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.

References

1. Wall ME, Wani MC (1996) Camptothecin and taxol: from dis-

covery to clinic. J Ethnopharmacol 51:239–254

2. Weiss JF, Landauer MR (2003) Protection against ionizing

radiation by antioxidant nutrient and phytochemicals. Toxicology

189:1–20

3. Arora R, Gupta D, Chawla R, Sagar R, Sharma AK, Prasad J,

Singh S, Kumar R, Sharma RK (2005) Radioprotection by plants

products: present status and future prospects. Phytother Res

19:1–22

4. Arora R, Chawla R, Sagar R, Prasad J, Singh S, Kumar R,

Sharma A, Sharma RK (2005) Evaluation of radioprotective

activities of Rhodiola imbricata Edgew—a high altitude plants.

Mol Cell Biochem 273:209–223

5. Arora R, Chawla R, Puri SC, Sagar RK, Chawla R, Singh S,

Kumar R, Sharma AK, Prasad J, Singh S, Kaur G, Chaudhary P,

Qazi GN, Sharma RK (2005) Radioprotective and antioxidant

properties of low altitude Podophyllum hexandrum (LAPH).

J Environ Pathol Toxicol Oncol 24:299–314

6. Karawita R, Siriwardhana N, Lee Ki-Wan, Heo Moon-Soo,

Yeo In-Kyu, Lee Young-Don, Jeon You-Jin (2005) Reactive

oxygen species scavenging, metal chelation, reducing power

and lipid peroxidation inhibition properties of different solvent

fractions from Hizikia fusiformis. Eur Food Res Technol

220:363–371

7. Cao G, Prior RL (1999) Measurement of oxygen radical absor-

bance capacity in biological samples: methods in enzymology,

vol 299. Academic Press, San Deigo, pp 50–62

8. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006)

Free radicals, metals and antioxidants in oxidative stress-induced

cancer. Chem Biol Interact 160:1–40

9. Tian B, Xu Z, Sun Z, Lin J, Hua Y (2007) Evaluation of the

antioxidant effects of carotenoids from Deinococcus radioduransthrough targeted mutagenesis, chemiluminescence, and DNA

damage analyses. Biochim Biophys Acta 1770:902–911

10. Siriwardhana N, Lee KW, Jeon YJ, Kim SH, Haw JW (2003)

Antioxidant activity of Hizikia fusiformis on reactive oxygen

species scavenging and lipid peroxidation inhibition. Food Sci

Technol Int 9:339–346

11. Zhang HY, Li XJ, Gao N, Chen LL (2009) Antioxidants used by

Deinococcus radiodurans and implications for antioxidant drug

discovery. Nat Rev Microbiol 7:476–481

12. Dal MJ (2009) A new perspective on radiation resistance based

on Deinococcus radiodurans. Nat Rev Microbiol 3:237–245

13. Ferreir AC, Nobre MF, Moore E, Rainey FA, Battista JR,

da-Costa MS (1999) Characterization and radiation resistance of

new isolated of Rubrobacter radiotolerans and Rubrobacterxylanophilus. Extremophiles 3:235–238

14. Kumar R, Patel DD, Bansal DD, Mishra S, Mohammed A, Arora

R, Sharma A, Sharma RK, Tripathi RP (2010) Extremophiles:

sustainable resource of natural compounds-extremolytes. In:

Singh OV, Harvey SP (eds) Sustainable biotechnology sources of

renewable energy. Springer, New York, pp 279–294

15. Oyaizu M (1986) Studies on products of browning reaction:

antioxidative activities of products of browning reaction prepared

from glucosamine. Jpn J Nutr 44:307–315

16. Garrat DC (1964) The quantitative analysis of drug. Chapman

and Hall, Hirakawachi

17. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-

Evans C (1999) Antioxidant activity applying an improved ABTS

radical cation decolourization assay. Free Radic Biol Med

26:1231–1237

18. New RRC (1990) Preparation of liposomes. In: New RRC (ed)

Liposomes: a practical approach. Oxford University Press,

Oxford

19. Halliwell B, Gutteridge JM, Aruoma OI (1987) The deoxyribose

method: a simple test tube assay for determination of the rate

constants for reactions of hydroxyl radicals. Anal Biochem

165:215–219

20. Vankar PS, Tiwari V, Srivastava J (2006) Extracts of stem bark of

Eucaliptus globulus as food dye with high antioxidant properties.

Electron J Environ Agric Food Chem 5(6):1664–1669

21. Giambarresi L, Jacobs AJ (1987) Radioprotectant. In: Conklin JJ,

Walker RI (eds) Militory radiobiology. Academic Press, London,

pp 265–301

22. Sies H (1985) Oxidative stress. Academic press, New York

23. Pin-Der-Duh X (1998) Antioxidant activity of burdock (Arctiumlappa Linne): it’s scavenging effect on free radical and active

oxygen. J Am Oil Chem Soc 75:455–461

24. Gordon MH (1990) The mechanism of the antioxidant action

in vitro. In: Hudson BJF (ed) Food Antioxidants. Elsevier,

London

25. Kumar PI, Goel HC (2000) Iron chelation and related properties

of podophyllum hexandrum, a possible role in radioprotection.

