Industrial Crops and Products 74 (2015) 434439

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Industrial Crops and Products

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Biological efcacy and radical scavenging potential of shikonin in

Arnebia benthamii (Wall ex. G Don) Johnston
Javid A. Parray , Rehana Hamid, Azra N. Kamili, Nowsheen Shameem, Sumira Jan,
Bashir A. Ganai
Centre of Research for Development, University of Kashmir, Srinagar 190006, J&K, India

a r t i c l e

i n f o

Article history:
Received 1 December 2014
Received in revised form 16 April 2015
Accepted 17 April 2015
Keywords:
Antioxidant
HPLC
Kahzaban
Shikonin
Dye
DNA damage

a b s t r a c t
Free radicals produced due to various environmental stresses and numerous metabolic processes ultimately lead to complete cell impairment. To combat this cellular damage organisms require dynamic
defense system. In addition to inherent defense machinery, we can supplement the defense system of
organisms with natural resources like herbs. The main objective of our research had been the investigation of the radical scavenging potential of folklore medicinal herb Arnebia benthamii and its competence
in protection against DNA damage. The presence of shikonin (5,8-dihydroxy-2-(1-hydroxy-4-methyl-3pentenyl)-1,4-naphthoquinone) in the plant was conrmed by HPLC quantication from its roots. The
ethyl acetate extract of 50 g/ml yields the 5.19 g/g shikonin. This ethyl acetate extract exhibited complete protection of DNA by quenching of hydroxyl radicals. The activity of plant extract was also compared
with the synthetic shikonin which also validates our study that the presence of dye like substance can
also be potential candidate for the augmenting antioxidant defense system.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Nutraceuticals refers to assets of medicinal herbage which
could harbor extensive variety of nutritional and pharmaceutical
adjuncts which helps to regulate good health and combat disease (Ivanova et al., 2005). The impending function of curative
herbs in disease anticipation has been ascribed to their microbial inhibition or the antioxidant properties of their constituents
(Quinming et al., 2010). Reactive oxygen species (ROS) like OH,
O2 , H2 O2 , O3 , HOCI, RO2 , and RO are generated during various
metabolic activities as well as by environmental factors with in
cells (Balz, 1994). Once initiated these free radicals get involved in
chain reaction with steady molecular species and the compounds
thus formed have longer stability in body and increase the potential for cellular damage causing serious disorders (Sun, 1990). After
the propagation of radicals, the termination or neutralization of

Abbreviations: DPPH, 1,1-diphenyl-2-picryl-hydrazyl; EA, ethyl acetate; NWH,

North Western Himalaya; NBT, nitro blue tetrazolium chloride; ROS, reactive oxygen
species; SOD, superoxide dismutase.
Corresponding author at: Centre of Research for Development/Department of
Environmental Science, University of Kashmir, Srinagar 190006, India.
Tel.: +91 9797878884.
E-mail addresses: javid06@gmail.com, cordjavid06@gmail.com (J.A. Parray).
http://dx.doi.org/10.1016/j.indcrop.2015.04.040
0926-6690/ 2015 Elsevier B.V. All rights reserved.

free radicals is achieved by antioxidants or enzymatic mechanism

(Halliwell, 1994). However, it is necessary to maintain the balance
rate of the radical generation and their termination by antioxidant supplements derived from medicinal plants (Helmut, 1997).
Most phytochemicals derived from plants have antioxidant activity and protect our cells against oxidative damage and reduce
the risk of developing certain types of cancer (Jiang et al., 2006).
In this regard, the Arnebia benthamii was screened out among
the immense medicinal herbage that thrive across North Western
Himalaya (NWH) to explore and evaluate its potential as nutraceutical. This medicinal herb has extensive pharmaceutical properties
like antibacterial, antifungal, anti-inammatory, wound-healing
properties. It yields red colored dye shikonin sold under market
name Gaozaban and Ratanjot (Kashiwada et al., 1995; Kaul, 1997;
Ganie et al., 2012). Besides being medicinal herb it is used as cardio tonic and febrifuge (Kaul, 1997). The tribal people often used
this herb for the treatment of pharyngitis and laryngitis (Dar and
Khuroo, 2013). Though, the antioxidant potential of shikonin and
its derivatives was explored by numerous researchers (Nishizawa
et al., 2005; Jin and Bai, 2012) however, no reports are found on
bio efcacy of shikonin from A. benthamii NWH via HPLC. In this
context, the current investigation is the rst report in this region
for evaluation of bioactivity and quantication of root ethyl acetate
extract of this plant.

