Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens.

Abstract

Rubia cordifolia L. (Rubiaceae) is an important medicinal plant used in the Ayurvedic medicinal system. Its use as a traditional therapeutic has been related to the treatment of skin disorders and cancer. Besides its medicinal value, anthraquinones from this plant are used as natural food colourants and as natural hair dyes. Dyes derived from natural sources have emerged as important alternatives to synthetic dyes. Alizarin (1,2-dihydroxyanthraquinone) was isolated and characterized from R. cordifolia L. and evaluated for its antigenotoxic potential against a battery of mutagens viz. 4-nitro-o-phenylenediamine (NPD) and 2-aminofluorene (2-AF) in Ames assay using TA98 tester strain of Salmonella typhimurium; hydrogen peroxide (H(2)O(2)) and 4-nitroquinoline-1-oxide (4NQO) in SOS chromotest using PQ37 strain of Escherichia coli and in Comet assay using human blood lymphocytes. Our results showed that alizarin possessed significant modulatory role against the genotoxicity of mutagens.

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Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens
Prabhjit Kaura, Madhu Chandela, Subodh Kumarb, Neeraj Kumarc, Bikram Singhc, Satwinderjeet Kaura,*
aDepartment of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India
bDepartment of Chemistry, Guru Nanak Dev University, Amritsar, Punjab 143005, India
cDivision of Natural Plant Product, Institute of Himalayan Bioresource Technology, Palampur, HP 176061, India
a r t i c l e i n f o
Article history:
Received 6 August 2009
Accepted 14 October 2009
Keywords:
Rubia cordifolia
Alizarin
Antigenotoxic
Ames test
Comet assay
SOS chromotest
a b s t r a c t
Rubia cordifolia L. (Rubiaceae) is an important medicinal plant used in the Ayurvedic medicinal system. Its
use as a traditional therapeutic has been related to the treatment of skin disorders and cancer. Besides its
medicinal value, anthraquinones from this plant are used as natural food colourants and as natural hair
dyes. Dyes derived from natural sources have emerged as important alternatives to synthetic dyes. Aliz-
arin (1,2-dihydroxyanthraquinone) was isolated and characterized from R. cordifolia L. and evaluated for
its antigenotoxic potential against a battery of mutagens viz. 4-nitro-o-phenylenediamine (NPD) and
2-aminofluorene (2-AF) in Ames assay using TA98 tester strain of Salmonella typhimurium; hydrogen
peroxide (H2O2) and 4-nitroquinoline-1-oxide (4NQO) in SOS chromotest using PQ37 strain of Escherichia
coli and in Comet assay using human blood lymphocytes. Our results showed that alizarin possessed
significant modulatory role against the genotoxicity of mutagens.
? 2009 Elsevier Ltd. All rights reserved.
1. Introduction
It is well established that mutations in somatic cells play a key
role in cancer initiation and other stages of the carcinogenesis pro-
cess (De Flora and Ferguson, 2005). A large number of mutagens
have been identified and are known to be potentially deleterious
to human health. To minimize human exposure to different envi-
ronmental mutagens, it is pertinent to identify various mutagenic
substances as well as to enhance the exposure to antimutagenic
agents, such as those naturally occurring in plants as secondary
metabolites (Edenharder et al., 2002; Ikuma et al., 2006; Jeong
et al., 2006). Plant derived medicines are based on the fact that
they contain natural substances whose consumption may provide
health benefits and diminish illness. Interactions among bioactive
compounds are complicated and ubiquitous, making the detection
and identification of natural mutagens and antimutagens impor-
tant (Greenwald et al., 2001). Knowledge of sources of natural anti-
mutagens will help people to make selections of food or drink
containing substantial amounts of active compounds, thereby
enhancing their health status. In the last two decades, a wide range
of evidence from epidemiological and laboratory studies have
demonstrated that some plants eaten whole, or some of their
active principles taken in isolation, have substantial protective
effects against human carcinogenesis and mutagenesis (Surh and
Ferguson, 2003). Several plant extracts have proved to contain a
wide variety of antimutagenic/antigenotoxic substances (Versha-
eve et al., 2004; Scassellati-Sforzolini et al., 1999; Kaur et al.,
1998, 2000, 2001, 2009) and some can prevent cancer (Nishino,
1998; Nagpal et al., 2000; Saleem et al., 2005). Rubia cordifolia L.
is a well known medicinal plant and is commonly known as Indian
madder, belonging to the family Rubiaceae. It has been used widely
in traditional Chinese medicine for its antibacterial, antioxidant
and anti-inflammatory activities. This plant contains substantial
amounts of anthraquinones, especially in the roots. Literature re-
ports showed that anthraquinone molecules possess antigeno-
toxic/antimutagenic activities (Huang et al., 1985; Choi et al.,
1997; Yen et al., 2000; Jasril et al., 2003; Lee et al., 2005). In the
present investigation, we isolated an anthraquinone fraction (aliz-
arin) from R. cordifolia and evaluated its antigenotoxic/antimuta-
genic potential.
