction of DPPH radicals at 6.4% concentration.
EPR spectral analysis of SQGD was revealed an appearance
of very strong EPR signal of 2.00485 (crystalline form) and
2.00520 (solution form) gy tensor value, which were an
established characteristic of o-semiquinone radicals.
Therefore, it can be concluded that SQGD is a natural stable
o-semiquinone-type radical, possessing strong antioxidant
activities and can effectively neutralize radiation induced
free radicals in biological system.
Keywords Antioxidant � Electron paramagnetic
resonance �Membrane protection � Free radical scavenging
Introduction
Natural products represent an invaluable gold mine of
pharmacophores for pharmaceutical sector [1]. A plethora
of natural products from microbial, plant, and animal
sources have been evaluated for their diverse pharmaco-
logic activities for use as promising radioprotective drugs
[2–5]. The growing knowledge about the role of free rad-
icals, i.e., reactive oxygen species (ROS) in various clinical
situations stimulate interest of various researchers to screen
novel free radical scavenging compounds from natural
origin. Exogenous factors such as toxicants, radiation, and
other stress conditions are known to produce extremely
dangerous ROS, which are capable enough to oxidize the
biomolecules, and induce oxidative stress in the biological
system [6].
Oxygen-derived free radicals species, particularly
superoxide (O2-), hydroxyl (OH-), hydroperoxyl (HOO-),
peroxyl (ROO-), alkoxyl (RO-) nitric oxide (NO-), per-
oxynitrite anion (ONOO-) radicals, etc., are known to
react with various vital biomolecules like functional pro-
teins, enzymes, membrane lipids, and DNA by extracting
an electron from them to become stable [7, 8].
R. Kumar (&) � D. D. Bansal � D. D. Patel � S. Mishra �R. K. Sharma
Institute of Nuclear Medicine and Allied Sciences,
Brig. S. K. Mazumdar Road, Delhi, India
e-mail: rajkumar790@yahoo.com
Y. Karamalakova � A. Zheleva � V. Gadjeva
Department of Chemistry and Biochemistry, Medical Faculty,
Trakia University, Stara Zagora, Bulgaria
123
Mol Cell Biochem (2011) 349:57–67
DOI 10.1007/s11010-010-0660-x
Bacterial, fungal, and algal metabolites are excellent
source of antioxidants and antimicrobials. Deinoxanthin,
an intermediate product of carotenoid biosynthesis path-
way has been reported to posses strong oxygen free radical
scavenging and DNA-protecting ability against lethal
irradiation that could be attributed to the radioresistance of
Deinococcus radiodurans [9]. Similarly, Hizikia fusiformis
has also been identified as a valuable natural source of
water and fat-soluble antioxidant components [10].
In the eukaryotic system, even lower doses of ionizing
radiation (1–4 Gy) may induce free radicals production and
initiate a chain reaction to oxidize the biological mem-
brane, vital proteins and induce single- and double-strand
break in the chromosomes lead to lethal mutation and cell
death. However, various prokaryotic organisms such as
Deinococcus radiodurans, Rubrobacter radiotolerans, and
a gigantic group of Bacillus sp. showed remarkable resis-
tance against supralethal doses of gamma radiation
[11–13]. This unprecedented radioresistance in microor-
ganisms suggested the existence of an efficient DNA and
other vital molecule’s repair machinery with them [14].
One of the several hypothesis of radioresistant mechanism
exist in the radioresistant microbes can be explained by
their ability to synthesize specific antiradiation/antioxidant
biomolecules before, during or post-irradiation period to
neutralize the free radicals generated by irradiation in the
biological system. A novel radioresistant bacterium was
isolated and characterized by 16S ribosomal RNA analysis,
fatty acid methyl ester analysis, and biochemical analysis
as Bacillus sp. INM-1. The type strain (Bacillus sp. INM-1,
MTCC No. 1026) was deposited at Microbial Type Culture
Collection, Institute of Microbial Technology, Chandigarh,
India as reference. SQGD was isolated from the fermen-
tation broth of Bacillus sp. INM-1 and its antioxidant and
radioprotective activities in terms of hydroxyl, nitric oxide,
total antioxidant capacity, liposomal membrane protection
against gamma radiation and DPPH radical scavenging
activities were evaluated in vitro models.