J.A. Parray et al. / Industrial Crops and Products 74 (2015) 434439

2. Materials and methods

435

of reaction mixture was measured at 517 nm after incubation for

30 min.

2.1. Chemicals required

All the chemicals like procurement of standard shikonin, HPLC
grade solvents like methanol, water and ethyl acetate was made
through authentic rms including Sigma Aldrich, Merck and Himedia Mumbai Pvt. Ltd.
2.2. Collection and identication of plant material
A. benthamii (wall ex. G Don) Johnston roots were collected
from Sinthan Top (3748 m), Kashmir Himalaya, J&K, India in
JulySeptember, 2013. Sampling was carried out immediately after
inorescence formation and plants were collected manually in bulk
from the area. The Kashmir University Herbarium (KASH), Center
of Plant Taxonomy identied herb under accession No. 1748.
2.3. Preparation of plant extracts
Dried roots of A. benthamii were nely powdered and 50 g of
this powder was rendered to thorough mining via Soxhlet apparatus. The extract was concentrated via rotary evaporator, which
were later completely dried, weighed and kept for further usage in
sterilized sealed vials at 4 C.
With the aim of optimization in the extraction of samples, effects
of different extraction solvents were premeditated via ultrasoundassisted extraction as a fast and procient extraction tool. Five
solvents with diverse polarity such as hexane, chloroform, ethanol,
ethyl acetate, and methanol had been selected based on prior studies (Papageorgiou et al., 1999; Bozana et al., 1999; Hu, 2004; Sharma
et al., 2009). Further, it has been reported from previous studies
that shikonin was rst isolated as acetate from roots of Lithospermum erythrorhizon by Japanese chemists (Majima and Kuroda,
1922). Moreover, they had conrmed that naphthoquinones exist
as racemic mixture of two isomers alkannin (Natural Red) and
shikonin which were identied as enantiomers (Brockmann, 1936).
He has further conrmed that the ethylacetate was able to resolve
the both enantiomers effectively in A. benthamii without overlapping of peaks.
2.4. Chromatographic system
The instrument used was Nexera UHPLC (130 MPa) system
consisted of a quaternary pump, auto sampler, thermocouple column compartment and diode array detector. Data was acquired
using lab solution software. The chromatographic separation was
carried out on RP C18 column (250 mm 4.6 mm; particle size
5 m; Merck, Germany) at 30 C. Samples were prepared by dissolving crude extracts and standard in methanol (HPLC grade),
followed by ltration through 0.2 micron nylon lter. The mobile
phase consisted of water (A) and methanol (B) run in an isocratic
mode 85:15 at a ow rate of 1 ml/min. Total run time for each sample was 25 min. Injection volume was 5 l. The detection was made
at 222 nm. By using peak height and area of the standard (Shikonin,
Sigma-Aldrich), the amount of shikonin was calculated in sample.
2.5. Anti-oxidant activity
2.5.1. DPPH assay
Quantitative measurement of radical scavenging properties of
extracts was carried out according to the method (Parray et al.,
2015). 0.1 mM solution of 1,1-diphenyl-2-picryl-hydrazyl (DPPH )
in methanol was prepared and 1 ml of this solution was added to
3 ml of plant extract (100300 g/ml) and shikonin (300 g/ml).
-Tocopherol was used as a reference antioxidant. Discoloration