2. Material and methods
2.1. Bacterial strains and chemicals
2.1.1. Escherichia coli
PQ37 strain was purchased from Institut Pasteur, France. Salmonella typhimuri-
um TA98 strain was kindly provided by Professor B.N. Ames, University of Califor-
nia, Berkeley, USA.Nicotinamide adenine
glucose-6-phosphate (G6P), normal melting point agarose (NMPA), low melting
point agarose (LMPA), ethidium bromide and ortho-nitrophenyl-b-D-galactopyran-
oside (ONPG), were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India.
Para-nitrophenylphosphate (PNPP), were procured from Sisco Research Laborato-
ries Pvt. Ltd., Mumbai, India; polyethyleneglycol-4-tetraoctylphenolether (Triton
dinucleotidephosphate (NADP),
0278-6915/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.10.019
* Corresponding author. Address: Department of Botanical and Environmental
Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India. Tel.: +91 183
2259732/2451048; fax: +91 183 2258819/20.
E-mail addresses: sjkaur@rediffmail.com, sjkaur2001@yahoo.co.in (S. Kaur).
Food and Chemical Toxicology 48 (2010) 320–325
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Page 2

X-100), hydrogen peroxide, dimethyl sulphoxide from Qualigens Fine Chemicals,
Mumbai, India. Histopaque 1077, from Sigma Chemicals (St. Louis, MO, USA). All
other chemicals used were of analytical grade.
2.2. Plant material and isolation
The roots of R. cordifolia were purchased from local market at Amritsar, Punjab,
India. Voucher specimen No. 0342-B-03/2006, has been submitted to the Herbar-
ium of Department of Botanical and Environmental Sciences, Guru Nanak Dev Uni-
versity, Amritsar, Punjab, India. The roots were washed with running water to
remove any dust impurities and dried at 40 ?C. They were finely powdered and per-
colated with 80% methanol to obtain the methanol extract. The dried methanol ex-
tract was made aqueous with distilled water in a separating funnel and further
fractionated with series of organic solvents to obtain the fractions, viz. hexane frac-
tion and chloroform fraction (Flow Chart 1). The chloroform fraction was subjected
to aluminium oxide column chromatography which yielded the fraction (‘RUC-1’)
when eluted with ethyl acetate:methanol (10:90).
2.3. Phytochemical analysis
The thin layer chromatography (TLC) of the fraction (‘RUC-1’) revealed it to be a
single compound, which was characterized structurally using1H NMR,13C NMR and
mass spectroscopy.
2.4. Anti-genotoxicity assays
2.4.1. Ames assay
The plate incorporation assay (Maron and Ames, 1983) with little modification
(Grover and Bala, 1993) was used for the present investigation. To check the anti-
mutagenic potential, two sets of experiments were designed.
2.4.2. Co-incubation and preincubation
Bacterial culture (0.1 ml), 0.1 ml of direct-acting mutagen NPD and 0.1 ml of
non-toxic concentrations of ‘RUC-1’ fraction were added in the above order into
sterile test tubes containing 2 ml of soft agar and poured onto minimal agar plates.
In the case of indirect-acting mutagen, 2-aminofluorene (2-AF), 0.1 ml of bacteria,
0.1 ml of 2-AF, 0.5 ml of S9 mix and 0.1 ml of ‘RUC-1’ fraction were added into
2 ml of soft agar, mixed and poured onto minimal glucose agar plates. In case of pre-
incubation mode of treatment the mutagen was incubated with test fraction at
37 ?C for 30 min in gyrorotary incubator. After solidification, the plates were placed
in incubator at 37 ?C in an inverted position for 48 h.
