Chemical composition and anti-proliferative properties of flowers of Clitoria Ternatea

Abstract

Aqueous and methanol extracts of the flowers of Clitoria ternatea (CT), a popularly plant consumed for blue colour in Nasi Kerabu was selected to explore its cytotoxic effect on six types of normal and cancer-origin cell lines. These included the hormone-dependent breast cancer cell line (MCF-7), non-hormone-dependent breast cancer cell line (MDA-MB-231), human ovary cancer cell line (Caov-3), human cervical cancer cell line (Hela), human liver cancer cell line (HepG2) and human foreskin fibroblast cell line (Hs27). The anti-proliferation activities of the extracts were examined by employing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay through time periods of 24, 48 and 72 hours. Preliminary results showed that the water extracted of CT had significant effects (p < 0.05) against MCF-7 with an IC 50 value of 175.35 μg/ml. Furthermore, the aqueous and methanolic extracts were investigated by Gas Chromatogram-Mass spectrometry (GC-MS). The GC-MS chromatogram analysis of the water extracted had shown five peaks that represented components in the water extract namely mome inositol (38.7%) and pentanal (14.3%). Fifteen chemical constituents were identified in the methanol extract and the major chemical constituents were mome inositol (33.6%), cyclohexen, 1-methyl-4-(1-methylethylideme)-(7.1%), acetic acid, cyano-(6.5%) and hirsutene (5.7%). Heavy metals tested were at very low levels. The analysis conducted on the flowers provides a strong basis for emphasizing the medicinal and nutritional value of CT.

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*Corresponding author.
Email: rabeta75@yahoo.com
International Food Research Journal 20(3): 1229-1234 (2013)
Journal homepage: http://www.ifrj.upm.edu.my
1
Neda, G. D.,
1*
Rabeta, M. S. and
2
Ong, M. T.
1
Food Technology Division, School of Industrial Technology,
2
Institute for Research in
Molecular Medicine, Universiti Sains Malaysia
Chemical composition and anti-proliferative properties of
owers of Clitoria Ternatea
Abstract
Aqueous and methanol extracts of the owers of Clitoria ternatea (CT), a popularly
plant consumed for blue colour in Nasi Kerabu was selected to explore its cytotoxic
effect on six types of normal and cancer-origin cell lines. These included the hormone-
dependent breast cancer cell line (MCF-7), non-hormone-dependent breast cancer cell
line (MDA-MB-231), human ovary cancer cell line (Caov-3), human cervical cancer
cell line (Hela), human liver cancer cell line (HepG2) and human foreskin broblast
cell line (Hs27). The anti-proliferation activities of the extracts were examined by
employing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium
bromide) assay through time periods of 24, 48 and 72 hours. Preliminary results
showed that the water extracted of CT had signicant effects (p < 0.05) against MCF-7
with an IC
50
value of 175.35 μg/ml. Furthermore, the aqueous and methanolic extracts
were investigated by Gas Chromatogram-Mass spectrometry (GC-MS). The GC-MS
chromatogram analysis of the water extracted had shown ve peaks that represented
components in the water extract namely mome inositol (38.7%) and pentanal (14.3%).
Fifteen chemical constituents were identied in the methanol extract and the major
chemical constituents were mome inositol (33.6%), cyclohexen, 1-methyl-4-(1-
methylethylideme)- (7.1%), acetic acid, cyano- (6.5%) and hirsutene (5.7%). Heavy
metals tested were at very low levels. The analysis conducted on the owers provides
a strong basis for emphasizing the medicinal and nutritional value of CT.
Introduction
The World Health Organization (WHO) has
declared that total mortality due to cancer will increase
to 12 million in 2030 whereas this number was only
7.6 million in 2005 (Farooqui et al., 2011). In year
2006, National Cancer Registry had reported about
21,773 cancer cases were diagnosed in Peninsular
Malaysia (Zainal et al., 2006). Recently, cancer
chemoprevention has developed as a major attention
for researchers (Kim et al., 2010).
Treatment used against cancer such as
chemotherapy, radiation, hormone therapy and
immunotherapy can kill both cancer and normal cells
(Cooper, 1993). Hence the potential usage of natural
products as anticancer treatment has been explored
intensively by the scientists (Wan-Nor Izzah et al.,
2009).
