Vol.2, No.6, 595-602 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Inhibition of H2O2-induced DNA damage in single cell gel
electrophoresis assay (comet assay) by castasterone
isolated from leaves of centella asiatica
Nishi Sondhi1, Renu Bhardwaj1*, Satwinderjeet Kaur1, Madhu Chandel1, Neeraj Kumar2,
Bikram Singh2
1Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India;
*Corresponding Author: renubhardwaj82@gmail.com
2Division of Natural Plant Product, Institute of Himalayan Bioresource Technology, Palampur, India
Received 10 December 2009; revised 16 February 2010; accepted 20 February 2010.
Brassinosteroids (BRs) are a large group of
polyhydroxy steroids, which regulate numerous
aspects of plant growth and development, in-
cluding stem elongation, leaf bending, tracheary
element differentiation, stress protection and
photomorphogenesis. Recent studies indicate
antigenotoxic and anticancerous activities of
these compounds. The role of natural BRs in
H2O2 (hydrogen peroxide) -induced DNA damage
in human lymphocytes is still unknown. The
present study reports the presence of Cas-
tasterone from leaves of Centella asiatica, an
important medicinal herb commonly used as a
memory enhancer and immunomodulator. CA50
fraction isolated from Centella asiatica was
characterized as Castasterone by electrospray
ionization mass spectral data with standard
Castasterone. An attempt has been made to
study antigenotoxic activity of the isolated
Castasterone against H2O2 -induced DNA dam-
age in human blood lymphocytes using Single
cell gel electrophoresis assay (Comet Assay).
Castasterone at 109 M concentration proved to
be effective in diminishing the DNA damage by
89.42 %.
Keywords: Brassinosteroids; Castasterone;
Comet Assay; Hydrogen Peroxide
In living system, oxidative stress results in the produc-
tion of reactive oxygen species (ROS) like superoxide
radical (O2
-), hydroxyl radical (HO) and hydrogen per-
oxide (H2O2). H2O2 in Fenton reaction is spontaneously
converted to the highly reactive hydroxyl radicals (HO).
These hydroxyl radicals oxidize proteins, lipids and nu-
cleic acids leading to even mutations at the cellular level
[1]. Several plant hormones are implicated in modulating
the response to oxidative stress like ethylene [2], ab-
scisic acid [3], auxins and plant steroids [4]. Brassinos-
teroids are a group of naturally occurring plant hormones,
which are structurally similar to animal steroid hor-
mones. They influence diverse physiological processes
by regulating the expression of genes like their animal
counterparts [5]. Recent studies indicate antiviral active-
ties of BRs against various viruses, like herpes simplex
virus type I (HSVI), arena virus, measles virus and ve-
sicular stomatitis virus [6-8]. The treatment of BRs to
these viruses was 10-18 folds more effective than ri-
bavirin towards HSV-I and arenavirus. It has further
been reported that 24-epibrassinolide can increase the
mitochondrial membrane potential, reduce intercellular
antibody levels, increase the proportion of cells in G0/G1
phase, reduce the population of cells in s-phase and in-
crease the population of viable hybridoma mouse cells at
subnanomolar concentrations [9]. Anticancerous active-
ties of 28-homocastasterone and 24-epibrassinolide were
studied in several normal and cancer cell lines. The
anticancer and antiproliferative activities have been
documented very recently [10]. The BRs used showed
high cytotoxic activity in breast (MCF-7/MDA-MB-468)
and prostate cancer cell lines (LNCaP/DU-145) [11].
Centella asiatica (L.) (Family Apiaceae) commonly
known as urban herb regarded as rasayana or rejuvenat-
ing herb reputed to increase intelligence and memory in
Ayurvedic medicines. The methanol extracts of whole
plants of Centella asiatica showed a significant increase
in the phagocytic index and total WBC count thereby
showing immunomodulatory activity. [12] isolated a
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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water soluble arabinogalactan, HBN and traced remark-
able immunoenhancing activities on T-and B-lymphocytes
in vitro and vivo tests. The antioxidative properties of
Centella asiatica were evaluated by [13,14]. The C. asi-
atica extract has a chemopreventive effect [15]. [16]
studied the healing effects of C. asiatica when orally
administered to rats with acetic acid induced gastric
ulcers, It reduced the size of ulcers in dose-dependent
manner. Chemical studies reveal that triterpene saponins
Asiaticoside and Madecassoside are the main active
constituents of Centella asiatica. The other saponins and
triterpene acids present in this plant are brahmoside,
brahmnoside, brahmic acid, isobrahmic acid, betulic acid,
centelloside and cetillic etc. The presence and role of
brassinosteroids in this plant is yet to be studied. The
present study was therefore planned to study the pres-
ence of BRs and inhibition of H2O2-induced DNA dam-
age by Castasterone isolated from Centella asiatica
which is the first report in this direction.
