Indian Journal of Experimental Biology
Vol. 40, June 2002, pp. 727-734
Free radical scavenging and metal chelation by Tinospora cordifolia, a possible
role in radioprotection
H C Goel &' I l'i-em Kumar
Department of Radiation Biology, Institute of Nuclear Medicine and Allied sciences, Brig SK Majumdar Marg, Delhi 110054
and
S V S Rana
Department of Zoology, C.C.S. University, Meerut
Aqueous extract of T. cordi/alia inhibited Fenton (FeS04) reaction and radiation mediated 2-deoxyribose degradation
in a dose dependent fashion with an IC 50 value of 700 ~glml for both Fenton and radiation mediated 2-DR degradation.
Similarly, it showed a moderate but dose dependent inhibition of chemically generated superoxide anion at 500 ~glml concentration and above with an IC 50 value of 2000 ~g/ml. Aqueous extract inhibited the formation of Fe2+-bipiridyl complex
and formation of comet tail by chelating Fe 2+ ions in a dose dependent manner with an IC 50 value of ISO ~glml for Fe 2+_
bipirydyl formation and maximally 200 ~glml for comet tail formation, respectively . The extract inhibited ferrous sulphate
mediated lipid peroxidation in a dose-dependent manner with an IC 50 value of 1300 ~glml and maximally (70%) at
2000 ~glml. The results reveal that the direct and indirect antioxidant actions of T. cordi/alia probably act in corroboration
to manifest the overall radioprotective effects.
Radiation having low linear energy transfer like
gamma rays, X-rays etc. generate damage mainly by
producing reactive oxygen species (. OH , . O 2 , H20 2,
singlet oxygen etc.) which on interaction with different biomolecules incur damage and lead to oxidative
stress and ultimately the deathl,2. Important consequences of free radical damage include peroxidation
of lipids, oxidation of proteins and enzymes, strand
breaks and adduct formation in nucleic acids 3-5 . Oxidative stress has also been implicated in several
pathological conditions6 • Under normal conditions the
cell operates a strong and effective defence system
against free radical mediated damage, which includes
both direct and indirect antioxidant mechanisms7 . Direct defence system includes molecules like reduced
glutathione, vitamin E, ascorbic acid etc, and antioxidant enzyme systems like sUferoxide dismutase, catalase, glutathione peroxidase etc, Indirect system includes metal chelating molecules like ferritin, transferrin, ceruloplasmin etc which on binding with free
iron and other metal ions reduce the generation of free
radicalsB,
levels of free radicals increase and the balance between · prooxidants and antioxidant shifts towards
former, resulting in severe oxidative stress B• Any
agent, which can modulate the shift towards antioxidant can be of immense help to the cellular system in
dealing with the oxidative stress and in augmenting
recovery9, During radiotherapy and chemotherapy of
tumours and also under post-irradiation conditions the
levels of free metal ions (iron) have been reported to
increase in body fluids 10, 11 . These free metal ions on
interaction with radiation or chemical generated hydrogen peroxide produce most reactive ·OH radicals,
which in tum interact with several biomolecules and
exacerbate the initial damage 12 • Chronically increased
oxidative stress from elevated levels of iron ions in
the body may increase radiation sensitivity by decreasing cellular oxygen radical scavenging capacit/ 2, Any agent which can chelate these free iron ions
can be helpful in minimising the damage. Therefore
any attempt to develop good biological radioprotector
should address the problem of scavenging free radicals, specifically iron ions,
Under several pathological conditions, on exposure
to radiation apart from post irradiation conditions the
A large number of chemicals have been investigated during last 50 years to overcome radiation damage, Among them some are, natural amino acids like
cysteine and their derivative cysteamjne, AET, vitamins like A, E, C, small proteins like glutathione etc.
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728
INDIAN J EXP SIOL, JUNE 2002
and synthetic molecules like WR-2721 ete D . However, the inherent toxicity of these agents at the useful
concentration has warranted further exploration 14 . In
the last decade natural herbal products have received
maximum attention owing to their wide utility in traditional medicine system like Ayurveda. A number of
herbal preparations both in crude form and their fractionated components or combination of herbal preparations from different plants have been shown to render radioprotection both in vivo and in vitro mammalian systems I5- 18 . Among different isolated components
the flavones, flavonoids, tannins, glucosides, other
polyphenols, catechins, epigallactocatechins etc. have
been reported to render radioprotection because of
their free radical scavenging potentials 19.20 .