CT is originally related to the Leguminosae
(Fabaceae) family (Morris, 2009). The vines of this
plant are climbing, herbaceous, tall and slender with
ve leaets, while the ower colour ranges from
white to blue with a white or yellowish center (Jain et
al., 2003). It is known as buttery pea and commonly
known as Bunga telang in Malaysia. Flowers of
CT are consumed to make Nasi Kerabu blue in
colour, which is a famous local dish in Malaysia.
All parts of CT are useful for medical treatments
and have been used in folk medicines and for curing
different diseases (Mukherjee et al., 2008). The
primary objectives of this study were to determine
the chemical composition of the CT’s owers and
examine the effect of its aqueous and methanolic
extracts on normal and cancer cell lines.
Materials and Method
Flowers of CT, harvested in October 2010
Keywords
Proximate analysis
mineral and heavy metal
content
MTT assay
cancer cell line
Gas Chromatogram-Mass
Spectrometry
Article history
Received: 4 October 2012
Received in revised form:
7 January 2013
Accepted: 11 January 2013

1230
Neda et al./IFRJ 20(3):1229-1234
were obtained from Kampung Seronok, Bayan
Lepas, Pulau Pinang, Malaysia. Hs27 (ATCC
®
CRL-1634™, human foreskin broblast cell line),
MCF-7 (ATCC
®
HTB-22™, hormone-dependent
breast cancer cell line), MDA-MB-231 (ATCC
®
HTB-26™, non-hormone-dependent breast cancer
cell line) Caov-3(ATCC
®
HTB-75™, human ovary
cancer cell line), Hela (ATCC
®
CCL-2TM, human
cervical cancer cell line) and HepG2(ATCC
®
HB-
8065™, human liver cancer cell line) were purchased
from the American Type Culture Collection (ATTC),
USA. Phosphate Buffer Solution (PBS) tablets were
obtained from AMRESCO INC, Cleveland, Ohio,
USA. The media used was Dulbecco’s Modied
Eagle Medium (DMEM with low glucose and high
glucose) and Foetal Bovine Serum (FBS), penicillin–
streptomycin and trypsin were obtained from Gibco
®
,
InvitrogenTM, USA. MTT (3-(4,5-dimethylthiazol-
2-yl) 2,5 diphenyltetrazolium bromide) labelling
reagent was obtained from Molecular Probes
®
,
InvitrogenTM, Oregon, USA.
Sample preparation
Flowers of CT were separated from the stem and
sun dried at room temperature (20
o
C ± 5
o
C) for 4
days. Plant powders were kept in an airtight polyester
container at -20°C before use.
Proximate analysis
Proximate Analysis was determined by using the
method stipulated by Ofcial Agricultural Chemists
(AOAC) International (1990). The six analyses are
including moisture, ash, fat, protein content, ber and
carbohydrate as well.
Determination of mineral and heavy metals content
by Inductively Coupled Plasma Optical Emission
Spectrophotometer (ICP-OES)
The mineral and heavy metal content determined
in the owers of CT included calcium (Ca), zinc
(Zn), iron (Fe), sodium (Na), manganese (Mn),
copper (Cu), nickel (Ni), chromium (Cr), lead (Pb),
boron (B), calcium (Ca), cobalt (Co), potassium (K),
magnesium (Mg), selenium (Se), arsenic (As) and
cadmium (Cd) as well. This analysis was performed
by using Inductively Coupled Plasma Optical
Emission Spectrophotometry (ICP-OES) (OPTIMA
7000DV, Perkin Elmer, USA).
Digestion and sample preparation
Powder dry samples were weighted 1 gram (n =
3) in microwave digester tubes and digested in (10:1)
mixture of nitric acid 65% (HNO
3
) and perchloric acid
(HCLO
4
). Samples were made to undergo digestion
in mineral digester for 60 minutes. After digestion,
a few drops of concentrated hydrochloric acid
(HCl) were added. The solution was heated gently
and then ltrated. Subsequently, the entire ltrate
were transferred into a 100 mL volumetric ask and
marked up with de-ionized water. The dilute ltrate
solutions was transferred into medicine bottles and
then injected into the ICP-OES.