Non-toxic concentrations were determined to be those where there was no sta-
tistically significant difference in the (1) number of spontaneous revertant colonies,
(2) size of colonies, and (3) intensity of the background lawn, as compared to the
control where no extract/fraction was added. Concurrently, positive control (muta-
gen but no fraction) was also set. Each concentration was tested in triplicate and the
entire experiment was repeated twice.
The antimutagenic activity of each fraction was expressed as percent decrease
of reverse mutations as follows:
Inhibitory activityð%Þ ¼ a ? b=a ? c ? 100
a = number of histidine revertants induced by mutagen (NPD/2-AF)
b = number of histidine revertants induced by mutagen in the presence of
fraction
c = number of histidine revertants induced in the presence of fraction alone and
solvent (negative control)
2.4.3. SOS chromotest
For the SOS chromotest, an overnight culture of E. coli PQ37 (100 ll) was added
to 5 ml of fresh La medium and incubated for 2 h at 37 ?C. One ml of this culture was
diluted with 9 ml of La medium (Quillardet and Hofnung, 1985). Aliquots of 600 ll
of bacterial suspension were distributed to series of glass test tubes, each contain-
ing 20 ll of genotoxicant [H2O2(1.0 mM)/4NQO (20 lg/ml)] and 20 ll of ‘RUC-1’ of
different concentrations. Positive control was prepared by exposure of bacteria to
either hydrogen peroxide or 4NQO alone. After incubation of 2 h at 37 ?C, 300 ll
samples each were used for assay of b-galactosidase and alkaline phosphatase
activities respectively. The activity of the constitutive enzyme alkaline phosphatase
was used as a measure of protein synthesis and toxicity. In order to determine the
b-galactosidase activity, 2.7 ml of B-buffer (adjusted to pH 7.5) was added and after
10 min, 600 ll of 0.4% 4-nitrophenyl-b-galactopyranoside (ONPG) solution was
added to each of the test tubes of one set. To determine the constitutive alkaline
phosphatase activity, P-buffer (adjusted to pH 8.8) was added and after 10 min,
600 ll of 0.4% 4-nitrophenyl phosphate (PNPP) solution was added to another set
of tubes. All mixtures were incubated at 37 ?C and observed for the colour develop-
Rubia cordifolia L.
Root Powder (3Kg)
80% Methanol Extract
Dried by rotary vaccum evaporator
80% Methanol: Water
Aqueous Methanol extract
Distilled water (1000ml)
Marc
Hexane extract
Hexane 3 X300 ml
Chloroform 10 X300 ml
Residue
Chloroform extract
Column chromatography [Aluminium oxide (brockman’s
activity) in 2.5cm x 50cm column]
RUC-1 (39mg)
Ethyl acetate: methanol (10: 90) fraction.
Chart 1. Schematic representation of isolation of ‘RUC-1’fraction from chloroform extract of Rubia cordifolia L.
P. Kaur et al./Food and Chemical Toxicology 48 (2010) 320–325
321

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ment. After 30 min, the conversion of ONPG was stopped with 2 ml of 1 M sodium
carbonate and that of PNPP with 2 ml of 1.5 N sodium hydroxide. The mixtures
were centrifuged individually and absorption was measured at 420 nm using a ref-
erence solution in which culture is replaced by La medium.
The enzyme activities were calculated according to the simplified method:
Enzyme unitsðUÞ ¼ A420 ? 1000=t
(A420= optical density at 420 nm; t = substrate conversion time in minutes).
ðIFÞ ¼ Rc=R0
Rc= b-galactosidase activity/alkaline phosphatase activity determined for the
test compound at concentration c,
R0= b-galactosidase activity/alkaline phosphatase activity in the absence of the
test compound.
Anti-genotoxicity was expressed as percentage inhibition of genotoxicity
according to the formula:
Inhibitionð%Þ ¼ 100 ? ðIF1? IF0=IF2? IF0Þ ? 100
where:
IF1is the induction factor of the test compound
IF2is the induction factor of positive control (H2O2/4NQO)
IF0the induction factor of the blank (without any test compound).
2.4.4. Single cell gel electrophoresis assay (Comet assay)
The alkaline comet assay was performed on human blood lymphocytes (Singh
et al., 1988). Heparinized blood samples were obtained by venipuncture from a
non-smoking; healthy male donor aged 25–40 years. Lymphocytes were isolated
(Boyum, 1968) and the viability of lymphocytes was determined by trypan blue
exclusion test (Nath and Hanjan, 1983).