Tinospora cordifolia Meirs (Menispermiaceae)
commonly known as Guduchi (Sanskrit) and Giloy
(Hindi); is a glaborous, climbing, succulent herb
commonly found in hedges and is a native of India
and thrives easily in tropical regions. It is widely used
in Ayurveda as vitaliser, antidiabetic, hepatoprotective, antipyretic, antistress, antiulcer and immunomodulatory agent2 1 It was therefore considered that
antioxidant properties of T. cordifolia may be
substantially responsible for these medicinal effects.
In view of this its radioprotective action against
gamma radiation was studied in experimental models.
Aqueous extract of T. cordifolia has shown significant
radioprotection (75%) against 10 Gy whole body
gamma radiation in experimental animals and elicited
moderate free radical scavenging under in vitro conditions (unpublished data). The present study has been
undertaken to understand probable mode of action
with special reference to their metal chelating and free
radical scavenging abilities individually and in combination.
Materials and Methods
Chemicals-AR grade trichloroacetic acid from
BDH (UK), ferrous sulfate, ferric chloride, ferrous
ammonium sulfate, 2-2- bipirydyl, 2-deoxyribose
from SRL (India). Tertiary butyl hydroperoxide,
NaCl, KCl, EDT A, sodium lauryl sarcosine, TritonX-lOO, agarose, phenazine methosulfate, nitroblue
tetrazolium, thiobarbituric acid and NADH from
Sigma (USA) were procured.
Plant material and herbal preparations -Dried
stem of T. cordifolia supplied by Institute of Himalayan Bioresource Technology, CSIR, Palampur, HP,
India was powdered mechanically. Powder (lg) was
mixed in distilled water and kept at 37° ± 1°C in an
incubator for 24 hr and filtered thereafter using
Whatman filter paper no.l. The filtrate passed through
millipore filter (0.22 ).!m) was vacuum dried and lyophilized. Resulting powder was suitably diluted in
triple distilled water for obtaining desired concentrations required for evaluating free radical scavenging
and metal chelating potentials. For al l experiments the
quantity taken was the lyophilized powder of the respective preparations.
Irradiation -Different solutions were delivered
radiation doses (Gamma chamber 5000 BRIT,
INDIA) as per the requirement of experiment. The
dose rate during the course of experiment was about
6.43 K Gy/hr.
Preparation of liver homogenate and thymocytesRandomly selected 6-8 weeks old strain A male mice
were sacrificed by cervical dislocation, dissected and
abdominal cavity was perfused with 0.9% saline;
whole liver was taken out and visible clots were carefuJIy and maximally removed and weighed amount of
liver was processed to get a 10% homogenate in cold
. phosphate buffer saline, pH 7.4, using Potter Elvjham
homogenizer and filtered to get a clear homogenate.
Preparation of thymocytes -From above mentioned
animals thymic lobes were taken out and visible clots
over them were removed carefully. The thymic lobes
were placed on frosted slides, minced gently and the
resulting cell suspension was passed through a 25gauge needle to avoid cell aggregates. The thymocytes were resuspended in standard buffer saline.
Assay for estimating antioxidant activity-All
measurements were done on chemito 2500 UV -Vis
spectrophotometer. Estimations were expressed as
inhibition (%) with respect to control considered as
zero inhibition. Total protein level of 10% homogenate was estimated by Lowry's method 22 .
Lipid peroxidation-Liver homogenate (2 ml of
10%) was taken in a series of 35 mm petri dishes to
which desired amount of different plant extracts and
10 ).!M butylated hydroxy anisole (BHA) were added
and mixed gently to form homogeneous solution.
Lipid peroxidation was initiated by adding 100 ).!l ferrous ammonium sulfate (0.5mm) and thereafter petri
dishes were incubated at 37°C for 30 min. Homogenate (l00 ).!l) was pi petted out for estimating lipid
peroxidation levels. The degree of lipid peroxidation
in terms of thiobarbituric acid reactive substance
(TBARS)_was measured following Beuge and Ause 3
method.
