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Int. J. Pharm & Ind. Res Vol - 01 Issue - 04 Oct Dec 2011
Original Article
ISSN
Print 2231 3648
Online 2231 3656
CYTOTOXIC ACTIVITY OF ETHANOLIC EXTRACTS OF
CAESALPINIA SAPPAN LINN AND ANAONA SQUAMOSA LINN.
IN A-549 CELL LINE
*1
Hemalatha K,
1
Sunitha D,
2
Satyanarayana D
*1
Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally,
Secundrabad, Hyderabad, Andhra Pradesh. India - 500 014.
2
N.G.S.M.Institute of Pharmaceutical Science, Paneer, Deralakatte,
Mangalore, Karnataka, India - 574 160.
Introduction
Caesalpina sappan Linn (Caesalpiniceae) is commonly
known as Sappan wood or patang (Hindi). It is
spreading tree or shrub upto 10 m in height found in
India (West Bengal, Orissa, Kerala) Malaya, China
and Sri Lanka.
1
. The seeds contain n-triactone, lupeol,
-amyrin, stigmosterol and diterpenoidal alcoholic
compounds
2
. The chloroform extract of C. sappan on
cell death in head and neck cancer cell lines. The results
suggest that the chloroform extract of C. sappan may
increased cell death in HNSCC4 and HNSCC31 cells,
which are liked to increased cellular levels of p53 and
p21WAF/CIPI
3
.
Author for Correspondence:
Hemalatha K,
Malla Reddy College of Pharmacy, Secundrabad,
Hyderabad, Andhra Pradesh. India - 500 014.
Email: kamurthy18@gmail.com
The methanol extract and two purified compounds,
brazilin and hematoxylin, isolated from the wood
showed a significant and dose dependant
vasorelaxing effect
4
. Several triterpenoids, flavanoids,
and steroids have been isolated from the heartwood of
Caesalpinia sappan
5
. Annona squamosa Linn
(Annonaceae) is commonly called custard apple in
English and Sharifa in hindi
6
. The plant is reputed to
possess varied medicinal properties like, cytotoxic
7
and
antioxidant activities
8
. Numerous Annonaceous
acetogenins have been shown antimalarial, cell growth
inhibitory
9
, antiparasitic and antimicrobial activities.
From the leaves of Annona squamosa, a
tetrahydroisoquinoline alkaloid with cardio tonic
activity
10
and a bioactive acetogenins like bulatacin
and bullatacinone from its bark have been isolated
11
.
Squamocin, another Annonaceous has been reported to
exert antiproliferative effect on HL-60 cancer cells via
Abstract
The present study was carried out to evaluate the in vitro cytotoxic activity of unexploited plants, heartwood
of Caesalpinia sappan Linn and roots of Annona squamosa Linn. on A-549 lung cancer cell line, which are
indigenous to India. Different concentrations of the methanolic extracts of heartwood and root parts of the
plant (1000, 500, 250, 125, 50, 25, 12.5 g/ml) were subjected to cytotoxic study against A-549 lung
cancer cell lines by Trypan blue dye exclusion technique. In addition, a phytochemical screening of the
ethanolic extracts was done. The phytochemical screening demonstrated the presence of different types of
compounds like flavonoids, triterpenoids, alkaloids, acetogenins, phenols and sterols. The maximum reducing
power of the Caesalpinia sappan and Annona squamosa extract at 680nm was found to be 0.976 0.051 at
1000 g/ml and 0.953 0.037 at 1000 g/ml respectively. The inhibition percentage with regard to
cytotoxicity was found to be 87 % at 1000 g/ml with IC50 value of 49 0.03 g/ml for Caesalpinia sappan
and 85 % at 1000 g/ml with IC50 value of 47 0.02 g/ml for Annona squamosa respectively. The
ethanolic extracts of Caesalpinia sappan and Annona squamosa are showing potent cytotoxic activity against
A-459 lung cancer cell line.
Key words: Caesalpinia sappan Linn., Annona squamos Linn., Ethanolic extracts.
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Int. J. Pharm & Ind. Res Vol - 01 Issue - 04 Oct Dec 2011
activation of caspase-3
12
. In the Ayurvedic system of
medicine, herbal extracts but not purified compounds
have been used from centuries because many
constituents with more than one mechanism of action
are considered to be beneficial. The present study
aimed to evaluate the possible cytotoxic activity of the
heartwood of Caesalpinia sappan and roots of Annona
squamosa against lung cancer cell line.
Material and Methods
Plant Material
Heartwood of Caesalpinia sappan Linn and roots of
Annona squamosa Linn were collected from Medicinal
garden of SDM College of Ayurveda, Udupi and
authenticated by Dr. T. Shridhar Bairy by comparison
with the standard specimens deposited at the
department of Drava Guna. SDM college of ayurveda.
Udupi. Voucher specimens are kept at the NGSM
Institute of Pharmaceutical Sciences, Deralakatte,
Mangalore, Karnataka. India. The powdered shade
dried plant materials were exhaustively extracted with
ethanol using soxhlet apparatus. The extract was
concentrated to dryness. A phytochemical screening of
the ethanolic extracts was performed. Further more;
the dried ethanolic extract was used for evaluation of
cytotoxicity activities.