Material and method
Chemicals
Trypton Yeast Glucose (TYG) media, 2,20-azinobis-
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2-deoxy-
D-ribose, soya-lecithin, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) was purchased from HiMedia, India. Thiobarbituric
acid (TBA), sodium nitroprusside, naphthylethylenedi-
amine and sulfanilamide were purchased from Sigma
Chemicals (St. Louis, MO, USA). Chloroform, hexane,
hydrogen peroxide, and cholesterol were purchased
from Qualigen, India. Potassium ferricyanide, EDTA,
trichloroacetic acid, and ferric chloride were procured from
Ranbaxy, India.
Isolation and characterization of semiquinone glucoside
derivative (SQGD)
Fresh inoculums was transferred to 1.0 l sterile TYG culture
medium and incubated for 96 h at 32�C with continuous
shaking. On completion of this fermentation process, bac-
terial growth was terminated and broth was centrifuged at
5000 rpm for 10 min to separate the microbial mass.
Remaining supernatant were lyophilized. This lyophilized
powder was then fractionated by a series of non-polar to
polar organic solvents with the sequence of hexane–chlo-
roform–ethylacetate–methanol–water (1:6 ratio). The last
fraction separated with water was collected and lyophilized
again. This water fraction was further separated by column
chromatography using silica gel matrix (mess size 200–400
mess) and methanol–water (70:30 ratio) as mobile phase.
Fraction collected from the column was further checked on
TLC using silica gel as stationary phase and methanol–
water (70:30 ratio) as mobile phase. The single spot (Rf
value 0.73) detected on the silica gel plate was collected and
the purity of it was further checked by HPLC using reverse
phase C18 column and water–methanol (35: 65 ratio) as
mobile phase. Chemical characterization of the compound
separated by column chromatography was carried out by
UV–visible spectroscopy, Fluorescence spectroscopy, FTIR
spectroscopy, HNMR spectroscopy, LCMS spectroscopy,
and electron-paramagnetic resonance spectroscopy.
Measurement of reducing power
The reducing power of SQGD was determined according to
method described by Oyaizu [15] with slight modification.
Briefly, 50 ll SQGD in varied concentration was mixed
with 200 ll of 0.2 M phosphate buffer (pH 6.6) and 0.1%
potassium ferricyanide followed by incubation at 50�C in a
water bath for 20 min. The reaction was stopped by adding
250 ll of 10% trichloroacetic acid (TCA) and the mixture
was centrifuged at 30009g for 10 min at room tempera-
ture. Supernatant was then taken and mixed with 500 ll of
deionized water and 100 ll of 0.1% ferric chloride and
allowed to stand for 10 min. The absorbance was recorded
at 700 nm as reducing power using a spectrophotometer
(PowerWave, Biotek, USA). Reducing power of SQGD
was estimated and compared based on their respective
concentrations (lg/ml) corresponding to unit absorbance
expressed as mean ± SD, using the following formula:
Concentration ðlg/mlÞunit Abs: value
¼ C1/Abs:C1 þ C2/Abs:C2 þ C3/Abs:C3
58 Mol Cell Biochem (2011) 349:57–67
123
where C1, C2, and C3 are three randomly selected con-
centrations (mg/ml) from their linear response curve.
Higher absorbance is indicative of higher reducing power.
Nitric oxide scavenging activity assay
Nitric oxide radical scavenging activity of SQGD was
determined according to the method reported by Garrat
[16]. Sodium nitroprusside in aqueous solution at physio-
logical pH (7.4) spontaneously generates nitric oxide, which
interacts with oxygen to produce nitrite ion, which can be
determined by the use of Griess Illosvoy reaction. Varied
concentrations of SQGD were mixed with sodium nitro-
prusside (5 mM), and the volume made up to 1.0 ml using
phosphate buffer saline (pH 7.4). The reaction mixture was
incubated at 25�C for 150 min followed by addition of
Griess reagent (6% sulphanilamide in 3 M HCl ? 0.3%
napthylethylene diamine dihydrochloride ? 7.5% ortho-
phosphoric acid in 1:1:1 ratio). Pink colored chromosphere
was formed. The nitric oxide scavenging activity was ana-
lyzed as percent decreased in the absorbance of the complex
formed by diazotization of nitrite with sulfanilamide and
subsequent coupling with naphthylethylenediamine at
546 nm. Decrease in optical density is an indicative of
higher nitric oxide potential of the SQGD.