Extraction of sample for anti-proliferative properties
Hot water was used for the aqueous extracts
and methanol was used for the organic extracts.
Extraction was done by soaking the CT owers in
boiling distilled water in the proportion of 1:20 (w/v)
for 4 hours. The resulting crude extracts were ltered
and lyophilized (Huang et al., 2003).
The methanol extract was obtained by maceration
of the powdered owers in 95% methanol for 24
hours. The methanol fraction was collected and the
residual solvent eliminated by reduced pressure
at 40
o
C by using a rotary evaporator. The residue
obtained was dried in a desiccator until it reached a
constant weight (Wicaksono et al., 2009). The extract
produced was used to screen the antiproliferative
properties and stored at -20
o
C until use. The extract
was diluted in PBS and then sterilized before assays.
Final serial dilution was contained in DMEM with
20% FBS.
Cell culture
MCF-7, MDA-MB-231, Caov3, Hela and
HepG2 were grown in DMEM with low glucose,
and the Hs27 was grown in DMEM supplemented
with additional 4.5 g/L of glucose that was used as
a comparison. The cells were cultured in the growth
medium (supplemented with 10% FBS and 1%
penicillin-streptomycin) and incubated overnight at
37
o
C with 5% CO
2
using 25 cm
2
tissue culture asks
(McAteer and Douglas, 1979).
Cell subculture
The cells were grown to 70-80% conuency by the
method of Freshney (1994). Firstly, the old medium
was removed, and subsequently the cells were rinsed
with PBS twice to wash the cells. The subconuent
monolayer was trypsinized and incubated at 37
o
C
and 5% CO
2
for 5 minutes. About 1-2 ml of medium
was added into the ask and the cells were collected
in growth medium containing serum. The cells were
then re-suspended in growth medium, and counted.
The total number of viable cells was counted by a
haemocytometer to prepare the cell suspension. Sixty
microlitres of suspension containing 3 x 10
3
cells/ ml
was added to each well of a 96-well microtiter plate.
The plate was then incubated overnight at 37
o
C with
5% CO
2
.

Neda et al./IFRJ 20(3):1229-1234
1231
Measurement of the growth inhibitory effect
Each of the cancer cell lines was grown in a 96-
well microtiter plate (Nunc, Denmark) in a volume
of 60 μL culture medium per well. The normal and
cancer cells were then treated with 60 μL extracted of
CT owers, which contained a serial dilution at doses
of 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 μg/
mL and the temperature was maintained at 37
o
C with
CO
2
for 24–72 hours. The cells in the rst row of the
96-well microtiter plate were feed with fresh growth
medium for control. After the incubation period, 24
μL of MTT - formazan labelling reagent was added
to each well. The microtiter plate was then incubated
again for 4 hours at 37
o
C with 5% CO
2
. At the end of
the drug period, the medium and MTT were removed
from all of the wells. Subsequently, the remaining
MTT- formazan crystals were solubilised with 100
μL of acidied-isopropanol. One hundred microlitre
distilled water was added into each well for further
colour development. The absorbance of viable cells
was measured using a spectrophotometric plate reader
(Multiskan spectrum, Thermo Electron Co., Waltham,
Massachusetts, USA) at 570 nm immediately, due to
unstable product. To calculate the IC
50
, the processes
giving below were followed.
Cell viability (%) = OD of drug- tested sample - OD of Blank x100
OD of Control – OD of Blank
Dose response curve were constructed using
probit analysis (Finney, 1962) on a nney computer
program Bio Stat
TM
2009 (AnalystSoft Inc.,
Vancouver, Canada) to obtain IC
50
value.
Gas Chromatogram- Mass Spectrometry analysis
GC-MS analyses were conducted to analyze
volatile compounds by using a GC system coupled
to a mass selective detector. The column was VB-1
(30 m x 320 µm). The temperature programming for
the operating condition was: initial oven temperature,
50
o
C for 0 min increased up to 260
o
C at a rate of 10
o
C/
min and held for 9 min; Injector temperature, 260
o
C;
split ratio, 100:1; carrier gas, helium, solvent delay
for 1.70 min; transfer temperature, 260
o
C; ion source
temperature, 260
o
C and mass range 28 to 400 Da.