Indian J Exp Biol 38:1003–1006

66 Mol Cell Biochem (2011) 349:57–67

123

26. Miller MJ, Sadowska-Krowicka H, Chotinaruemol S, Kakkis JL,

Clark DA (1993) Amelioration of chronic ileitis by nitric oxide

synthase inhibition. J Pharmacol Exp Ther 264:11–16

27. Guo X, Wang WP, Ko JK, Cho CH (1999) Involvement of

neutrophils and free radicals in the potentiating effects of passive

cigarette smoking on inflammatory bowel disease in rats.

Gasteroenterol 117:884–890

28. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physi-

ology, pathophysiology, and pharmacology. Pharmacol Rev

43:109–142

29. Yermilov V, Rubio J, Becchi M, Friesen MD, Pig-Natelli B,

Ohshima H (1995) Formation of 8-nitroguanine by the reaction of

guanine with peroxynitrite in vitro. Carcinogenesis 16:2045–2050

30. Matthew B, Grisham D, David AW (1999) Nitric oxide I.

Physiological chemistry of nitric oxide and its metabolites:

implications in inflammation. Am J Physiol Gastrointest Liver

Physiol 276:G315–G321

31. Salvatore C (2004) Effect of inhibitors of nitric oxide in animal

models and future directions for therapy in inflammatory disorders.

Curr Med Chem Anti-Inflam Anti-Allergy Agents 3:261–270

32. Devasagayam TPA, Boloor KK, Ramasarma T (2003) Methods

for estimating lipid peroxidation: an analysis of merits and

demerits. Indian J Biochem Biophys 40:300–308

33. Aruoma OI (1999) Free radicals, antioxidants and international

nutrition. Asia Pacific J Clin Nutr 8:53–56

34. Yen G, Duh P (1994) Scavenging effect of methanolic extracts of

peanut hulls on free-radical and active-oxygen species. J Agric

Food Chem 42:629–632

35. Kitts DD, Wijewickreme AN, Hu C (2000) Antioxidant proper-

ties of a North American ginseng extract. Mol Cell Biochem

203:1–10

36. Zoete V, Bailly F, Vezin H, Teissier E, Duriez P, Fruchart JC,

Catteau JP, Bernier JL (20004) 4-Mercaptoimidazoles derived

from the naturally occurring antioxidant ovothiols 1. Antioxidant

properties. Free Rad Res 32:515–524

37. Matthaus B (2002) Antioxidant activity of extracts obtained from

residues of different oilseeds. J Agric Food Chem 50:3444–3452

38. Rajaram D, Nazeer RA (2010) Antioxidant properties of protein

hydrolysates obtained from marine fishes Lepturacanthus savalaand Sphyraena barracuda. Int J Biotechnol Biochem 6:435–444

39. Polovka M, Brezova V, Stasko A (2003) Antioxidant properties of

tea investigated by EPR spectroscopy. Biophys Chem 106:39–56

40. Khan N, Swartz H (2002) Measurements in vivo of parameters

pertinent to ROS/RNS using EPR spectroscopy. Mol Cell Bio-

chem 234(235):341–357

41. Gao Z, Huang K, Yang X, Xu H (1999) Free radical scavenging

and antioxidant activities of flavonoids extracted from the radix

of Scutellaria baicalensis Georgi. Biochim Biophys Acta

1472:643–650

42. Bors W, Michel C, Stettmaier K, Lu Y, Foo LY (2004) Antiox-

idant mechanisms of polyphenolic caffeic acid oligomers, con-

stituents of Salvia officinalis, antioxidant mechanisms of

polyphenolic caffeic acid oligomers, constituents of Salvia offi-cinalis. Biol Res 37:301–311

43. Sophia MY, Kuppala V, Narasimhulu RI, Samoilova RB, Gennis-

Sergei AD (2010) Characterization of the semiquinone radical

stabilized by the cytochrome aa3-600 menaquinol oxidase of

Bacillus subtilis. J Biol Chem 285:18241–18251

44. Akiyama N, Nakanishi I, Ohkubo K, Satoh K, Tsuchiya K,

Nishikawa T, Fukuzumi S, Ikota N, Ozawa T, Tsujimoto M,

Natori S (2007) A long–lived o-semiquinone radical anion is

formed from N-b-alanyl-5-S-gluthationyl-3, 4-dihydroxyphenyl-

alanine (5-S-GAD), an insect-derived antibacterial substance.

J Biochem 142:41–48

Mol Cell Biochem (2011) 349:57–67 67

123