Evaluating the antioxidant potential of aqueous and alcoholic extracts

of Ficus religiosa using ORAC assay and assessing their cytotoxic

activity in cervical cancer cell lines

Amit Subhash Choudhari, Snehal Suryavanshi, Harshad Ingle, Ruchika Kaul-
Ghanekar

Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University Medical
College Campus, Dhankawadi, Pune-Satara Road, Pune, Maharashtra 411043, India; Email:
ruchika.kaulghanekar@gmail.com, kaul_r@yahoo.com


ABSTRACT

Ficus religiosa L. (Moraceae) has been known to have numerous therapeutic uses in traditional medicine
wherein it has been used for different disease conditions related to central nervous system, endocrine system,
gastrointestinal tract, reproductive system, respiratory system and infectious disorders. Here, we have
evaluated the antioxidant potential of aqueous and ethanolic preparations of F. religiosa bark material by
Oxygen Radical Absorbance Capacity (ORAC) method and assessed their cytotoxic activity in cervical cancer
cell lines, SiHa and HeLa. The aqueous (FR
aq
) and ethanolic (FR
et
) extracts of the bark exhibited significant
total antioxidant capacity as determined by ORAC method, however the FR
aq
showed higher Oxygen Radical
Absorbance Capacity than FR
et
. Both the extracts exhibited a significant increase in anti-lipid peroxidative
(ALP) activity with IC
50
values of 29.06 and 34.39g/ml, respectively for FR
aq
and FR
et
. However, FR
aq

showed a slightly higher ALP activity than FR
et
. The total phenol content present in one milligram of FR
aq
and
FR
et
was found to be around 497.77 and 375.23g, respectively, equivalent to gallic acid control. Interestingly,
both the aqueous and ethanolic extracts showed significant cytotoxicity in cervical cancer cell lines SiHa
(HPV16 positive) and HeLa (HPV18 positive) wherein FR
et
showed cytotoxicity at much lower doses as
compared to FR
aq
. All these results suggest that Ficus religiosa could be explored further for its anticancer
potential with special reference to cervical cancer.

Keywords: Ficus religiosa, antioxidant, oxygen radical absorbance capacity, cytotoxicity, cervical cancer, cell
lines, anti-lipid peroxidation, chemopreventive


INTRODUCTION

Oxidative stress resulting from imbalance between oxidants and antioxidants can cause oxidative
damage to large biomolecules that include lipids, proteins and DNA. This in turn leads to an
increased risk for various pathological conditions such as cancer and cardiovascular diseases [1]. To
prevent or slow down the oxidative stress induced by free radicals, our body needs to consume
sufficient amounts of antioxidants. Besides, fruits, vegetables, whole grains and spices, medicinal
plants also have wide variety of natural antioxidants or phytochemicals [2]. Consumption of
antioxidants may help protect cellular systems from oxidative damage and also may lower the risk
of chronic diseases [1].
Ficus religiosa Linn. (Moraceae), commonly known as peepal, is widely cultivated in south-
east Asia and has been known for many medicinal properties in traditional system. It has been used
to treat respiratory disorders, ulcers, stomatitis, hiccup, arthritis, gout, skin diseases, allergies,
inflammatory disorders, bone fracture, diabetes, gynaecological disorder, etc. [3,4]. Numerous
therapeutic uses in folk medicine have encouraged looking for its effect in disease management
Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(4):443-450
2011 Society for Applied Biotechnology. Printed in India; ISSN 2249-9075

444
such as diabetes [5], brain-related disorders [6,7], kidney and urinary disorders [8]. Recently, lot of
work has been done to evaluate the antioxidant properties of different parts such as leave, fruit, latex
and barks of Ficus religiosa (FR) [9-12] wherein antioxidant assay such as DPPH radical
scavenging assay, phosphor-molybdenum scavenging assay, reducing power assay, hydrogen
peroxide scavenging activity have been studied.
In the present study, we have compared the total antioxidant capacity of aqueous (FR
aq
) and
ethanolic (FR
et
) extract of bark of Ficus religiosa by ORAC method. We have also evaluated the
anti-lipid peroxidation (ALP) activity as well as cytotoxic potential of both the extracts in cervical
cancer cell lines. Our findings suggest that both the extracts exhibit significant antioxidant potential;
however, the aqueous extract showed significantly higher total antioxidant capacity that is supported
by its high ALP activity as well. Both FR
aq
and FR
et
extracts showed significant cytotoxicity in
cervical cancer cell lines, thereby suggesting the chemopreventive potential of Ficus religiosa in
cervical cancer.