2.5.2. Superoxide anion radical scavenging activity

Measurement of superoxide anion scavenging activity of the all
extracts based on the method described by (Liu et al., 1997) with
slight modication. 100 l riboavin solution (20 g), 200 l EDTA
solution (12 mM), 200 l methanol and 100 l nitro-blue tetrazolium (NBT) solution (0.1 mg) were mixed in test tube and reaction
mixture was diluted up to 3 ml with phosphate buffer (50 mM). The
absorbance of solution was measured at 590 nm using phosphate
buffer as blank after illumination for 5 min. Different concentrations (50 l) i.e. 100 g, 200 g, 300 g of plant extracts were taken
and diluted up to 150 l with methanol. To each of these, add
100 l riboavin, 200 l EDTA, 200 l methanol and 100 l NBT
was mixed in test tube and further diluted up to 3 ml with phosphate buffer. Absorbance was measured after illumination for 5 min
at 590 nm. Decreased absorbance of the reaction mixture indicates
increased super oxide anion scavenging activity.
2.5.3. Hydroxyl scavenging activity-deoxyribose assay
The colorimetric deoxyribose (TBARS) method by Padmaja et al.
(2011) was applied as the reference procedure of comparison
for determining the hydroxyl radical scavenging activity of plant
extracts. The reaction mixture for the deoxyribose assay contained in a nal volume of 1 ml of the following reagents: 200 l
KH2 PO4 -KOH (100 mM), 200 l deoxyribose (15 mM), 200 l ferric chloride (500 M), 100 l EDTA (1 mM), 100 l ascorbic acid
(1 mM), 100 l hydrogen peroxide (10 mM), and 100 l of plant
sample (100300 g/ml). Reaction mixtures were incubated at
37 C for 1 h. At the end of the incubation period, 1 ml of 1%
(w/v) thiobarbituric acid (TBA) was added to each mixture followed by the addition of 1 ml of 2.8% (w/v) trichloroacetic acid
(TCA). The solutions were heated on a water-bath at 80 C for
20 min to develop pink colored malondialdehydethiobarbituric
acid (MDATBA) adduct and the absorbance of the resulting solution (total volume = 3 ml) was measured at 532 nm.
2.5.4. Ferric thiocyanate (FTC)
Ferric thiocyanate (FTC) assay was done following the method
of Kikuzaki and Nakatani (1993). 2 ml of extract (100300 g/ml)
was mixed with 2.88 ml of linoleic acid (2.51%, v/v in 4 ml of 99.9%
ethanol), 0.05 M phosphate buffer (pH 7.0, 8 ml) and distilled water
(3.9 ml). The whole reaction mixture was followed by 0.1 ml of 30%
ammonium thiocyanate. Immediately, after 3 min, 0.1 ml of 3.5%
(v/v) HCl was added to the reaction mixture, the absorbance at
500 nm of the resulting solution was measured and it was recorded
again after 24 h, until the day when the absorbance of the control
reached the maximum value. -Tocopherol was used as reference
antioxidant substance.
2.5.5. Thio-barbutiric acid assay
Thiobarbituric acid was added to the reaction mixture which
interacts with malondialdehyde and TBARS produced was measured spectrophotometrically (Kishida et al., 1993). To 2 ml of
reaction mixtures of ferric thiocyanate assay, 2 ml of TCA (20%) and
2 ml TBA (0.67%) was added and kept in boiling water for 10 min
and were later on cooled under tap water. The reaction mixture
was centrifuged at 3000 rpm for 20 min and the supernatant was
read at 500 nm. -Tocopherol was taken as reference antioxidant
substance.
2.5.6. Lipid peroxidation method
A modied thiobarbituric acid reactive species (TBARS) assay
(Soobrattee et al., 2008) was used to measure the lipid peroxide