Total antioxidant capacity estimation of SQGD
To determine the total antioxidant potential of SQGD, a
modified ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid)] assay was performed [17]. ABTS radicals
were generated by mixing ABTS (7.0 mM) stock solution
with potassium persulfate (2.45 mM), incubating for
12–16 h in the dark at room temperature until the oxidation
of ABTS was completed, and the absorbance was stabi-
lized. The absorbance of the solution was equilibrated to
0.70 ± 0.02 by diluting with PBS at room temperature.
The ABTS radical formed was incubated with different
concentrations (0–500 lg/ml) of SQGD for 5 min and
absorbance was measured at 734 nm. The percentage
inhibition in the absorbance was calculated and plotted
against SQGD concentrations.
Preparation of liposomes
Liposomes was prepared in a round bottom flask using a
mixture of soya lecithin (phospholipid content above 70%)
and cholesterol (2:1 ratio) mixed in chloroform and was
dried under vacuum as reported by New [18]. A thin film
developed on the bottom surface of flask after vacuum
drying was suspended in phosphate buffer saline (0.1 M)
for hydration and incubated in water bath for 3–4 h at 40�C
with continuous shaking at 50 rpm. Resulted liposomes
were further used to study the inhibition of lipid peroxi-
dation potential of SQGD.
Membrane protection index
Thiobarbituric acid reactive substances (TBARSs) are the
most commonly used method to detect lipid peroxidation.
This procedure measures the MDA formed as the split
product of an endoperoxide of unsaturated fatty acids
resulting from oxidation of a lipid substrate. The MDA is
reacted with thiobarbituric acid (TBA) to form a pink
pigment (TBARS) that is measured spectrophotometrically
at its absorption maximum at 535 nm. Different concen-
trations of the SQGD (0–100 lg/ml) were prepared in
distilled water. The volume of the SQGD was diluted to
1.0 ml using liposomal solution. The reaction mixture was
then incubated at 37�C for 60 min. After incubation,
radiation dose of 0.25 kGy at a dose rate of 1.93 kGy/h was
subjected to reaction mixtures. Followed by an incubation
of 1 h at 37�C, the equivalent volume of 10% trichloro-
acetic acid (TCA) and 0.5% thiobarbituric acid (prepared
in 0.025 N NaOH) were added. The resultant mixture was
then subjected to incubation at of 80�C for 1 h in water
bath. A pink-colored chromogen complex formed was read
at 535 nm. The protection index of the SQGD was com-
pared based on percentage inhibition in the absorbance.
The membrane protection index was estimated by using
following formula:
% inhibition of membrane degradation
¼ Abs535control � Abs535test=Abs535Control � 100
Hydroxyl radical scavenging activity of SQGD
The generation and detection of non-site-specific hydroxyl
radical scavenging were carried out according to a Fenton
method described by Halliwell and Gutteridge [19]. Dif-
ferent concentrations of SQGD were mixed with 0.1 mM
ferric chloride solution, 0.104 mM EDTA-Na2, and
0.1 mM L-ascorbic acid along with 1 mM H2O2, 3.6 mM
2-deoxy-D-ribose in potassium phosphate buffer (pH 7.4) in
a total assay volume of 1 ml, followed by incubation at
37�C temperature for 1 h and then treated with equal vol-
umes of 10% TCA and 0.5% thiobarbituric acid (in 0.025 M
NaOH). The samples were incubated (55�C) for 15 min.
The pink reactive chromogen of TBA and the malondial-
dehyde from deoxyribose degraded by OH• was measured
at 532 nm. The decrease in absorbance at a particular
concentration indicates higher hydroxyl ion scavenging
potential with respect to control. The percentage inhibition
of degradation of deoxyribose or hydroxyl ion scavenging
potential will be calculated as follows:
Mol Cell Biochem (2011) 349:57–67 59
123
% Inhibition ¼ Abscontrol � Abstest=Abscontrol � 100
The procedure for evaluating the site-specific hydroxyl ion
scavenging potential was similar to the above-mentioned
assay with a small change that in lieu of using EDTA, a
similar volume of buffer was used in a 1 ml reaction
mixture.
Electron paramagnetic resonance (EPR) studies
For all EPR experiments an EMXMICRO EPR spectrometer
(Bruker, Germany) equipped with standard resonator
working at a frequency in X-band region was used. Quartz
capillaries (2 mm diameter 9 5 cm length) were used as
sample tubes. All EPR spectra were recorded at room
temperature (22�C) in triplicate. EPR spectral processing
including g value calculation was performed using Bruker
WinEPR� and simulations were performed with Bruker
SimFonia�.