2.1. Extraction and Purification of
Study material for the present investigation included
leaves of Centella asiatica procured from Dehradun
(M/s Gautam globals, Dehradun, India). Fresh leaves of
Centella asiatica (2 kg) were homogenized and percolated
with 80% methanol (3 × 1000 ml). The combined metha-
nol extract was dried under vacuum using rotary evapo-
rator (Strike 202, Stereoglass, Italy). 80% methanol ex-
tract (449.6 g) was partitioned between chloroform and
water. Chloroform extract was then partitioned between
80% methanol and hexane. The resulting 80% methanol
extract (28.3 g) was partitioned between ethyl acetate
and distilled water. The ethyl acetate fraction (20 g) was
dried and subjected to silica gel (60-120 mesh) column
chromatography with step-gradient elution from 0, 1, 2,
3, 4, 5, 6, 7, 10, 15, 20, 50, 100% (each 500-1000 ml).
All the fractions were subjected to radish hypocotyl bio-
assay with the aim to find the bioactive fraction. Four
fractions CA5, CA10, CA50 and CA60 were found to be
active (Figure 1). CA50 fraction was directly subjected
to ESI-MS and MS/MS analysis (Figures 2(a), 3(a)).
2.2. Radish Hypocotyl Bioassay
The bioactivity of isolated fractions was determined us-
ing intact plants of Raphanus sativus as described by
[17]. 5 days-old seedlings were placed into the test solu-
tions (0.03 ml of fraction diluted with distilled water to
get the final volume 3 ml). 3 ml solution was poured in
each petriplate containing radish seedlings and kept in
01256710 15 20 50 60 70100
percent increase
Figure 1. Biological activity of fractions of Centella asiatica
in Radish hypocotyls bioassay after silica gel column chroma-
the dark for 24 h at 25 ± 2˚C. After 24 h, the length of
hypocotyls were measured and compared to control.
Percent increase over control was calculated.
2.3. Electrospray Ionization Mass
Spectrometry of CA 50 Fraction
ESI-MS analysis of CA50 and standard Castasterone
was carried out by the addition of 10 μl of concentrated
aqueous formic acid solution to the sample mixture to a
total volume of 1000 μl making 0.1% as final concentra-
tion. ESI-QTOF-MS was performed in positive ioniza-
tion mode in QTOF Mass Spectrometer (Micromass,
Manchester, UK). The general conditions were: Source
temperature of 280˚C, capillary voltage of 2.1 kV and
cone voltage of 23 V. ESI-MS was performed by direct
infusion with a flow rate of 10 μl/min using a syringe
pump and mass spectra were acquired and accumulated
over 60 s. MassLynx 4.0 (Waters, Manchester, UK) was
used for data analysis. Tandem mass spectrometry of
single molecular ion in the mass spectra was performed
by mass-selecting the ion of interest, which was in turn
submitted to 15-35 eV collisions with argon in the colli-
sion quadrupole.
2.4. Comet Assay
DNA damage was determined by alkaline single cell
microgel electrophoresis (comet assay) assay following
the method proposed by [18] with minor modifications
as suggested by [19]. Heparinized blood samples were
obtained by venipuncture from a non-smoking, healthy
male donor aged 30-40 years. Lymphocytes were iso-
lated by the method of [20] and mixed with equal vol-
ume of Phosphate Buffer Saline (PBS) pH 7.2. This
mixture was then overlayed to double volume of Histo-
paque 1077 and centrifuged at 1500 rpm for 20 minutes.
The layer containing lymphocytes was aspirated very
carefully with the help of pasture pipette. The lympho-
cytes were diluted in PBS and centrifuged at 2000 rpm
for 15 min. The supernatant was discarded and pellet
was again suspended in PBS and centrifuged at 2000 rpm
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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Figure 2. (a) ESI-QTOF-MS analysis of Castasterone fraction (CA50) isolated from Centella asiatica; (b) ESI-QTOF-MS of Stan-
dard Castasterone.
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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Figure 3. (a) ESI-QTOF-MSMS analysis of Castasterone fraction (CA50) isolated from Centella asiatica; (b) ESI-QTOF-MSMS
analysis of Standard Castasterone.