Statistical analysis
Results for percentage cell viability were reported
as means ± standard error of triplicate measurements.
Signicant differences for multiple comparisons
were determined by one-way analysis of variance
(ANOVA) followed by Duncan test with α = 0.05
using the SPSS statistical package (ver.19.0).
Results and Discussion
Results of the proximate analysis, mineral and
heavy metal content of the CT owers are presented
in Tables 1. In proximate analysis, the parameters
determined were moisture content, ash, crude fat,
crude protein and crude ber as well. The owers of
CT contained appreciable amount of crude ber (2.1
± 0.2) and fat (2.5 ± 0.1). A dietary pattern containing
low-fat and high-ber products has been associated
with reduced risks of breast cancer (Kushi et al.,
2012; Rabeta et al., 2009). Results also indicate
that the owers of CT are rich in calcium (3.09
mg/g) and magnesium (2.23 mg/g). The potassium,
zinc, sodium and iron concentrations of the owers
on this plant were clearly high (1.25, 0.59, 0.14
and 0.14 mg/g), respectively (p < 0.05) than most
of the other parameters analyzed (< 0.01 mg/g). In
addition, minerals such as calcium and magnessium
are necessary for growth, skeletal development and
other vital processes within the body. Iron is useful
for the prevention of anemia and other related
diseases (Oluyemi et al., 2006) while zinc plays a
role in protein synthesis, normal body development
and recovery from illness (Muhammad et al., 2011).
Deciency of these nutrients and minerals can be
detrimental to human health. The heavy metals
tested were at very low levels thus making the plants
relatively safe for consumption.
Figure 1 shows the percentage inhibition exerted
by the water extracted on normal cell line (Hs27)
and various human cancer-origin cell lines such as,
MCF-7, MDA-MB-231, Caov-3, Hela, and HepG2.
Based on The inhibition of cancer-origin cell lines
increased steadily with increasing concentrations
of the water extract and time duration. Growth
Proximate
Mineral
Mineral
Heavy metal
Moisture
92.4±0.1
Boron
0.0150±0.00 2
Magnesium
2.2306±0.13 4
Cadmium
< 0.0001
Ash
0.45±0.15
Calcium
3.0953±0.09
Manganese
0.0249±0.00 3
Arsenic
< 0.0001
Fat
2.5±0.1
Cobalt
< 0.0001
Molybdenum
0.0001±10
-4
×5.7
Lead
0.002333±0.0002
Protein
0.32±0.03
Chromium
0.0007±0.0
Sodium
0.1413±0.00 3
Nickel
0.001267±0.0001
Crude Fibre
2.1±0.2
Cupper
0.0103±0.00 04
Selenium
< 0.0001
Carbohydrate
2.23±0.3
Iron
0.1441±0.00 7
Zinc
0.5980±0.00 6
Potassium
1.2506±0.23 5
Table 1. Proximate analysis, mineral and heavy metal content of owers of CT
Data are mean values ± standard deviation (SD) of triplicate results; for proximate analysis, dry basis and are expressed in percentage (%) and results for
mineral and heavy metal (mg/g).

1232
Neda et al./IFRJ 20(3):1229-1234
inhibition of the cancer origin cell line was most
signicant (p < 0.05) at 72 hours. The water extract
had no effect on normal (non cancer-origin) cell
growth. After treatment with the water extracted,
the inhibition showed more anti proliferations for
MCF7, Caov3, HepG2 and MDA-MB-231 with IC
50
values of 175.3 µg/mL, 224.5 µg/mL, 236.3 µg/mL,
and 304.7 µg/mL at 72 hours, respectively but the
growth of Hela wasn’t effected with the water extract
(Table 2). Figure 2 emphasizes the growth inhibitory
effect of the methanol extract on MCF-7 cell lines
(IC
50
= 536.01 µg /mL), and MDA-MB-231 cell lines
(IC
50
= 561.3 µg/mL) at the 72 hours mark (p < 0.05).
In contrast, the methanol extracted had no effect
on the growth of Caov-3, Hela and HepG2 (Table
2). The water extracted of CT was found to have a
stronger antiproliferative effect in comparison with
the methanolic extract (p < 0.05). This may be due
to the presence of more active compounds into the
water extract.