Radical scavenging capacity of SQGD toward stable
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)
Determination of radical scavenging capacity was based on
the decay of DPPH EPR signal after addition of SQGD to
the stock ethanol solution of DPPH. Briefly, 250 ll of
DPPH (80 lM) was added to 10 ll of 6.4% water solution
of SQGD and stirred for 5 min at room temperature. The
mixture was then incubated for 10 min in the dark. After
incubation, the mixture was transferred into a quartz cap-
illary and placed in the microwave cavity of EPR spec-
trometer. The decay of EPR signal of DPPH in presence of
different concentrations of SQGD (from 0.2 to 6.4%) was
monitored and compared to that of the blank sample,
containing water instead of SQGD. Percent scavenged
DPPH radical by SQGD was calculated according to the
equation:
Scavenged DPPH radicals %ð Þ ¼ I0 � Ið Þ=I0½ � � 100
where I0 is the double integrated intensity of DPPH signal
for blank sample; I is the double integrated intensity of
DPPH signal for studied sample measured after addition of
the SQGD to DPPH solution.
EPR settings were as follows: center field 3516.00 G,
microwave power 0.632 mW, modulation amplitude
5.00 G, sweep width 200.00 G, receiver gain 5.02 9 103,
sweep time 167.936 s.
Direct EPR spectroscopy detection of free radicals
in solid and solution form of SQGD
Quartz capillaries were filled with crystalline powder or
6.4% solution of SQGD in deionized water and placed in
the cavity of the EPR spectrometer. EPR spectra were
recorded at the following spectrometer settings: center field
3514.00 G, receiver gain 2 9 103, microwave power
0.632 mW, modulation amplitude 8.00 G, sweep width
61.23 G, sweep time 5.243 s. Recording of the EPR
spectrum of SQGD in solution form was performed at the
same spectrometer settings, with exception of the modu-
lation amplitude, receiver gain and sweep width, that were
correspondingly settled to 10.00 G; 1 9 105 and 97.37 G.
Statistical analysis
The experimental results were performed in triplicate. The
results are expressed as mean values and standard error
(SE) or standard deviation (SD). The SQGD antioxidant
activity was analyzed using one-way analysis of variance
(ANOVA) with significance of \0.05 using SPSS version
7.5.
Results
Reducing power assay
SQGD exhibited a dose-dependent increase in reducing
power activity. Maximum reducing power activity of
SQGD (1.267 ± 0.03356) was observed at highest con-
centration (1000 lg/ml) as compared to the control.
Therefore, this study indicated that the reducing power of
the SQGD in the aqueous phase is significantly increased
along with increasing concentrations and found compara-
ble with ascorbic acid, a well-known antioxidant (Fig. 1).
Nitric oxide scavenging activity
Oxidative stress induced by irradiation generates nitric
oxide, which play a very critical role in initiation and
progression of oxidative damage in the biological system.
This study indicated significant (P \ 0.05) nitric oxide
inhibitory activity of SQGD in in vitro models. The max-
imum nitric oxide inhibitory activity of SQGD
(34.68 ± 2.13%), was observed at maximum tested con-
centration (i.e., 1000 lg/ml) as compared to drug negative
control (Fig. 2).
Total Antioxidant capacity estimation of SQGD
ABTS radicals generated after reaction with potassium
persulfate and their neutralization by SQGD was estimated.
The results of the study indicated significantly higher
antioxidant activity in SQGD at all tested concentration as
compared to the Quercetin-derived ABTS activity. SQGD
showed 40% increase in the ABTS radicals scavenging
60 Mol Cell Biochem (2011) 349:57–67
123
activity at 0.25 lg/ml concentration as compared to
Quercetin. Further increase in the concentration of
SQGD enhanced its ABTS radical scavenging activity.
SQGD-mediated ABTS radical neutralization was found
significantly higher with all the tested concentrations as
compared with Quercetin (Fig. 3). Maximum activity of
SQGD was found to be 74.09 ± 0.08% as compared to
positive control Quercetin which showed only 50.12 ±
3.94% at 500 lg/ml concentration (Fig. 3).
Membrane protection index
Free radicals, formed via different mechanisms, induce
peroxidation of membrane lipids. Although peroxidation in
model membranes may be very different from peroxidation
in biological membranes, the results obtained in model
membranes (liposome) may be used to advance our
understanding of issues that cannot be studied in biological
membranes. Maximum percentage inhibition in formation
of radiation-induced TBARS radicals by SQGD was
observed to be 39.54 ± 0.37% at 100 lg/ml concentration.