Human peripheral blood lymphocytes (2 [ 106-cells/ml) suspended in 1 ml
phosphate buffer saline (PBS), were incubated for 30 min at 37 ?C in BOD incubator
with 20 ll of hydrogen peroxide (25 lM)/4NQO(5 lg/ml) in the presence 20 ll of
different concentrations of ‘RUC-1’. Each test compound/genotoxicant combination
was tested thrice in each experiment along with positive controls.
To evaluate the extent of DNA damage, images of 100 randomly selected cells
stained with ethidium bromide, were analysed from each sample using an Epifluo-
rescent Nikon microscope connected with a digital camera. Imaging was performed
by using a computerized image analysis system (Lucia Comet Assay Software 4.8 of
Laboratory Imaging Ltd.) which acquires images, computes the integrated intensity
profile for each cell, estimates the comet cell components (head and tail) and eval-
uates a range of derived parameters. Different concentrations of ‘RUC-1’ were tested
without hydrogen peroxide/4NQO (negative control).
Antigenotoxic activity of ‘RUC-1’ was determined as
Inhibitionð%Þ ¼ ðT1? TCÞ=ðT1? T0Þ ? 100
where:
T1= tail moment induced by H2O2/4NQO (positive control).
TC= tail moment of fraction in presence of H2O2/4NQO.
T0= tail moment of negative control (fraction only).
2.4..5. Statistical analysis
The results are presented as the mean ± standard deviation/standard error of
three experiments. The data in all the experiments were analysed for statistical sig-
nificance using analysis of variance (one-way ANOVA). The difference among aver-
age values was compared by high-range statistical domain (HSD) using Tukey’s test
(Mayers and Grossen, 1974). The significance was checked at *p < 0.05. IC50was cal-
culated from the regression line.
3. Results
3.1. Analysis of ‘RUC-1’ fraction
‘RUC-1’ was obtained as a red amorphous powder (0.001%). The
ESI–MS of ‘RUC-1’ showed molecular ion peak [M+H]+at m/z
241.255 and corresponded to the molecular formula C14H8O4.
The1H and13C NMR values are given in Table 1.1H NMR spectrum
of ‘RUC-1’ showed signals for unsubstituted ring A at d 8.00 (2H, m,
H-5 and H-8) and 7.73 (2H, m, H-6 and H-7). Other ortho-coupled
aromatic protons of ring C were displayed at d 7.04 (1H, d,
J = 7.8 Hz, H-3) and 7.46 (1H, d, J = 7.8 Hz, H-4).13C NMR spectrum
showed two ortho-hydroxyls substituted carbons, C-1 and C-2, at d
151.1 and 153.2, respectively. The presence of two carbonyl func-
tions was demonstrated at d 181.2 and 189.0 for C-10 and C-9,
respectively. Thus, on the basis of NMR spectral data and compar-
ison with reported values (Siddiqui et al., 2007), ‘RUC-1’ was eluci-
dated as Alizarin (Fig. 1). Small difference in d values are due to
change in solvent. In the present study DMSO-d6has been used
as solvent. In earlier reports CDCl3was used as solvent.
3.2. Anti-genotoxicity
3.2.1. Ames assay
The antimutagenic activity of alizarin (‘RUC-1’) was examined
in plate incorporation assay against mutagens NPD and 2-AF (S9-
dependent) using S. typhimurium tester strain TA98. As evident
from Table 2, alizarin was quite effective in inhibiting the mutage-
nicity of NPD in TA98 tester strain of S. typhimurium. His + rever-
tants induced by NPD were reduced by 65.75% and 75.82%
during co-incubation and preincubation mode of experiments at
the maximum tested dose of 103.70 ? 103lM. Alizarin was very
effective in reducing the his + revertants induced by 2-AF, S9-
dependent mutagen (Table 3). Antimutagenic activity at the dose
of 103.70 ? 103lM was observed to be 97.32% and 96.76% in co-
incubation and preincubation experiments respectively.
3.2.2. SOS chromotest
In the SOS chromotest, it was ascertained that different concen-
trations of alizarin added to the indicator bacteria were not geno-
toxic as the induction factor induced by the tested doses was below
1.5. The compounds are classified as non-genotoxic, if the induc-
tion factor (IF) remains <1.5 and genotoxic if IF exceeds 2.0. Doses
of 1.0 mM of H2O2and 20 lg/ml of 4NQO were chosen for present
studies since these doses were not toxic and induced a significant
SOS response without affecting the alkaline phosphatase activity.