Several studies have shown that the cytotoxicity
and anticancer properties of natural plant are mainly
due to the presence of avonoids. Phenolic compounds,
including avonoids are especially promising
candidates for cancer prevention. This could have
contributed to the susceptibility of the cells to be
aqueous extract of CT owers. Furthermore, Rajan et
al., (2011) reported similar result that showed extracts
of petroleum ether from CT had cytotoxic activity
against HepG2 cell line. Shyam Kumar and Bhat
(2011) also added that petroleum ether extracted and
ethanolic extract of CT owers had cytotoxic activity
against the Dalton’s Lymphoma Ascites (DLA) cell
Figure 1. Inhibition of water extract of owers from CT
on MCF-7, MDA-MB-231, Caov3, Hela, HepG2 and
Hs27 respectively. A) Treated in 24 hours. B) Treated in
48 hours. C) Treated in 72 hours. Values are expressed as
mean ± standard error (SE) of triplicate measurements. (a,
b, c) Same letters in each concentration are not statistically
signicant from each other at p < 0.05
Table 2. The IC
50
of water and methanol extract from
owers of CT on cancer cell lines
Cell lines
24 hr
48 hr
72 hr
Water
Methanol
Water
Methanol
Water
Methanol
Caov3
8386.5
41333.1
857.1
2109.9
224.5
947.2
Hela
42274.3
20381.7
18835.4
6281.1
51513.7
2095.7
HepG 2
1438512
40674.6
481.5
23880
236.3
5214.1
MDA-MB-231
55355.3
-
481.5
4343.6
304.7
536.01
MCF-7
42567.6
-
1159.2
1983.4
175.35
561.3
Using Probit analysis on a nney computer program Bio Stat
TM
2009.
Probit analysis is a type of regression used to analyze binomial response variables.
Figure 2. Inhibition of methanol extract of owers from
CT on MCF-7, MDA-MB-231, Caov3, Hela, HepG2 and
Hs27 respectively. A) Treated in 24 hours. B) Treated in
48 hours. C) Treated in 72 hours. Values are expressed as
mean ± standard error (SE) of triplicate measurements. (a,
b, c) Same letters in each concentration are not statistically
signicant from each other at p < 0.05

Neda et al./IFRJ 20(3):1229-1234
1233
line. Based on the growth inhibitory properties of the
extracts observed, GC-MS analysis were obtained.
The components presented in the water extract
from owers of CT were mome inositol (38.7%)
and pentanal (14.3%) (Table 3). The GC-MS
chromatogram of the methanol extracte (Table 4)
showed 15 peaks indicating the presence of three
phytochemical constituents. The components
presented in the methanol extract from owers of CT
were mome inositol (33.6%), cyclohexen, 1-methyl-
4-(1-methylethylideme)- (7.1%), acetic acid, cyano-
(6.5%) and hirsutene (5.7%). Furthermore, Studies
from in vitro experiments, animal studies, and limited
clinical experiences, claim that inositol may be used
effectively against some types of cancer, particularly
when used in combination with phytic acid (Vucenik
and Shamsuddin, 2003). These compounds may
be responsible for the anti-cancer activity observed
during in this study. However, further in vivo study is
needed to conrm our ndings and evaluating actual
anti-proliferative properties in the CT owers.
Conclusion
Conclusively, better inhibitions of cancer cell
lines were observed in the water extract (IC
50
of 175.3
μg/ml for MCF7). Knowing the exact compounds
responsible for the plant’s anticancer properties will
Table 3. Potocomponents identied in the water extract CT owers by GC-MS
Table 4 . Potocomponents identied in the methanol extract from owers of CT by GC-MS

1234
Neda et al./IFRJ 20(3):1229-1234
help in formulating anticancer agents. In addition, it
results from the proximate and mineral constituent
analysis at the plant has provided pertinent information
for food formulations.
Acknowledgements
We would like to acknowledge the excellent
technical guidance and support Ms. Lam Kit
Lay from Institute for Research in Molecular
Medicine (INFORMM). The authors are thankful
to Universiti Sains Malaysia Short Term Grant
304/PTEKIND/6310065 and School of Industrial
Technology, USM.
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