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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Human blood lymphocytes suspended in 1 ml PBS,
were incubated in a shaking water-bath for 30 minutes at
37˚C with 5 × 10–5 M H2O2 in the presence of different
concentrations of CA50 fraction tested in duplicate. To
evaluate the extent of DNA damage, 100 randomly se-
lected cells were analysed from each sample by Nikon
Epifluorescent Microscope (Nikon Eclipse E200) con-
nected to 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 compo-
nents (head and tail) and evaluates a range of derived
parameters. These include % Head DNA, % Tail DNA
and tail moment (an index of DNA damage that consid-
ers both the tail length and fraction of DNA in comet
The Antigenotoxic activity of CA50 was expressed by
percent decrease of Tail moment:
Inhibition (%) = a – b/a – c × 100
a = Tail moment induced by H2O2 (positive control).
b = Tail moment of the fraction in the presence of
c = Tail moment of the negative control.
The results were obtained as the mean and standard error
of three experiments. The data in all the experiments
were analyzed for statistical significance using analysis
of variance (one-way ANOVA). The difference among
average values was compared by high-range statistical
domain (HSD) using Tukey’s Test [21].
3.1. Results and Discussion
CA50 fractions was isolated and characterized as cas-
tasterone by ESI-QTOF-MS/MS analysis. Electrospray
ionization mass spectroscopy of standard castasterone
and CA50 fraction (Figures 2(a) and 2(b)) showed the
pseudomolecular mass ion peaks at m/z 465 [M + H]+
and 487 [M + Na]+ corresponded to the molecular
weight 464 [M]+ and molecular formula as C28H48O5.
ESI-QTOF-MS/MS analysis of m/z 465/487 revealed
similar kind of fragmentations for CA50/ standard cas-
tasterone (Figures 3(a) and 3(b)). The fragments at m/z
447/469,429/451 were observed due to the sequential
loss of two H2O molecules. The fragments at m/z
393/415 and 305/327 were observed due to the C23-C24
and C17-C20 bond fissions. Other fragments were de-
tected at m/z 411/433,297/319,281/303, 269/291,173/195
and 161/183. Figure 4 shows the important fragmenta-
tion pattern of castasterone. We report for the first time,
the presence of Castasterone in Centella asiatica. The
m/z 393/415
m/z 305/327
m/z 281/303
m/z 269/291
m/z 161/183
m/z 173/195
Figure 4. Structure of Castasterone and major mass fragmenta-
presence of brassinosteroids in medicinal plant like
Centella asiatica suggests a possible medicinal applica-
tion of these compounds. Brassinosteroids are found in
gymnosperms, monocotyledonous and dicotyledonous
plants, and in algae. Studies [22-24] confirmed that BRs
are obligatory plant constituents, the highest concentra-
tion being found in the reproductive organisms and in
growing tissues (pollen, immature seeds and shoots).
H2O2-induced DNA damage was studied using various
parameters. These parameters were measured using Lu-
cia Comet Assay Software. Percent inhibition was cal-
culated on the basis of comet evaluation i.e. Tail moment.
The observations made on various parameters of DNA
indicated that 10–9, 10–10 and 10–11 M concentrations of
Castasterone were not toxic as no significant change was
noticed when compared with negative control i.e. dis-
tilled water.
Percent head DNA in the treatments of Castasterone
revealed no significant change when compared with
negative control i.e. only distilled water. The H2O2
treatment however reduced %Head DNA. But supple-
mentation of Castasterone with H2O2 to lymphocytes
revealed an observable enhancement in % Head DNA. It
increases from positive control (83.08%) to (93.66%) at
10–9 M concentration (Table 1). Similar observations
were made for % Tail DNA and Tail moment. BRs
treatments showed amelioration of toxicity. % Tail DNA
decreased significantly with the increase in concentra-
tion of castasterone. A significant decrease in Tail mo-
ment was observed with the increase in the concentration
of castasterone (Figure 5). It varied from 1.35 (10–11M),
1.02 (10–10 M), 0.48 (10–9 M). Percent inhibition was
maximum in 10-9 M concentration i.e. 89.42% (Table 1).
The reports obtained on the toxicity of BRs suggested
that they do not have negative influences in mammals,
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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Table 1. Inhibition of H2O2-induced DNA damage in Human blood lymphocytes by CA50 fraction isolated from Centella asiatica
using Comet assay.