729
GOEl et al. : FREE RADICAL SCAVENG ING AND METAL CHELATION BY T1NOSPORA CORD/FOLIA
Direct antioxidant defence system
Scavenging of hydroxyl radicals - The radiati on
and FeS04 generated OH radi cals quenched by T.
cordiJolia (aq) were est imated using 2-deoxy ribose ~s
24
the marker substrate .
Scavenging of superoxide anions -The superoxide
quenching ability of T. cordiJolia (aq) was estimated
using nitroblue tetrazolium as the marker substrate 25 .
Indirect defence system
Chelation of Fe 2+ ions by Tinospora cordiJoliaConcentration of free iron ions (Fe 2+) was estimated
by usi ng chelating agent 2-2' bipiridyl26.
Single cell gel electrophoresis (Comet assay)
Prepa ration of solutions-Stock so lutions of
t B-OOH was prepared in standard buffered saline
(0.8 12 gil NaCI , 0.372 gil KCl , 0.366g/l NaHC0 3 and
0.9g11 glucose). Butylated hydroxy toulene (BHT)
was dissolved in minimum amou nt of 95 % ethanol
and 1-10 phenanthroline was di ssolved in dimethyl
sulfoxide (DMSO).
Cell treatment-Thymocytes (l x 106/ml ) were
suspended in standard buffered saline and centrifuged
at 200 g for 10 min at 4°C. Supernatant was di scarded
and the pellet was suspended in 1 ml of t B-OOH
(200 ~ prepared in standard buffered saline and
kept at 4°C for 30 min. After the incubation period the
cells were again pelleted by centrifugation (200 g for
10 min) the resulting supernatant was decanted and
the cells were washed in standard buffered saline to
wash off the remaining t B-OOH. Resulting thymocytes were used for assaying DNA damage using single cell gel electrophoresis (Comet assay). Antioxidants, iron chelators and different concentrations of T.
cordiJolia were added to cell preparation 5 min prior
to addition of t B-OOH.
Preparation of comets-DNA strand breaks in individual cells was detected using Single cell gel electrophoresis (Comet assay)27 with minor modifications.
After different treatments thymocytes were resus4
pended at a concentration of 2 x 10 /400 ~l of low
melting agarose in phosphate buffered saline containing EDT A and immediately pipetted on to a normal
agarose precoated microscopic slides and spread to
form a uniform layer. Thereafter slides were tran sferred to cold surface to hasten the gelling. After 5
min the slides were immersed in ice-cold lysis solution (2.5 M NaCl , 100 mM EDTA, 10 mM tri s, 1%
sodium lauryl sarcosine, 5% DMSO and 1% Triton
X-100 , pH 10) and left in refrigerator for 30 min. The
slides were transferred to an electrophoreti c tray containing freshly prepared alkali buffer (300 mM NaOH,
ImM EDTA, pH 13) for 20 min to allow DNA to unwind . The slide!> were electrophoresed at 1.5 V Icm fo r
another 20 min under similar conditions. The slides
were then washed thrice by adding O. IM tri s, pH 7.4
on to the slides and DNA was stained with propidium
iodide (50 ~M) for 10 min . The DNA was visuali zed
using LEITZ fluorescence microscope and photographs were taken using CCD camera. The length of
the comet tai l was measured using micrometer and at
least 50 comets were measured for each treatment and
averaged.
Statistical analysis-The data has been presented
as mean ± SD of 3 separate estimations and significance was tested using student t test; probability level
of < 5% was considered significant.
Results
Antioxidant properties
Inhibition of lipid peroxidation -Inhibition of
Fenton reaction mediated lipid peroxidation by diffe rent concentrations of herbal preparation as expressed
by inhibition of TBARS formation has been shown in
Fig. 1. T. cordiJolia rendered a dose-dependent inhibition of lipid peroxidation; 50% inhibition concentration was 1300 ~g/ml. With increasing doses the inhibition continuously enhanced but remained lower than
the level of 100% even at a concentration 2000 ~g/ml
(70%) . Standard anti-lipid peroxidative compound
butylated hydroxy anisole (BHA) rendered 50% inhibition at a concentration of 6.25 ~M.