Preliminary Phytochemical Screening
Conventional standard protocols
13, 14
for detecting the
presence of different chemical constituents in the plant
extracts were employed. The tests for the secondary
metabolites viz. alkaloids, tannins, sterols, saponins,
amino acids, glycosides, proteins, sterols/terpenes,
reducing sugars, non-reducing sugars, resins flavonoids
and phenols were carried out with the ethanolic
extracts of heartwood of Caesalpinia sappan and roots
of Annona squamosa using preliminary phytochemical
screening.
Assay of Cytotoxic Activity
The A-549 cell lines (lung carcinoma cells) used for the
assay were obtained from Christian Medical College,
Vellore. Tamil nadu. India. The stock cells were cultured
in DMEM with 10% Fetal Bovine Serum (FBS), Penicillin
(100 IU/ml) Streptomycin (100 g/ml) and
Amphotericin-B (5 g/ml) in a humidified atmosphere
of 5 % CO2 at 37C. The cells were dissociated with
0.2 % trypsin in phosphate buffer saline solution. The
stock cultures were grown in 25cm
2
tissue culture flasks
and all cytotoxicity experiments were carried out in 6
well plates.
Viability Staining by Trypan blue dye exclusion
method
Cytotoxic activity of ethanolic extracts of Caesalpinia
sappan and Annaona squamosa were analysed by
Trypan Blue dye exclusion method
15
. Cell lines in
exponential growth phase were washed with
phosphate buffer saline (PBS) solution and trypsinized
and re-suspended in complete culture media. Cells
were plated at 30,000 cells/well in 6 well plates and
incubated for 24 hours during which a partial
monolayer forms. After incubation the cells were
exposed to various concentrations of the drugs, which is
the plant extract (1000g/ml, 500g/ml, 250g/ml,
150g/ml, 125g/ml, 50g/ml and 25g/ml). The
control well received only maintenance of medium. The
plates were incubated at 37C in a humidified
incubator with 5% CO2 for a period of 24 hours.
Morphological changes of drug treated cells were
examined using an inverted microscope and compared
with the cells serving as control. At the end of 24 hours
incubation, cell viability was determined.
Calculations and statistics
Experiments were performed in six replicates. Results
were expressed as percentage growth inhibition of
control. IC50 values were derived from a nonlinear
regression model (curvefit) based on sigmoidal dose
response curve (variable) and computed using
Graphpad Prism version 3.00 were expressed as
meanS.E.M.
Table 01: Cytotoxic Activity of ethanolic extract of
Caesalpinia sappan and Annona squamosa
against A-549.
Concentration
(g/ml)
Cytotoxic activity (%) IC50 (g/ml)
C. Sappan A. squamosa C. Sappan A. squamosa
1000 87 86
490.03 470.02
500 71 67
250 62 55
125 58 52
50 56 47
25 50 46
12.5 40 36
Each value represents mean S.E.M. of six replicates (n=6).
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Int. J. Pharm & Ind. Res Vol - 01 Issue - 04 Oct Dec 2011
Results and Discussion
The results of the phytochemical screening of the
investigated methanolic extracts showed the presence
of different types of active constituents. Ethanolic
extract of heartwood of C. sappan showed presence of
flavonoids, triterpenes, polyphenols and sterols,
ethanolic extract of roots of A. squamosa showed
presence of alkaloids, acetogenins, and absence of
flavonoids and terpenoids. The maximum reducing
power of the C. sappan and A. squamosa extract at
680nm was found to be 0.9760051 at 1000 g/ml
and 0.9530037 at 1000 g/ml respectively (Fig.
1a, Fig. 1b & Fig. 1c). The inhibition percentage with
regard to cytotoxicity was found to be 87 % at
1000 g/ml with IC50 value of 490.03 g/ml for C.
sappan (Table-1) and 85 % at 1000 g/ml with IC50
value of 470.02g/ml for A.squamosa (Table-01)
respectively. The in-vitro screening of the ethanolic
extracts of C. sappan and A. squamosa showed
potential cytotoxic activity against the breast cancer
cells. The results obtained are shown in table no-1. The
results obtained from the present study showed that the
C. sappan and A. squamosa are moderately cytotoxic
activity. The cytotoxic activity may be due to the
presence of flavonoids, alkaloids, acetogenins, sterols,
polyphenols and terpenoids present in the heartwood
of C. sappan and roots of A. squamosa respectively.
Conclusion
The results of the study revealed that the plant extracts
have strong anticancer activity. Our phytochemical
screening revealed the presence of terpenoid,
flavonoids, alkaloids and acetogenins in the ethanolic
extracts of C. sappan and A. squamosa respectively,
which could be responsible for these noteworthy
activities. It also justifies the folklore medicinal uses and
claims about the therapeutic values of this plant as
curative agent against cancer and we therefore,
suggest further, the purification and characterization of
the phytochemicals along with investigations are
needed to provide some additional insight into the in-
vivo cytotoxic activity of the plants with a view to
obtaining useful chemotherapeutic agent. In nutshell,
extracts of C. sappan and A. squamosa have
remarkable anticancer potentials against A-549 Lung
cancer cell lines. Drug prepared from extracts of
C.sappan and A. squamosa could be an excellent drug
for treating lung cancer.
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