Inhibition of H2O2-induced DNA damage in single cell gel electrophoresis assay (comet assay) by castasterone isolated from leaves of centella asiatica

doi:10.4236/health.2010.26088

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Vol.2, No.6, 595-602 (2010) Health

doi:10.4236/health.2010.26088

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.

ABSTRACT

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 10–9 M concentration proved to

be effective in diminishing the DNA damage by

89.42 %.

Keywords: Brassinosteroids; Castasterone;

Comet Assay; Hydrogen Peroxide

1. INTRODUCTION

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

596

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. MATERIALS AND METHODS

2.1. Extraction and Purification of

Brassinosteroids

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

100

150

200

250

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-

tography.

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

597

(a)

(b)

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

598

(a)

(b)

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

599

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

tail).

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

H2O2.

c = Tail moment of the negative control.

3. STATISTICAL ANALYSIS

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-

tions.

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

600

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

Castasterone

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

(50M)

10–9M 93.66 ± 0.98* 6.34 ± 0.98* 0.48 ± 0.07* 89.42%

*indicates significant values at p ≤ 0.05.

0.5

1.5

2.5

1234

Tail moment

Concentrations

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

601

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.

4. ACKNOWLEDGEMENTS

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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