Further increase in the SQGD concentration did not induce
its TBARS radical inhibiting activity in vitro (Fig. 4).
Hydroxyl radical scavenging potential
The hydroxyl radical scavenging potential of SQGD was
evaluated in terms of % inhibition of deoxy D-ribose deg-
radation. Maximum SQGD mediated % inhibition in deoxy
D-ribose degradation as estimated by non-site-specific
Fig. 1 Evaluation of reducing
power of SQGD. The
absorbance at 700 nm was
recorded in triplicate and each
experiment was repeated thrice.
The value was expressed as
mean ± SD. Ascorbic acid, a
standard synthetic antioxidant
was used as control. High
absorbance at 700 nm indicates
high reducing power.
Significance (*P \ 0.05)
Fig. 2 The nitric oxide radical
scavenging of SQGD. Ascorbic
acid was used as standard. The
data represent the percentage
nitric oxide inhibition. The
SQGD Showed maximum
activity of 34.68 ± 2.13% at
1000 lg/ml. Significance
(*P \ 0.01)
Mol Cell Biochem (2011) 349:57–67 61
123
assay was observed to be 54.01 ± 1.01% at 250 lg/ml
concentration of SQGD. Whereas, site-specific % inhibi-
tion in the deoxy D-ribose degradation was observed to
be 46.36 ± 0.5% at same (250 lg/ml) concentration of
SQGD (Fig. 5).
Radical scavenging capacity of SQGD by EPR
spectroscopy
EPR spectrum of alcoholic solution of DPPH in the blank
sample was characterized by its five lines of relative
intensities 1:2:3:2:1 [20] (Fig. 6a). The same quintet EPR
signals were registered in all samples containing DPPH and
different concentrations of SQGD (Fig. 6b). When the
concentration of SQGD increased the percent of the scav-
enged DPPH radicals was also found to be increased
(Fig. 7). The maximal percentage (88; P \ 0.05) of the
DPPH radicals scavenged by SQGD was calculated at its
highest concentration i.e., 6.4% w/v (Fig. 7). Therefore,
present EPR study clearly demonstrates that SQGD
exhibits tremendous antioxidant activity.
Detection of stable free radicals in solid and solution
form of SQGD by EPR spectroscopy
To explore possibility of a stable natural free radical
structure in SQGD, we have recorded its EPR spectra in
powdered and solution form. A sharp characteristic EPR
singlet signal of very high intensity (Fig. 8a) was registered
Fig. 3 The effects of
concentration of the SQGD on
the inhibition of ABTS•?.
Significance difference for
SQGD compare to control
(0 lg/ml) (*P \ 0.01)
Fig. 4 Effect of different concentrations of SQGD on radiation
(0.25 kGy)-mediated lipid peroxidation in liposomes. Each experi-
ment was performed in triplicate and was repeated three times. Lipid
peroxidation in control represents 0% inhibition (maximal activity).
Maximal decrease in activity at 100 lg/ml SQGD versus standard
significance (*P \ 0.001 and #P \ 0.05)
Fig. 5 Hydroxyl radical scavenging activities of the SQGD and the
reference compound quercitin. The data represent the percentage
inhibition of deoxy-D-ribose degradation. The results are mean ± SD.
SQGD exhibited significant percentage inhibition of site-specific
versus non-site specific (*P \ 0.01)
62 Mol Cell Biochem (2011) 349:57–67
123
in powdered form of SQGD. The g value of the EPR signal
was calculated to be 2.00485. The aqueous solution of
SQGD expressed almost the same stable characteristic EPR
singlet signal (Fig. 8b) with a g value of 2.00520. EPR
signals registered in both (powder/solution) forms of
SQGD were not altered with the time and temperature.