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J.A. Parray et al. / Industrial Crops and Products 74 (2015) 434439

Table 1
HPLC method variation parameters of shikonin in ethylacetate extract of Arnebia benthamii.
Compound

Linearity range (g/ml)

Regression equationy = ax + b

r2

Shikonin

3.12550

y = 606.9x 872.4

0.999

5.19 0.03

Amount

Recovery (%)

RSD%(n = 3)

LOD (g/g)

LOQ (g/g)

85.9

0.540

0.0152

0.0462

RSD, relative standard deviation; r , coefcient of determination; LODs, limits of detection; LOQ, limit of quantitation; y, peak area; x, concentration.
a
Mean amount of shikonin (g/g) based on dry weight of plant extract (n = 3).

formed using the egg yolk homogenate as lipid rich media. Malondialdehyde, a secondary end-product of oxidation of polyunsaturated
fatty acids reacts with two molecules of thiobarbituric acid (TBA)
yielding a pinkish red chromogen. Egg homogenate (0.5 ml of 10%,
v/v) and 0.1 ml of plant extracts were added to a test tube and volume was made up to 1 ml with distilled water. The peroxidation
was induced by adding 0.05 ml of 0.07 M FeSO4 . The reaction mixture was vortexed and then heated at 95 C for 60 min. After cooling,
5 ml of butanol was added to each tube and centrifuged at 3000 rpm
for 10 min. The absorbance of the upper organic layer was measured
at 532 nm.
2.6. Calculations
The capacity to scavenge the radicals was calculated using the
following equation:
% inhibition =

Ac As
100
Ac

where Ac is the absorbance of the controlled reaction (reaction

mixture without any antioxidant substance) and As is absorbance
of reaction mixture with reference substance or plant extract. The
whole experiments were repeated thrice.
2.7. DNA damage assay
Hydroxyl radical generated by Fenton reaction were used to
induce oxidative damage to DNA (Sun et al., 2004). The reaction mixture (15 l) contained 25 mg of calf thymus DNA (Sigma)
in 20.0 mM phosphate buffer saline (pH 7.4) and 500 g of test
compounds were added and incubated with DNA for 15 min at
room temperature. The oxidation was induced by treating DNA
with 1 l H2 O2 30 mM, 1 l 20 mM ferric nitrate and 1 l 100 mM
ascorbic acid and incubated them for 1 h at 37 C. The reaction
was terminated by the addition of loading dye (40% sucrose and
0.25% bromophenol blue) and the mixture was subjected to gel
electrophoresis in 0.7% agarose/TAE buffer runat 100 V. DNA as
visualized by Gel Doc system.
3. Results and discussion
3.1. HPLC analysis
In the current studies, an appropriate, consistent, quick and easy
HPLC method was recognized for competent partition of shikonin
from the crude EA extract. The processed plant extract was instilled
to assess the resolution of diverse components present in it. The
acidic mobile phase was found mainly appropriate for the characteristic chromatogram improvement with smooth baseline. The
mobile phase (consisting of water and methanol) was very favorable, because its use resulted in short time analysis (retention times
7.5 min) and single symmetrical peaks resolution for shikonin. The
recognition of the marker compound from the plant extract was
well-known by contrast of the UV spectra and retention time with
that of authentic standard. HPLC method was validated for various parameters as per ICH guidelines including specicity, linearity
range, accuracy, precision, LODs, and LOQ. The specicity of an analytical method is its capability to evaluate the analyte evidently