MATERIALS AND METHODS

All the chemicals used were of analytical grade. Fluorescein sodium salt, AAPH (2,2-azobis-2-
methyl-propanimidamide, dihydrochloride), Trolox was obtained from Sigma Chemicals, USA.
Napthyl ethylene diamine dihydrochloride (NEDD) was obtained from SISCO Research
Laboratories Pvt. Ltd., Mumbai, India. Folin-Ciocalteus phenol reagent (FCR) was obtained from
SD Fine-Chemicals Limited, Mumbai. Sodium nitroprusside (SNP), sulphanilamide, phosphoric
acid, ferric chloride (FeCl
3
), ferrous sulphate (FeSO
4
), trichloroacetic acid (TCA), thiobarbituric
acid (TBA), potassium chloride (KCl), Tris hydrochloride buffer were obtained from Qualigens.
Tissue culture plasticware was purchased from BD Biosciences, CA, USA; Axygen Scientific Inc,
CA, USA and Nunc, Roskilde, Denmark. Dulbecco's Modified Eagles Medium (DMEM) was
obtained from Himedia Corporation, Mumbai, India. Penicillin and streptomycin were obtained
from Gibco BRL, CA, USA. Fetal bovine serum was purchased from Moregate Biotech, Australia,
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazolium bromide (MTT) was purchased from
Sigma-Aldrich, St. Louis, MO, USA.

Plant material and extract preparation

Bark of Ficus religiosa L. was collected from Pune District, Maharashtra, India. Botanical
identification of plant material was carried out with the help of standard flora [13] and a voucher
specimen (MPCC 2417) of authentic plant species have been deposited at the herbarium of
Medicinal plants Conservation Center (MPCC), Pune, Maharashtra, India. Bark was chopped into
small pieces, shade dried at ambient temperature and ground into coarse powder in a grinder.
Aqueous and ethanolic extracts were prepared as per standard Indian Pharmacopoeia and soxhlet
method, respectively. The extract obtained, was centrifuged at 13000 rpm for 15min and the
supernatant was filtered through Swiney filter (pore size, 0.45 m) and the extract was stored at -
80C until further use.

Oxygen radical absorbance capacity (ORAC) assay

The total antioxidant capacity of the extract was determined using ORAC method [14]. A freshly
prepared fluorescein (150 l of a 5 nM solution) was mixed with 25l of various concentrations of
the extract (0-200 g/ml) in the flat-bottom black 96-well plate and incubated for 30 min at 37C.
After incubation, fluorescence measurements (excitation, 485 nm; emission, 520 nm) were taken
every 100 seconds to determine the background signal. After 3 cycles, 25 l (250 mM) of AAPH
445
(2,2-azobis-2-methyl-propanimidamide) was added manually with a multi-channel-pipette. The test
was resumed and fluorescent measurements were taken upto 150 min using FLUOstar omega
multiplate reader, BMG labtech (Offenburg, Germany). The net area under the curve (AUC) of the
standards and samples was calculated. The standard curve was obtained by plotting Trolox
concentrations against the average net AUC of the three measurements for each concentration. The
AUC was calculated as AUC = 1 + f1/f0 + ... fi/f0 + ... + f149/f0 + (f150/f0), where, f0 = initial
fluorescence reading at 0 min and fi = fluorescence reading at time i. The data were analyzed in
Microsoft Excel to calculate the AUC. The net AUC was obtained by subtracting the AUC of the
blank from that of the sample.

Anti-lipid peroxidation activity by TBARS method

Inhibition of lipid peroxidation activity by both aqueous and ethanolic extracts was determined
using goat liver homogenate and thiobarbituric acid-malondialdehyde (TBA-MDA) [15]. Animal
tissue (goat liver) was perfused with KCl (0.15M in H
2
O) and the perfused tissue (10%w/v) was
homogenized in ice cold KCl (0.15M) using mortar and pestle. The reaction mixture was set up
using 0.5 ml of homogenate, 0.5 ml of different concentrations (0-200g/ml) of F
aq
and F
et
extracts
and 1 ml KCl (0.15 M). Lipid peroxidation was induced by adding 100l FeCl
3
(1mM in H
2
O) and
the reaction mixtures were incubated at 37C for 30 min. Trichloroacetic acid (15% in 0.25N HCl),
thiobarbituric acid (0.38%) and 200l butylated hydroxyl toluene (0.05%) were added to stop the
reaction. The reaction mixtures were heated at 80C for 60 min, cooled at room temperature and
centrifuged at 6000 rpm for 15 min. Supernatant was collected and measured at 532 nm. ALP %
was calculated using the formula: % inhibition of lipid peroxidation = [(OD of induced sample - OD
of test sample) / OD of induced sample] 100.