" 00
.,81
II
~ 90
go
80
"tI
:l:
70
°
60
~
~ 50
-?°
40
'"
'0
30
f5
..,
20
;e
.<:
10
~
fft.
O~-~-~-~--_
25
50
100
200
__--~--~--~--~
500
1000
1500
2000
Cone of herbal extract in}Jgtml
Fig. I-Effect of different concentrations of T.cordiJolia (aq) on
FeS04 medi ated lipid peroxidation expressed as qu antity of MDA
nm Img proteins formed ; control value was taken as zero perce nt
inhibition of lipid peroxidation . Results are mean ± SO of three
parallel measurements (P < 0.05) and compared to control.
INDIAN J EXP BIOL, JUNE 2002
730
Direct antioxidant properties
80
OH radical scavenging
Fenton reaction mediated OH radical generationThe effect of varied concentrations of T. cordifolia on
scavenging of Fenton reaction mediated OR radical as
determined by inhibition of 2-deoxyribose degradation has been depicted in Fig. 2. It rendered a dose
dependent inhibition of 2-deoxyribose degradation
and maximum inhibition was at a concentratior'l of
1.5 mg/ml (P< 0.05). The IC 50 values for T. cordifolia
and reduced glutathione, mannitol, the standard OR
radical scavengers, were 700 Ilg/ml, 0.6 and 6.5 mM
respecti vel y.
Fixed radiation dose and varied herbal concentrations -Effect of different concentrations of herbal
preparation on inhibition of 2-deoxyribose degradation by a radiation dose (200 Gy) has been depicted in
Fig. 3. Aqueous extract of T.cordifolia inhibited
radiation induced 2-deoxy ribose degradation in a
dose dependent fashion with an IC 50 values of 700
Ilg/ml. IC 50 values for reduced gl utathione and
mannitol were 0.56 and 6.35 mM, respectively.
Superoxide anion scavenging -T. cordifolia exhibited inhibition of superoxide anions only at concentrations of 0.5 mg/ml and above (Fig. 4). IC 50
value for T.cordifolia was 2000 Ilg/ml, while for
CUS04 and MnCh, the standard superoxide anion
scavengers it remained 0.006 and 0.008 mM, respectively.
Indirect antioxidant properties
Bipiridyl assay-Effect of different concentrations
of herbal preparation on inhibition ofFe2+-bipiridyl
,gc 80
.
...
""r! 70
.'"
""50
~
'0
c
2
V)
a:
50
«
...e!
40
0
c 30
.51
~
.=
20
'#I!
10
a
100
50
200
1000
500
1500
2000
Conc of herbal extract injJglml
Fig. 3-Effect of varying concentrations of T. cordifolia (aq) on
scavenging of OH radicals induced by 200 Gy. Percent Inhibition
of chromogen formation was calculated with respect to control
(considered as zero inhibition). Results are mean ± SO of three
parallel measurements (P < 0.05) and compared with control.
60
g 50
1l:J
"0
!
40
...
o
3~
:n
z
c
o
~
:c
20
.=
~ 1(;
50
25
lOa
200
500
1000
1500
2000
Conc of extract in}Jglm l
Fig. 4-Effect of varied concentrations of T. cordifolia (aq) on
scavenging of chemically induced superoxide anions expressed as
% inhibition ofNBT reduction (chromogen) which was calculated
with respect to control (considered as zero inhibition). Results are
mean ± SO of three paralle l measurements (P < 0.05) and compared with control.
70
2
>.
60
"0
40
·c
30
'a 50
20
:
40
10
'0c
30
~
2C
.E
1('
~
0
:;0
:a
:c
0
~ 60
.~E:1
]50
.;:
~
0
co
c 70
.=
~
o
25
50
100
200
500
1000
1500
2000
Conc of extract in),g/ml
Fig. 2-Effect of varied concentrations of T. cordifolia (aq) on
Fenton reaction induced OH radical mediated 2-deoxyribose degradation as expressed by the inhibition of chromogen formation.
Results are mean ± SO of three parallel experiments (P < 0.05) and
compared with control.