in the occurrence of additional components to be likely present

in analyte. Lack of any intrusive peak signied that the method
was specic. We have further evaluated peak purity by evaluating
the spectra at three diverse stages, i.e. peak start, peak apex, and
peak end. The calibration curves were established to be linear in
the range of 3.12550 g/ml for shikonin. Regression equation and
coefcient of correlation ranging from 0.9985 to 0.9997 demonstrated an excellent linearity reaction for developed method and
are presented in Table 1. % RSD of the linear equation was obtained
as 0.540 for shikonin. LODs obtained for shikonin was 0.0152 g/g
while the LOQ obtained was 0.0462 g/g as represented in Table 1.
This indicated that the planned method exhibits a good sensitivity for the quantication of shikonin. The shikonin content of
5.19 g/g from 50 g/g ethyl acetate extract with recovery (85.9%)
was obtained (Fig. 1a and b).
It was obvious from the results that the suitable isocratic HPLC
method was developed for separation of pure shikonin from A. benthamii. Though shikonin has also been isolated from L. erythrorhizon
using HSCCC (Lu et al., 2004), however it is the rst report of determination of shikonin from A. benthamii from this region.
3.2. Antioxidant activity
The EA extract (100, 200 and 300 g/ml), shikonin (300 g/ml)
and -tocopherol (300 g/ml) were used in all the antioxidant
methods. The antioxidant activity of EA extract (100 g/ml) was
much lower than shikonin and -tocopherol but its activity
increased signicantly with the increment in absorption up to
300 g/ml. All the methods provide a better assessment of scavenging properties of the tested substances and results revealed
that inhibitory activity was concentration dependent (Parray et al.,
2015).
Free radical scavenging prospective of EA extracts at diverse
concentration was tested by the DPPH method. Antioxidant substance reacts with DPPH, which is a stable free radical, and converts
it to 1,1-diphenyl-2-picryl-hydrazine (Ting et al., 2008) and absorption strength is reduced by discoloration (yellow color) and more
the discoloration and more is the reducing ability. The percent
inhibition of EA extracts was 37%, 55% and 78% respectively. Comparatively, shikonin showed 72% inhibition while as -tocopherol
exhibited 65% inhibition of radicals at higher concentration of
300 g/ml (Table 2). Similar type of work has also been carried out
on Arnebia spp by (Orhan et al., 2008; Ganie et al., 2012). The superoxide radicals were produced by lighting up a solution enclosing
riboavin. NBT, a dye is reduced by superoxide radicals to diformazan and is detected by change in color in presence of extract
(Liu et al., 1997) and the decrease in absorbance of the reaction
mixture specied amplied super oxide anion scavenging activity.
The EA extracts were found to be more effective in scavenging the
superoxide radicals with percent inhibition of 40%, 60% and 83%
respectively. Shikonin showed 78% inhibition and -tocopherol
exhibited 83% inhibition of radicals at 300 g/ml concentration
(Table 2). Our results conrmed the presence of shikonin and its
derivatives in root extract of A. benthamii which is the possible
reason for effective scavenging or chelating of superoxide radicals
(Kessler et al., 2003).
Hydroxyl radical is exceptionally unstable species formed in biological systems. This radical has the ability to bond with nucleotides

J.A. Parray et al. / Industrial Crops and Products 74 (2015) 434439

437

Fig. 1. Representative HPLC chromatogram for HPLC determination of shikonin. (a) Standard chromatogram of shikonin; (b) HPLC chromatogram of root EA extract.

and shikonin (90% inhibition) at 300 g/ml concentration tested.

The inhibitory activity of -tocopherol was low 78% (Table 3). In
TBA method, formation of malondialdehyde is basis for evaluating
the extent of lipid peroxidation. At low pH and high temperature,
malondialdehyde which is the end product of lipid peroxidation
binds TBA to form red colored complex (Table 3). The concentration
used were 100300 g/ml same as FTC method because this assay
is based on same samples. The FTC method measures the amount
of peroxide produced during the initial stage of lipid peroxidation.
Subsequently, at later stage of the oxidation, peroxides decompose
to form carbonyl compounds that are measured by the TBA method.
The plant extract and shikonin exhibited signicant 83% and 82%
inhibition of radicals at 300 g/ml than -tocopherol (66% inhibition) (Table 3). Our study showed that EA root extract form a strong
hydroxyl radical, scavenging DPPH and superoxide anion and inhibition of yolk lipid peroxidation in a dose-dependent manner as
reported earlier (Parray et al., 2010, 2011) and its possible mechanism may be presence of the naphthoquinone substances like
shikonin that helps to capture lipid peroxidation chain reactions
triggered by reactive oxygen species, reduce the lipid peroxidation
chain length, block or slow down the lipid peroxidation (Huang
et al., 2006). In addition, the ability to scavenge the DPPH radical is related to the inhibition of lipid peroxidation (Rekka and
Kourounakis, 1991) which may be either due to the individual or
additive effect of the phytoconstituents present in it (Pirbalouti