Alizarin was effective in reducing IF induced by H2O2as well as
by 4NQO. Alizarin effectively reduced the genotoxicity of H2O2by
69.00% at a concentration of 40.96 lM with IC50of 24.79 lM. It re-
duced the genotoxicity of 4NQO by 63.48% at a concentration of
Table 1
1H and13C NMR values of ‘RUC-1’ fraction isolated from R. cordifolia L.
Position
1H d (J in Hz)
13C d
1
2
3
4
5
6
7
8
9
10
4a
8a
9a
10a
–
–
7.04 d (j = 7.8)
7.46 d (j = 7.8)
8.00 d (j = 7.8)
7.73 m
7.73 m
8.00 d (j = 7.8)
–
–
–
–
–
–
151.1
153.2
121.8
121.2
127.1
135.6
134.6
126.9
189.0
181.2
123.9
133.7
116.4
132.9
O
O
OH
1
OH
2
3
4
5
6
7
8
9
10
A
BC
Fig. 1. Structure of alizarin (‘RUC-1’) from R. cordifolia L.
322
P. Kaur et al./Food and Chemical Toxicology 48 (2010) 320–325

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40.96 lM with IC50of 28.39 lM. The IF of both the genotoxins de-
creased in a dose-dependent manner (Table 4).
3.2.3. Comet assay
In human blood lymphocytes as well, alizarin when tested alone
at different concentrations, did not show any genotoxic effect as
observed from the insignificant changes in the tail moment param-
eter. It showed significant inhibitory activity against H2O2 and
4NQO induced DNA damage at all the doses tested in Comet assay.
At the highest tested doses of 40.96 lM, alizarin reduced the DNA
damage induced by H2O2by 75.46% with IC50of 23.09 lM and that
of 4NQO by 65.82% with IC50of 27.91 lM (Figs. 2 and 3).
4. Discussion
Alizarin (‘RUC-1’) isolated from R. cordifolia showed effective
inhibition of the mutagenicity of NPD and S9-dependent mutagen,
2-AF in Ames assay. The activation of 2-AF involves the formation
of N-hydroxy-2-aminofluorene. This reaction is catalyzed by the
cytochrome P450 enzyme system. The remarkable inhibitory effect
of alizarin against 2-AF may be attributed to the inhibition of
activity of cytochrome P450-dependent enzymes involved in the
metabolic activation. Takahashi et al. (2002) attributed the anti-
genotoxic activities of the anthraquinone food pigments alizarin
and purpurin against heterocyclic amines and polycyclicaromatic
hydrocarbons to the inhibition of CYP activities. Huang et al.
(1985) reported the antimutagenic potential of several hydroxyl-
ated anthraquinones against benzo[a]pyrene in S. typhimurium
using TA100 tester strain. Anthraquinones have been shown to
substantially contribute to the prevention of food-derived carcino-
genesis. Anthraquinone rich extract of Rheum officinale exhibited
antimutagenic activity against food mutagen, 3-amino-1-methyl-
5H-pyrido [4,3-b] indole (Trp-P-2) through the inhibition of
CYP1A1 (Sun et al., 2000). In our results, alizarin reduced the muta-
genicity of NPD by 65.75% and 75.82% in co-incubation and prein-
cubationmodes ofexperiments
anthraquinone from the aerial parts of Rumex acetosa (Polygona-
ceae) was shown to possess potent antimutagenic activities against
NPD and sodium azide in TA98 and TA100 tester strains of S.
typhimurium (Lee et al., 2005). Edenharder and Tang (1997) re-
ported the antimutagenic effects of various anthraquinones against
the mutagenicity of air pollutants viz. 2-nitrofluorene (2-NF), 3-
nitrofluoranthene and 1-nitropyrene (1-NP) in S. typhimurium
TA98. However, Blomeke et al. (1992) showed the genotoxic activ-
ity of anthraquinone glycosides alizarinprimveroside and lucidin-
primveroside.Alizarin and purpurin
thraquinone) have been reported to exhibit strong inhibitory ef-
fects on the genotoxicity of several carcinogens (Hao et al., 1995;
Marczylo et al., 1999, 2000; Takahashi et al., 2001).
In humans, oxidative DNA damage is also considered an impor-
tant promoter of cancer (Slupphaug et al., 2003). H2O2is one of the
molecules that induce many human diseases including cancer. The
involvement of H2O2 in carcinogenesis has been shown both
in vitro and in vivo studies (Lee et al., 2003; Heo et al., 2004).