:c
~
- - - -,,- - ---,-- 25
50
100
200
500
Conc of herbal extract in )'9/m l
Fi~. 5-Effect of varied concentrations of T.cordifolia (aq) on
Fe +-bipiridyl complex formation as expressed by % inhibition of
chromogen formation (P < 0.05) as compared to control.
GOEL et al.: FREE RADICAL SCAVENGING AND METAL CHELATION BY TlNOSPORA CORD/FOLIA
revealed by shortening of the length of the comet tail.
T.cordifolia at 200 Ilg/ml concentration did not allow
the increase in the length of the tail which remained
comparable to that of the normal thymocytes
(7.8 ± 1.0 11m; Fig. 6E).
(Chromogen) formation has been depicted in Fig. 5.
The extract inhibited the chromogen formation in a
dose-dependent fashion with a IC50 value of 150 Ilglrnl.
Single cell gel electrophoresis (Comet assay)Normal thymocytes without any treatment, on single
cell gel electrophoresis rendered a basal tail length of
6.2 ± 0.75 11m (Fig. 6A). t Butyl hydroperoxide at a
concentration of 200 11M induced severe strand breaks
in the DNA of mice thymocytes (Fig. 6B) enhancil'\g
the tail length to 35 ± 1.2 11m (P < 0.05). Figure 6C
reveals the effect of standard free radical scavenger,
butylated hydroxy toulene (BHT) on DNA strand
breaks induced by t B-OOH. BHT could not inhibit
the strand breaks induced by t B-OOH (32.4 ±0.8 11m).
However, the standard iron chelator, 1-10 phenanthroline (25 11M) completely abolished the inhibition
of strand breaks (6.3 ± 1.2 11m; Fig 6 D). DNA strand
breaks induced by t B-OOH were inhibited by different concentrations of T.cordifolia (50, 100 and
200 Ilg/ml) in a dose-dependent fashion (P < 0.05) as
40
35
1i
30
i
;; 25
I
E
~
The comparison of tail formation as observed
through comet assay reveals that T. cordifolia rendered iron chelation efficiently in a dose-dependent
manner (Fig. 7).
I
iJ
o
~o
I
15
JI
~j 10 ~
I
5 .
A
B
c
o
E
F
G
Fig. 6-Effect of treatment with various agents on the length of
the comet tail. A) cells without any treatment ; B) 200 11M
t B-OOH ; C) 50 Ilg/ml T. cordifolia (aq) + 200 11M t B-OOH; D)
100 Ilg/ml T. cordifolia (aq) + 200 11M t B-OOH ; E) 200 Ilg/ml T.
cordifolia (aq) + 200 11M t B-OOH ; F) 200 IlM BHT + 200 11M t
B-OOH ; G) 25 11M 1-10 Phenanthroline + 200 11M t B-OOH.
Discussion
Scavenging of reactive oxygen species by an agent
plays a pivotal role in radioprotective action against
low LET radiation I. Extract of T. cordifolia (aq) under
present study has been observed to scavenge both radiation and Fenton reaction generated OH radicals.
Fenton reaction mediated 2-deoxyribose degradati on
is more vigorous than radiation induced OH radicals.
Extracts (in polar solvents) of plants like Podophyllum hexandrum, Ocimum scanctum, Hippophae
rhamnoides and similarly alcoholic preparation of T.
cordifolia rendered more inhibition of radiation mediated 2-deoxyribose degradation than Fenton reaction
mediated degradation (Table 1). The aqueous extract
of T. cordifolia rendered similar IC50 values for both
Fenton and radiation mediated 2-deoxyribose degradation. It indicates that the aqueous extract of T.
cordifolia apart from scavenging the end product,
(OH radicals) could be acting at some other juncture
(s) in the Fenton reaction cascade. Fenton reaction
mediated OH radical production involves generation
of superoxide anions which either spontaneously or
enzymatically dismutates to hydrogen peroxide that in
tum generates OH radicals in presence of iron ions.