in DNA and can cause strand breakage, which leads to carcinogenesis, mutagenesis and cytotoxicity (Kumarappan et al., 2012). The OH
radical is identied as major cause of DNA damage by degradation
of deoxyribose moiety (Kumar, 2011; Parray et al., 2015). However,
the scavenging or chelation of radicals by any substance attributes
the antioxidant capacity of that particular substance (Orhan et al.,
2008). In our study, EA extract exhibited signicant scavenging
activity of OH radicals. The EA extract also showed protective effect
of DNA from OH radicals in increasing order of concentration as 54%,
74% and 89% respectively. However, shikonin and -tocopherol
exhibited 80% and 75% inhibition of radicals respectively (Table 2).
Ganie et al. (2012) reported that the methanolic extract of A. benthamii exhibited 72% protection effect at 800 g/ml concentration.
Ferrous iron can initiate lipid peroxidation by the Fenton reaction as well as accelerating peroxidation by decomposing lipid
hydroperoxides into peroxyl and alkoxyl radicals (Christopher
et al., 1995). The inhibition of FeSO4 induced lipid peroxidation in
presence of EA extract was remarkably high about 90% at 300 g/ml
conc. The activity of shikonin and -tocopherol at 300 g/ml concentration was comparatively low with 78% and 68% inhibition
respectively (Table 2).
The FTC evaluates the effect of reference antioxidant and tested
substances on alleviating peroxidation of polyunsaturated fatty
acids, linoleic acid. The % protective effect of linoleic acid peroxidation was found almost in equivalent for EA extract (92% inhibition)

Table 2
Scavenging activity of ethyl acetate extracts of A. benthamii.
% scavenging of radicals
Shikonin300 g/ml

Ethyl acetate extract

100 g/ml
DPPH assay
SOD assay
Hydroxyl assay
Lipid peroxidation assay

37
40
b
54
c
65
a

2.89
4.94
4.05
4.36

200 g/ml
a

55
60
c
74
d
84
b

4.36
4.73
4.59
3.47

-Tocopherol300 g/ml

300 g/ml
a

78
83
c
89
c
90

3.52
3.06
5.04
4.94

72
78
bc
80
abc
78
ab

1.32
5.04
3.60
3.55

65
83
b
75
a
68
c

2.51
2.80
4.86
7.53

EA, ethyl acetate.

Values are represented as mean SD (n = 3), Data was analyzed by ANOVA using Duncans multiple range test (SPSS17.0); the values with superscript along the columns are
statically signicant at P < 0.005.

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J.A. Parray et al. / Industrial Crops and Products 74 (2015) 434439

Table 3
Ferric thiocyanate and thiobarbituric acid activity of ethyl acetate extracts of A. benthamii.
% scavenging of radicals

Tested
substances

100 g/ml
1st day
EA
100 g/ml
EA
200 g/ml
EA
300 g/ml
Shikonin
300 g/ml
-Tocopherol
300 g/ml

200 g/ml
2nd day

300 g/ml
3rd day

100 g/ml
1st day

200 g/ml
2nd day

300 g/ml
3rd day

56 4.09

63 70

74 5.56

43 4.35

58 7.63

65 3.09

69 5.83

75 4.58

82 4.16

54 4.58

67 2.64

75 4.50

81 4.93

89 2.64

92 3.055

73 5.23

80 5.56

83 4.35

75 5.04

86 5.29

90 4.58

66 4.65

74 5.34

82 5.29

55 3.05

70 3.75

78 2.17

49 4.87

58 6.08

66 4.67

EA, ethyl acetate.