Hydrogen peroxide interacts with DNA through highly reactive
oxygen and radical species causing extensive oxidative damage
(Ratnam et al., 2006). Our study showed that alizarin was able to
reduce the level of DNA damage induced by hydrogen peroxide
when tested in SOS chromotest and Comet assay.It effectively re-
duced the induction factor (IF) induced by H2O2in a dose-depen-
dent manner in E. coli PQ37 using SOS chromotest and DNA
damage in single cell gel electrophoresis assay. Wu and Yen
(2004) attributed the antigenotoxic activity of Cassia tora to its
anthraquinones viz. rhein, emodin and chrysophanol in Comet
and Ames Salmonella/microsome test. The antigenotoxic activity
exhibited by alizarin may in part be due to its antioxidative poten-
tial (Yen et al., 2000). Jasril et al. (2003) also reported the antioxi-
dant activities of anthraquinones isolated from cell suspension
culture of Morinda elliptica.
Alizarin was also effective in inhibiting the genotoxicity in-
duced by 4NQO in both the assays i.e. SOS chromotest and Comet
assay. 4NQO is an alkylating compound and potent mutagen that
induces DNA oxidative lesions via generation of reactive oxygen
species (Arima et al., 2006) and DNA adducts (Kim et al., 2006).
Reduction in DNA damage by alizarin may partly be ascribed to
respectively. Emodinan
(diand trihydroxyan-
Table 2
Effect of ‘RUC-1’ fraction from Rubia cordifolia on the mutagenicity of NPD in TA98
tester strain of Salmonella typhimurium.
TreatmentDose (lM) TA98
Mean ± SE Percent inhibition
Spontaneous– 30 ± 1.33–
Positive control
NPD 20 lg/0.1 ml
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
1668 ± 88.88–
Negative control
28 ± 1.05
24 ± 1.30
26 ± 1.67
28 ± 2.97
26 ± 1.94
29 ± 1.30
–
–
–
–
–
–
Co-incubation
1311 ± 106.70
1004 ± 39.78
767 ± 48.37
754 ± 60.41
658 ± 30.72
591 ± 34.23
21.79*
40.54*
55.01*
55.79*
61.66*
65.75*
Preincubation
1215 ± 99.57
1042 ± 110.50
761 ± 79.28
682 ± 42.34
492 ± 20.05
426 ± 15.60
27.66*
38.22*
55.37*
60.19*
71.79*
75.82*
Level of statistical significance; *p < 0.05 with respect to positive control.
Table 3
Effect of ‘RUC-1’ fraction from Rubia cordifolia on the mutagenicity of 2-AF in TA98
(with metabolic activation) tester strain of Salmonella typhimurium.
Treatment Dose (lM)TA98
Mean ± SE Percent inhibition
Spontaneous–28 ± 6.54–
Positive control
2-AF 20 lg/0.1 ml
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
0.414 ? 103
04.14 ? 103
10.35 ? 103
20.72 ? 103
47.46 ? 103
103.7 ? 103
4660 ± 112.40–
Negative control
34 ± 1.09
36 ± 2.15
41 ± 0.99
33 ± 1.25
38 ± 1.72
33 ± 1.20
–
–
–
–
–
–
Co-incubation
3581 ± 448.40
921 ± 61.25
599 ± 6.01
330 ± 23.68
319 ± 67.26
152 ± 3.61
23.29*
80.72*
87.67*
93.48*
93.72*
97.32*
Preincubation
2921 ± 109.40
794 ± 42.77
422 ± 61.67
387 ± 7.839
257 ± 43.36
178 ± 6.00
37.54*
83.46*
91.49*
92.23*
95.06*
96.76*
Level of statistical significance; *p < 0.05 with respect to positive control.
P. Kaur et al./Food and Chemical Toxicology 48 (2010) 320–325
323

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its ability to scavenge free radicals. Emodin was shown to possess
antigenotoxic activity against 4NQO in SOS chromotest (Lee et al.,
2005). Choi et al. (1997) reported the antimutagenic activity of
anthraquinones isolated from methanolic extract of
against the mutagenicity of N-methyl-N0-nitro-N-nitrosoguanidine
(MNNG). Demma et al. (2009) reported the genoprotective poten-
tial of anthraquinone rich plant extracts used in Ethiopian tradi-
tional medicine against the genotoxicity induced by 4NQO and
benzo[a]pyrene in mouse lymphoma L5178Y cells using Comet
assay.