Any substance or agent that can scavenge superoxide
anions or chelate metal ions can reduce the generation
of OH radicals. These two points of the reaction could
be the most probable targets for strong inhibition of
Table I-Comparison of preparations from different plants manifesting Fenton reaction and radiation mediated 2deoxyribose degradation, expressed as 50% inhibition concentrations (lC 50)
Name of the extract
Inhibition of Fenton reaction
mediated 2-DR degradation (lC 50)
(Ilg/ml )
Radiation induced OH radical
mediated 2-DR degradation (IC 50 )
(Ilg/ml )
Podophyllum hexandrum (aq)
. 970
700
Podophyllum hexandrum (ale)
Hippophae rhamnoides (ale)
200
320
200
700
200
<25
<25
<25
700
<25
Ocimum sanctum (aq)
Tinospora cordifolia (aq)
Tinospora cordifolia (ale)
731
732
INDIAN J EXP BIOL, JUNE 2002
Fenton reaction mediated 2-deoxyribose degradation
by T. cordifolia. The lack of significant efficiency of
extract to scavenge superoxide anions (Fig. 4) swings
the balance to iron as most probable target in the Fenton reaction cascade.
Single cell gel electrophoresis or comet assay is a
sensitive, simple and reliable technique to determine
DNA damage at si ngle cell level27. When cells embedded in agarose gel get exposed to electric cun'eni,
the negatively charged DNA migrates towards anode
at a particular velocity depending upon the mol.wt of
the DNA. The fragments of DNA migrate faster, ow-
ing to their lower mol.wt. The differential movement
of DNA of different molecular weight leads, to a
smear formation resembling a comet with head and
tail. Tertiary butyl hydroperoxide in presence of iron
ions generates reactive oxygen species like OH radicals which in turn interact with DNA and induce
strand breaks. The mechanism by wh ich I B-OOH
induces strand breaks is insensitive to free radical
scavenging antioxidants but can be completely abolished by iron chelators 28, 29. Th is system offers a reliab le method to assess the iron chelating ab ility of an
agent 30 . The chelation of metal ions results in reduc-
Fig. 7 - Thymocytes treated with different chemical agent s
and subjected to alkaline co met assay and we re viewed at
400 x under fluorescent microscope. A) Th ymocytes with
out any treatment (control) ; B) 200 11M t B-OOH ; C) 200
11M BHT+200 11M t B-OOH ; D) 25 11M 1-10 Phenanthroline + 200 11M t B-OOH ; E) 200 I1g/ml T. cordi/alia
(aq) + 200 ~lM t B-OOH.
GOEL et al.: FREE RADICAL SCAVENGING AND METAL CHELATION BY TINOSPORA CORDIFOLIA
ion of the OH radical generated damage and as a
onsequence the length of the comet tail gets shorter. .
'he present study with aqueous extract of T. cordifoia explicitly reveals the property of chelating Iron
ons (Figs 6 and 7).
2-2' bipiridyl on combining with free Fe2+ ions
orms Fe2+-bipiridyl (pink chromogen) which can be
luantified spectrophotometrically. This assay has also
)een widely used in metal chelating studies, since
:helation of iron ions by any agent will reduce the
luantity of the chromogen and correspondingly the
>ptical density. The results (Fig. 5) obtained with this
:ystem also corroborate comet assay studies that
lqueous extract of T. cordifolia is a strong iron chelator.
Lipid peroxidation and oxidative stress can be inluced by a variety of agents capable of generating
'eactive oxygen species. Aqueous extract of T. cordi'alia has been shown to inhibit lipid peroxidation
Fig. 1) because of both direct antioxidant properties
scavenging of free radicals) and indirect antioxidant
nechanisms (chelation of metal ions). These free
'adicals scavenging and metal chelation capabilities
)f T. cordifolia must be acting in a competitive and
:omprehensive mode to amelioarate the radiation iniuced oxidative stress. These mechanisms along with
)ther properties like immunomodulation and cell proiferation must be responsible for radioprotective
nanifestation as revealed by survival against radiation
nduced mortality.
The direct and indirect antioxidant mechanisms can
Je crucial in case of pathological conditions like acute
.eukemia, haemochromatosis, and rheumatoid arthriis. Therefore various fractions of T. cordifolia extract
leed to be isolated and bio characterized for their di:ect and indirect antioxidative potential. This may
Jermit exploitation of the plant for various clinical
Jses.
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