Discussion
Radiation-induced oxidative damage is a multi-facet phe-
nomenon that impaired the cell regulation and controls the
vital processes such as proliferation, repair, and recovery
[21]. Exposure of lethal ionizing radiation increased pro-
duction of reactive oxygen/nitrogen species (ROS/RNS)
such as superoxide radicals (O2-), hydroxyl radicals
(OH-), hydrogen peroxide (H2O2), and singlet oxygen,
etc., and induced formation of leaky membranes, DNA
fragmentation and lesions, oxidation and denaturation of
vital proteins [22]. To counter all these oxidative delete-
rious effect of ionizing radiation, search for an efficient
free radical scavenger (i.e., antioxidant) is in progress
globally. In this study, a molecule SQGD extracted from
the radioresistant bacterium Bacillus sp. INM-1 was
Fig. 6 EPR spectra of DPPH
radicals in the blank sample
(a) and in the sample containing
10 ll of 3.2% water solution of
SQGD (b)
Mol Cell Biochem (2011) 349:57–67 63
123
evaluated for its antioxidant activities using several bio-
chemical in vitro assays such as reducing power assay
(measuring the conversion of Fe3?/ferricynide complex to
the ferrous form), nitric oxide scavenging assay (measure
the decrease of nitrite), ABTS assay (measured the
decrease of ABTS? radical), membrane protection index
(measured by the color intensity of MDA–TBA complex),
hydroxyl ion scavenging assay (measured the inhibition of
hydroxy ion), and free radical scavenging properties using
EPR spectroscopic method.
The reducing ability of a compound generally depends
on the presence of reductants [23], which have been
exhibited antioxidative potential by breaking the free rad-
ical chain through donating a hydrogen atom/electron [24].
The presence of reductants in the SQGD may causes the
reduction of Fe3?/ferricyanide complex into the ferrous
(Fe2?) form, which was monitored by measuring the for-
mation of Perl’s Prussian blue at 700 nm. SQGD exhibits
significantly high reducing activity with increasing
concentration (Fig. 1). Iron acts as an amplifier of radia-
tion-induced free radical-mediated damage by its unique
existence in two oxidation states (i.e., Fe?2 and Fe?3) [25].
Radiation exposure increases iron load in the cellular
milieu lead to hemolysis [21]. Therefore, reduction of Fe?3
into less harmful Fe?2 by SQGD may be suggested as
prominent mode of action exhibited antioxidative and thus
radioprotective efficacy in vitro [3].
Nitric oxide (NO), a diffusible free radical, plays an
important role in various biological functions including
neuronal messenger, vasodilation, antimicrobial, and anti-
tumor activities [26]. However, over-production of nitric
oxide and superoxide radicals contributes to the patho-
genesis of some inflammatory diseases [27]. Moreover, in
the pathological conditions nitric oxide reacts with
superoxide anion and form peroxynitrite which lead to
serious toxic reactions with biomolecules, like protein,
lipids, and nucleic acids [28, 29]. Results of this study
indicated a significant increase (34%) in % inhibition of the
nitric oxide radicals by SQGD (Fig. 2) shows significant
role in various radiation-induced pathological situations.
Radiation-induced nitric oxide radicals-mediated inflam-
mation is one of the serious causes of increased lethality in
the irradiated cells and tissue [30]. Nitric oxide inhibitors
[31] have been known for their beneficial effects on some
aspect of inflammation and tissue damage seen in the
inflammatory diseases. SQGD-induced inhibition of nitric
oxide radicals may be one of the mechanisms for its
radioprotective effect.
Radiation-induced modifications of the carbons of fatty
acids of membrane phospholipids form unstable and highly
reactive peroxide radicals that get decomposed further to
lower-molecular-weight products including malondialde-
hydes, secondary ketones, and alcohols [32]. Thus, the
antioxidant potential of any natural product depends upon
the restriction of lipid peroxidation by scavenging peroxyl
radicals. In biological systems, peroxyl radicals undergo
cyclization to form the isoprostanes, which are similar to
prostaglandins and thus generate inflammatory responses
lead to patho-physiological consequences. Results of this
study indicated significantly high (P \ 0.05) anti-lipid
peroxidation activity of SQGD (Fig. 4). Thus, by inhibiting
lipid peroxide radicals, SQGD may play an anti-inflam-
matory role indirectly, which is an unavoidable necessity
for radioprotection [3].
Activity of the SQGD on hydroxy radical has been
shown in Fig. 5. Hydroxyl radical is the most reactive
oxygen centered radical formed from the reaction of vari-
ous hydroperoxides with transition metal ions and it bears
the shortest half-life compared with other ROS. OH-
radicals are known to attack on proteins, DNA, polyun-
saturated fatty acid in membranes, and most biological
molecules in the surrounding cellular milieu [33] and
capable enough to abstracting hydrogen atoms from
membrane lipids [34]. The hydroxyl ion scavenging
activity of SQGD was evaluated using deoxy-D-ribose
assay. It was used to study the non-site specific
[Fe2? ? H2O2 ? EDTA] as well as site-specific [Fe2? ?