Estimation of total phenolic content by Folin-Ciocalteu method

The total phenolic content of F
aq
and F
et
extracts was determined spectrophotometrically by Folin-
Ciocalteu method [16]. The extract (100 g mL
-1
) was mixed with 5 ml of Folin-Ciocalteu reagent,
previously diluted in distilled water (1:10), and 4 mL of sodium carbonate (1M in H
2
O). The
mixture was incubated at 37C for 15 min for the colour development. The absorbance was
measured at 765 nm using a Perkin Elmer spectrophotometer (lambda EZ201). Samples of the
extracts were evaluated at a final concentration of 1 mg mL
-1
. Total phenolic content was expressed
as mg g
-1
gallic acid equivalent using the equation obtained from the standard calibration curve: y =
0.004x+0.0201, R
2
= 0.9953.

Cell lines

The cervical cancer cell lines, HeLa and SiHa used in the study were obtained from National Centre
for Cell Science (NCCS), Pune, India. The cells were grown in DMEM containing 2mM L-
glutamine supplemented with 10% fetal bovine serum and 100 U/ml of penicillin-streptomycin. The
cells were incubated in a humidified 5% CO
2
incubator at 37C.

Cytotoxicity measurement

For the assessment of the anticancer activity of F
aq
and F
et
extracts, MTT dye uptake assay was
performed as described previously [17]. Briefly, SiHa and HeLa cells were seeded at 1 10
5
/ml
density in 96-well plates. An untreated group was kept as a negative control. The F
aq
and F
et
extracts
446
were added to the cells at following concentrations: 10, 20, 40, 80, 160, 320 and 640g/ml, in each
well in triplicates. The MTT solution (5mg/ml) was added to each well, and the cells were cultured
for another 4 h at 37C in 5% CO
2
incubator. The formazan crystals formed were dissolved by
addition of 90 l of SDS-DMF (20% SDS in 50% DMF). After 15 min, the amount of colored
formazan derivative was determined by measuring optical density (OD) with the ELISA microplate
reader (Biorad, Hercules, CA) at 570 nm (OD
570-630nm
). The percentage viability was calculated
as: % viability = [OD of treated cells/ OD of control cells] 100.

Statistical analysis

All the assays were performed in triplicates and repeated at least three times at different time points
and the data were presented as mean SD. Statistical analysis was conducted with the SigmaStat
3.5 program (Systat Software, Inc.) using one-way ANOVA.

RESULTS AND DISCUSSION
Comparison of total antioxidant capacity of FR
aq
and FR
et
by ORAC method

The oxygen radical absorbance capacity (ORAC) has been widely used to investigate the
scavenging activities of several natural compounds [14] or crude mixtures such as ethanolic or
water extracts of plants. The free radical scavenging/oxygen radical absorbance capacity of a
compound may serve as a significant indicator of its potential antioxidant activity. Compared to the
traditional methods used to evaluate the antioxidant status of medicinal plants, ORAC is the only
method so far that combines both inhibition time and degree of inhibition by an antioxidant into a
single quantity thereby giving accurate measurement of antioxidant capacity [18]. The ORAC
method is based on the principle of inhibition of the peroxyl-radical-induced oxidation, which is
initiated by thermal decomposition of azo-compounds. The assay measures the loss of fluorescence
of fluorescein with time due to peroxyl-radical formation by the breakdown of AAPH (2,2-azobis-
2-methyl-propanimidamide, dihydrochloride). Trolox [6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid], a water soluble vitamin E analog, serves as a positive control that inhibits
fluorescein decay in a dose-dependent manner. The ORAC assay is a kinetic assay measuring
fluroescein decay and antioxidant protection over time.
Recently, various groups have reported the antioxidant activity of Ficus religiosa [9-11],
however, we have for the first time compared the total antioxidant capacity of aqueous (FR
aq
) and
ethanolic (Fr
et
) extracts of bark of F. religiosa by ORAC method. It was found that both the extracts
possessed significant free radical scavenging capabilities, however, the FR
aq
inhibited fluorescein
decay in a dose-dependent manner closer to that of the positive control, Trolox. On the other hand,
FR
et
showed less antioxidant capacity than FR
aq
and didnt follow a dose-dependent pattern (Figure
1). The ORAC assay has been documented for analyzing the antioxidant capacity of fruits,
vegetables, dietary supplements, nutraceuticals, juices, wines, as well as plasma and urine samples
from clinical trials [18-23]. We have also tried to compare the antioxidant activities of aqueous and
ethanolic preparations of F. religiosa bark material by using ORAC method. We found that the
aqueous extract possesses better antioxidant capacity than the alcoholic extract.