Values are represented as mean SD (n = 3), Data was analyzed by ANOVA using Duncans multiple range test (SPSS17.0); the values with superscript along the columns are
statically signicant at P < 0.005.

Table 4
IC50 determination of ethyl acetate extract and shikonin.
Method

IC50 g/ml
Ethyl acetate
extract

DPPH
SOD
Hydroxyl scavenging
Lipid peroxidation
Ferric thiocyanate
Thiobarbituric acid

e
d

160
150
c
95
a
40
a
35
b
54

2.89
2.12
3.32
2.15
1.92
1.73

Shikonin
d

185
190
c
155
c
158
a
48
b
60

3.85
2.98
2.32
2.01
4.20
2.76

-Tocopherol
f

210
160
140
e
183
a
90
b
115

d
c

2.11
1.87
1.55
1.82
2.65
2.12

Values are represented as mean SD (n = 3). Data was analyzed by ANOVA using
Duncans multiple range test (SPSS17.0); the values with superscript along the
columns are statically signicant at P < 0.005.

et al., 2011). The A. benthamii extracts seems to have good potential

as a source for natural antioxidants.
The inhibitor concentration for scavenging of radicals (IC50
g/ml) was determined for all the antioxidant methods. From the
results, it was observed that IC50 values varied with the substance
as well as with the type of method employed. The ethyl acetate
extract showed lowest value of 35 g/ml in scavenging of thiocyanate radicals followed by 40 g/ml in scavenging 50% peroxide
radicals (Table 4). The shikonin was also effective in scavenging
thiocyanate radicals at 40 g/ml.
We have observed that the reaction mixture (ferric nitrate,
ascorbic acid and H2 O2 ) completely induce DNA strand breaks in

calf thymus DNA. In our study, ethyl acetate extract (300 g) were
found to scavenge the hydroxyl radicals generated by Fenton reaction to offered complete protection to DNA damage in calf thymus
DNA (Lane 4). Our results indicate that ethyl acetate extract showed
strong DNA damage protection compared with that of shikonin
used (Fig. 2). Our study is supported by few reports who have also
observed the same DNA protection effect (Ganie et al., 2012, 2014).
4. Conclusions
The results of this investigation, which evaluates the antioxidant
activity of EA root extract of A. benthami, demonstrates that it might
be proposed as a dietary supplement or traditional drug for the prevention and/or treatment of conditions that occur due to oxidative
damage and can protect DNA damage by hydroxyl radicals.
Conict of interest
The authors hereby declare that they have no conict of interest.
Authors contributions
All authors declare the equal contribution in crafting experiments, investigation and interpretation of data. All authors have
studied and approved the nal manuscript.
Acknowledgments
This study was supported by DST, GoI, New Delhi funded project
vide letter no: DST/SSTP/J&K/11/146(G) Dtd.26/04/2012, the assistance of which is highly acknowledged. The authors are thankful
to Taxonomy Department, University of Kashmir and IIIM, Sanat
nagar J&K, India for identifying the Plant and helping in carrying
HPLC analysis respectively.
References

Fig. 2. Protective effect of DNA through scavenging of radicals by EA extract of

Arnebia benthamii, shikonin and -tocopherol. Lane 1: Native Calf thymus DNA.
Lane 2: BHT + reaction mixture + native calf thymus (ct) DNA. Lane 3: Native calf
thymus (ct) DNA shikonin (300 g) + reaction mixture. Lane 4: Native calf thymus
(ct) DNA + EA extract (300 g) + reaction mixture. Lane 5: Native calf thymus (ct)
DNA + reaction mixture.

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