H2O2] hydroxyl ion scavenging activity in aqueous system.
In non-site-specific assay, the presence of EDTA makes
Fe2? non-available for attacking deoxyribose directly and,
therefore, hydroxyl generation predominates [19, 35]. The
quenching ability of SQGD to hydroxyl radicals seems to
be directly related to the prevention of propagation of the
process of lipid peroxidation and seems to be a good
scavenger of active oxygen species. This study indicated a
significantly (P \ 0.01) higher non-site-specific hydroxyl
ion scavenging potential of SQGD as compared to site
Fig. 7 DPPH radical scavenging capacity at different concentrations
of SQGD. Alcoholic suspension of DPPH (80 lM) was treated with
different concentrations (0.2–6.4%) of SQGD and incubated for
10 min in the dark at room temperature. The reduction of the DPPH
radicals was estimated as the function of decrease absorbance as
monitored at 517 nm along with increasing SQGD concentration
64 Mol Cell Biochem (2011) 349:57–67
123
specific. The higher hydroxyl ion scavenging activity of
SQGD possibly contributed to its ability to chelate transi-
tion metal ions by filling its aqua-coordination sites, sug-
gested that SQGD could boost the intrinsic defence system
by scavenging free radicals due to its exhibited antioxidant
activities [25].
EPR has been established as most efficient tool to
evaluate antioxidant and stable free radical properties of
any natural compounds [36–38]. 2,2-Diphenyl-1-picrylhy-
drazyl (DPPH) is one of the most widely used stable free
radicals for evaluation of the antioxidant activity of natural
isolated extracts, food supplements, and pure natural and
synthetic compounds by EPR spectroscopy [20, 39]. It is
known that the stable DPPH radical can react with
antioxidants that possess functional groups donating H and/
or free radicals and these interactions might be written as
[40]:
DPPH� þ AH ¼ DPPH-H þ A� or DPPH� þ R�
¼ DPPH-R
To investigate DPPH radical scavenging capacity of
SQGD, we have applied direct EPR spectroscopy and
found that SQGD expressed a good DPPH radical scav-
enging capacity in a concentration-dependent manner. This
finding was in accordance with all above presented studies
and characterized SQGD as an antioxidant. The fact that a
number of naturally isolated antioxidants possess free
radicals in their structures [41, 42] led us to study the
Fig. 8 EPR spectrum of SQGD
in powdered form (a) and EPR
spectrum of 6.4% solution of
SQGD in deionized water (b)
Mol Cell Biochem (2011) 349:57–67 65
123
possible of antioxidant properties of SQGD to be due to the
presence of free radicals in its structure. EPR spectroscopy
analysis of SQGD in powdered and solution form revealed
presence of free radical structure. Very recently, Sophia
et al. [43] have reported a semiquinone radical stabilized
by the cytochrome aa3-600 menaquinol oxidase of Bacil-
lus subtilis with gy tensor value closely similar to that we
have calculated for the radical registered in powder and
solution form of SQGD. Tensor (g) value specifically
reflects the nature of radicals present in the mixture/prep-
aration. Unpaired electron of specific radicals possesses
effective magnetic moment under high magnetic field
which can be calculated in terms of g tensor value. Based
on calculated g tensor values and stability of the registered
radicals we accept that an o-semiquinone radical anion is
present in SQGD structure. It is known that o-semiquinone
radical anion is much more stable than neutral radical [44]
further provided a gain support to this study.
Based on this study it can be concluded that because of
its strong antioxidant and stable free radical properties
SQGD would possess ability to scavenge and neutralize
radiation induced radicals. Therefore, SQGD can be used
clinically as potential radioprotector/mitigator to overcome
the toxic effect of radiation in the cancer patient under-
going radiotherapy regime. However, further studies to
evaluate in vivo radioprotective efficacy of SQGD will
certainly explore its future application in planned (radio-
therapy of cancer patients) and unplanned (accidently or
deliberately) radiation exposure.
Acknowledgments This study was supported by grants of Defence
Research and Development Organization, Ministry of Defence, India,
Department of Science and Technology, India, and Ministry of
Education, Youth and Science, Bulgaria. Indian Council of Medical
Research is also duly acknowledged for providing research fellowship
for this study.
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