Total phenolic content in FR
aq
and FR
et


In addition, the antioxidant activity may be due to phenolic compounds the largest group of
phytochemical present in the extract. The activity is believed to be mainly due to their redox
properties. Folin-Ciocalteu method was used to determine the total phenol content. Different
447
concentration of standard gallic acid was prepared and mixed with diluted Folin-Ciocalteu reagent
along with sodium carbonate. The mixture was incubated for colour development. Total phenol
content was determined in comparison with standard gallic acid (Figure 2) and the results were
expressed in terms of mg/g of extract. The total phenol content values for the FA and FE was
497.77 and 375.23 mg/g equivalent of gallic acid respectively.



Figure 1. Oxygen radical absorbance capacity (ORAC) assay. The graph represents the oxygen radical
absorbance capacity of the FR
aq
and FR
et
extracts which is expressed as Net Area Under Curve and
compared with Trolox, a well-known antioxidant standard (n=3 independent experiments).



Figure 2. Standard gallic acid curve. The graph represents the absorbance of gallic acid at known
increasing concentration for total phenol determination of FR
aq
and FR
et
extract (n=3 independent
experiments).
448
FR
aq
and FR
et
inhibited lipid peroxidation

Lipid peroxidation (LPO) is an important cellular mechanism that commonly occurs under normal
physiological conditions. However, under excessive oxidative stress, the levels of LPO become
more significant. Lipid peroxidation of the cell occurs due to the reaction of free radicals with lipids
and is considered as an important feature of cell injury leading to deterioration of cellular
constituents including lipids, proteins and nucleic acids. We used modified TBARS method to
analyze the anti-lipid peroxidation capacity of both FR
aq
and FR
et
extracts. Animal tissue (liver)
homogenate was mixed with different concentrations of the extracts and lipid peroxidation was
induced with FeCl
3
. It was observed that the aqueous extract showed greater anti-lipid peroxidation
activity as compared to the ethanolic extract (Figure 3). The higher ALP activity of aqueous extract
could be attributed to higher antioxidant capacity as well as higher polyphenolic compounds present
in it as compared to the ethanolic extract. Due to the presence of high amounts of polyphenols,
aqueous extract may show promise in preventing or alleviating the progression of chronic disease
conditions such as cancer.



Figure 3. Anti-lipid peroxidation by TBARS method. The graph represents the inhibition of lipid
peroxidation activity by the FRAQ and FRET extracts expressed in percentage (n=3 independent
experiments).

FR
aq
and FR
et
exhibited significant cytotoxic activity in cervical cancer cell lines

It is well known that phytochemicals present in the plants have strong antioxidant and
antiproliferative activities [24]. Thus, we evaluated the cytotoxic potential of both FR
aq
and FR
et

extracts in cervical cancer cell lines, SiHa and HeLa. It was observed that FR
aq
exhibited 100%
viability in both SiHa and HeLa cell lines until 160 g/ml (Figure 4) beyond which it was cytotoxic
to both the types of cell lines. However, FR
et
showed 100% survival till 160 and 80 g/ml in SiHa
and HeLa cell lines, respectively beyond which it was cytotoxic to both the cell types (Figure 5).
Thus our results showed that besides antioxidant potential, F. religiosa aqueous and ethanolic bark
extracts exhibited significant cytotoxic activity in both the cervical cancer cell lines.
449


Figure 4. Cytotoxicity on cervical cancer cell line by MTT assay. The graph represents the cytotoxicity
on cervical cancer cell line SiHa and HeLa when dosed with different concentration (0-320ug/ml) of
FR
aq
(n=3 independent experiments).




Figure 5. Cytotoxicity on cervical cancer cell line by MTT assay. The graph represents the cytotoxicity
on cervical cancer cell line SiHa and HeLa when dosed with different concentration (0-320ug/ml) of FR
et

(n=3 independent experiments).

CONCLUSION

In summary, we have demonstrated the total antioxidant capacity, anti-lipid peroxidation activity,
phenol composition as well as cytotoxic potential of aqueous and ethanolic preparations of bark of
Ficus religiosa. The results confirm that F. religiosa aqueous and alcoholic extracts are potential
450
source of natural antioxidant as investigated by other investigators [9-12]. However, till date, no one
had explored the cytotoxic potential of F. religiosa in cervical cancer. Our data suggest that F.
religiosa could prove to be a potent source of therapeutic drug against cervical cancer.
Acknowledgements: This work was supported by funding from the Interactive Research School
for Health Affairs (IRSHA), Bharati Vidyapeeth University, Pune.

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