The antigenotoxic properties elicited by the anthraquinone,
alizarin from R. cordifolia L. in the present study suggests that aliz-
arin may have several applications in human health care and needs
to be further exploited for its chemopreventive potential.
C. tora
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
The authors are thankful to the Department of Science and
Technology (DST), New Delhi (India) for funding the research
project.
References
Arima, Y., Nishigori, C., Takeuchi, T., Oka, S., Morimoto, K., Utani, A., Miyachi, Y.,
2006. 4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human
fibroblasts through reactive oxygen species. Toxicol. Sci. 91, 382–392.
Table 4
Effect of ‘RUC-1’ fraction from Rubia cordifolia L. on genotoxicity of H2O2and 4NQO in SOS chromotest using E. coli PQ37 as a tester strain.
TreatmentDose (lM)
b-gal units
Mean ± SD
AP units
Mean ± SD
IF Percent inhibition
Positive controls
H2O2
4NQO
1 mM
20 lg/ml
4.61 ± 0.04
4.20 ± 0.06
12.95 ± 0.16
13.10 ± 0.13
8.00
7.27
–
–
NC ‘RUC-1’0.00
01.28
02.56
05.12
10.24
20.48
40.96
0.58 ± 0.02
0.60 ± 0.03
0.62 ± 0.05
0.61 ± 0.08
0.62 ± 0.06
0.62 ± 0.03
0.64 ± 0.04
13.12 ± 0.09
13.00 ± 0.08
13.12 ± 0.06
13.10 ± 0.12
13.12 ± 0.11
13.09 ± 0.13
13.08 ± 0.12
1.00
1.05
1.07
1.07
1.07
1.07
1.11
–
–
–
–
–
–
–
H2O2+ ‘RUC-1’ 01.28
02.56
05.12
10.24
20.48
40.96
4.29 ± 0.08
3.86 ± 0.05
3.51 ± 0.03
3.19 ± 0.07
2.59 ± 0.06
1.82 ± 0.08
13.00 ± 0.14
12.99 ± 0.07
13.00 ± 0.09
12.97 ± 0.11
12.90 ± 0.10
13.01 ± 0.12
7.50
6.75
6.13
5.60
4.54
3.17
7.15*
17.86*
26.72*
34.29*
49.43*
69.00*
4NQO + ‘RUC-1’01.28
02.56
05.12
10.24
20.48
40.96
4.00 ± 0.05
3.81 ± 0.06
3.42 ± 0.04
3.00 ± 0.06
2.39 ± 0.07
2.24 ± 0.06
13.05 ± 0.08
13.00 ± 0.13
13.07 ± 0.12
13.00 ± 0.14
12.96 ± 0.20
13.09 ± 0.13
6.96
6.59
5.90
5.29
4.54
3.29
4.95*
10.85*
21.86*
31.58*
43.55*
63.48*
b-gal = b-galactosidase (b-gal) units.
AP = Alkaline phosphatase (AP) units.
IF = Rc/R0.
NC = negative control.
SD = standard deviation.
Data shown are mean ± SD of three independent experiments.
Level of statistical significance; *p < 0.05 with respect to positive control.
0
5
10
15
20
25
Control0 1.282.56
Alizarin (µM)
5.1210.24 20.48 40.96
Tail moment (Arbitrary units)
0
20
40
60
80
100
Cell Viability (%)
H2O2
Cell viability
*
*
*
*
*
*
Fig. 2. Decrease in tail moment by alizarin (RUC-1) fraction from R. cordifolia L. in
human blood lymphocytes in Comet assay. Level of statistical significance; *p < 0.05
compared to positive control (H2O2).
0
5
10
15
20
25
Control01.282.56
Alizarin (µM)
5.1210.24 20.48 40.96
Tail moment (Arbitrary units)
0
20
40
60
80
100
Cell Viability (%)
4NQO
Cell Viability
*
*
*
*
*
*
Fig. 3. Decrease in tail moment by alizarin (RUC-1) from R. cordifolia L. in human
blood lymphocytes in Comet assay. Level of statistical significance; *p < 0.05
compared to positive control (4NQO).
324
P. Kaur et al./Food and Chemical Toxicology 48 (2010) 320–325

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