Treatment Dose concentration %Head DNA (Mean ± SE)% Tail DNA (Mean ± SE)Tail moment (Mean ± SE) % Inhibition
Negative control D.W 96 ± 0.32 4.0 ± 0.32 0.30 ± 0.02
10–11M 92.28 ± 2.06 7.72 ± 0.40 0.24 ± 0.03
10–10M 96.09 ± 0.41 3.91 ± 0.41 0.26 ± 0.03
10–9M 95.82 ± 0.56 4.22 ± 0.58 0.26 ± 0.03
Positive control H2O2 (50 M) 83.02 ± 0.72 16.98 ± 0.72 2.34 ± 0.13
10–11M 88.68 ± 1.06* 11.32 ± 1.06* 1.35 ± 0.13* 47.14%
10–10M 91.69 ± 0.8* 8.28 ± 0.81* 1.02 ± 0.11* 63.46%
Castasterone + H2O2
10–9M 93.66 ± 0.98* 6.34 ± 0.98* 0.48 ± 0.07* 89.42%
*indicates significant values at p 0.05.
Tail moment
Figure 5. Effect of Castasterone isolated from Centella asiat-
ica on the genotoxicity induced by Hydrogen peroxide (5 ×
10–5 M) in human lymphocytes using comet assay. 1 = treat-
ment with H2O2. 2-4 = treatment with different concentrations
of Castasterone.
(a) (b) (c)
Digital images illustrating the inhibition of DNA damage by
Castasterone in the Comet assay (a) +ve control i.e. H2O2 (b)
–ve control i.e. Castasterone only (c) Castasterone + H2O2.
water organisms, soil microbiological processes and
plants [25]. Mutagenic studies carried out at the Scien-
tific Research Center of Toxicological and Hygienic
Regulation of Biopreparations of Russia showed that
Ames test, with or without metabolic activitation, was
negative with the tester strains of Salmonella typhi-
murium TA1534, TA1537, TA1950, TA98 and TA100
[25]. Antigenotoxic properties of EBL isolated from
A.marmelos had also been studied by [26].
Reactive oxygen species can damage the normal cel-
lular functions and can cause atherosclerosis in vessels
or malignant growth in other tissues and ageing proc-
esses [27]. The lymphocytes when treated with H2O2
showed the significant DNA damage. However this
damage was ameliorated significantly by the simultane-
ous application of different concentrations of this BR.
The H2O2 stress protective properties of BRs in human
lymphocytes are the first such study carried out with
plant steroids. In the present study, the protective effect
observed against the ROS may in part be responsible to
the anticancer activity of brassinosteroids reported by
some workers [11,28]. In the studies carried out by [29]
three types of 5α-androstane and ergostane analogues of
brassinolide, containing a fluorine atom in either the 3α
or the 5α positions or in 3α or the 5α positions, were
prepared using standard operations. The 5α fluorine was
found to effect chemical reactivity as well as physical
properties of the products. Cytotoxicity of the products
was studied using human normal and cancer cell lines
with 28-homocastasterone as positive control and their
brassinolide type activity was established using the bean
second-internode test with 24-epibrassinolide as stan-
dard. The equivalence of F and OH groups was observed
in some of the active compounds. Ergostane derivatives
were most active in the anticancer activity while andro-
stane derivatives were active in brassinolide type activity.
Brassinolide was found to induce a time and concentra-
tion dependent cytotoxicity in androgen–independent
human prostate cancer in PC-3 cells. The mode of cell
death appeared to be predominately apoptosis. Western
blot studies indicated that treatment with brassinolide
triggered a time dependent decrease in the expression of
N. Sondhi et al. / HEALTH 2 (2010) 595-602
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antiapoptotic protein Bcl-2. 24-epibrassinolide and 28-
homoCS were found to inhibit the growth, at micro-
molar concentrations, of several human cancer cell lines
without affecting the growth of normal cells [10]. Stud-
ies carried out by Malikova et al. (2008) indicate that
BRs may prove to be promising leads for the develop-
ment of new generation of anticancer drugs. Varoius
animal steroids have been found to exhibit antioxidant
properties [30]. BRs have also been reported to regulate
antioxidative defence system of plants under stress con-
ditions [31]. The reduction in DNA damage indicates
amelioration of oxidative stress generation by H2O2 in
lymphocytes. The tissues are protected from oxidative
damage by variety of mechanism including antioxidants
and antioxidative enzymes, repair enzymes and growth
regulators. Further studies are needed to understand the
mechanism of protective effect of these steroids in ani-
mal system which opens a field of study on possible
medical applications of these plant steroids.
This work is supported by grants from University Grants Commission,
Government of India. Dr. P.S. Ahuja, Director, Institute of Himalyan
Bioresource Technology (IHBT) Palampur (HP) is duly acknowledged
for providing necessary lab facilities. Dr. Chalev Pachthong, Depart-
ment of Chemistry, Faculty of Science and Technology Kanchanaburi
Rajabhat University, Kanchanaburi, Thailand is also acknowledged for
his kind gift of Castasterone.
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