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Original article
Butterfly pea (Clitoria ternatea) seed and petal extracts decreased
HEp-2 carcinoma cell viability
Yixiao Shen,
1
Liqing Du,
2
Haiying Zeng,
3
Xiumei Zhang,
2
Witoon Prinyawiwatkul,
1
Jose R. Alonso-Marenco
1
&
Zhimin Xu
1
*
1 School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA
2 The Key Laboratory of Tropical Fruit Biology of Ministry of Agriculture, The South Subtropical Crop Research Institute, Chinese
Academy of Tropical Agricultural Science, 20 Jiefang W Rd, Zhanjiang, Guangdong 524001, China
3 School of Liquor and Food Engineering, Guizhou University, Xueshi Rd, Huaxi District, Guiyang, Guizhou 550025, China
(Received 23 February 2016; Accepted in revised form 29 April 2016)
Summary The hydrophilic phenolics, lipophilic tocopherols, phytosterols and fatty acids in butterfly pea seeds and
petals were determined. The seeds had fifteen phenolics; of them, sinapic acid, epicatechin and hydrox-
ycinnamic acid derivative concentrations were above 0.5 mg g
1
. The petals contained a group of ter-
natins, flavone glycosides and delphinidin derivatives. Both the seeds and petals had four phytosterols
and a- and c-tocopherols. However, the level of b-sitosterol or c-tocopherol in the seeds was much higher
than in the petals. Linoleic acid was the most abundant fatty acid in the seeds and petals, while phytanic
acid was found in the petals. The effect of lipophilic and hydrophilic extracts of the seeds [lipophilic
extract of the butterfly pea seeds (LBS) and hydrophilic extract of butterfly pea seeds (HBS)] and petals
[lipophilic extract of the butterfly pea petals (LBP) and hydrophilic extract of butterfly pea petals (HBP)]
on decreased HEp-2 human carcinoma cell viability was evaluated. The effect of HBS or HBP on
decreased cancer cell viability was much higher than that of either LBS or LBP, while HBS showed signif-
icantly higher effect than HBP. The results indicated that butterfly pea seed and petal extracts could have
the potential in functional food development.
Keywords Cell viability, lipids, phytosterols, polyphenols, ternatins, tocopherols.
Introduction
Butterfly pea (Clitoria ternatea), a member of Faba-
ceae family and Papilionaceae subfamily, is a perennial
leguminous twiner. Approximately 60 butterfly pea
species are distributed within the tropical belt, while a
few species are found in temperate areas (Al-Asmari
et al., 2014). The anthocyanins abundant in butterfly
pea petals make the petals as a natural blue colorant
source for a variety of food products (Mukherjee
et al., 2008). Also, triterpenoids, flavonol glycosides
and alkaloids were found in butterfly pea leaves, while
pentacyclic triterpenoids, taraxerol and taraxerone
were identified in the roots (Singh & Tiwari, 2010).
However, the chemical composition of butterfly pea
seeds, especially its bioactive constituents such as phe-
nolics, tocols and phytosterols, has not been docu-
mented. In this study, nonpolar solvent and polar
solvent were used to obtain lipophilic and hydrophilic
extracts of butterfly pea seeds and petals, respectively.
The phytochemicals in each extract were identified for
exploring the potential bioactive components in the
butterfly pea seeds and petals. Those identified phyto-
chemicals are a group of antioxidants that may play
an important role in the bioactive functions of butter-
fly pea.
The reported health-promoting functions of butterfly
pea include antidiabetic, nootropic, anxiolytic, anticon-
vulsant, sedative, antipyretic, anti-inflammatory and
analgesic functions (Jain & Shukla, 2011). However,
the anticancer potential of butterfly pea has not been
studied. In this study, a laryngeal carcinoma cell line
was used to examine the anticancer effect of butterfly
pea seed and petal extracts. Laryngeal carcinoma
accounts for 25% of head and neck carcinoma and is
the second most common respiratory tract cancer fol-
lowing lung cancer (Mirunalini et al., 2011). The limit-
less replication of the cancer cells and the multiple
interactions with their microenvironments increase the
*Correspondent: Fax: 225-578-5300; e-mail: zxu@agcenter.lsu.edu
The first two authors contributed equally to this article.
International Journal of Food Science and Technology 2016, 51, 1860–1868
doi:10.1111/ijfs.13158
©2016 Institute of Food Science and Technology
1860
difficulty in treating the cancer (Pienta et al., 2008).
Primary clinical treatments for laryngeal cancer are
surgery, chemotherapy and radiotherapy. However,
they induce seriously adverse side effects or even result
in resistance to these therapies (Agostinis et al., 2011).
Recently, much attention to cancer treatment has been
focused on some plant-derived compounds that have
pharmacological functions on the tumour but have less
side effects (Veerabadran et al., 2013). In this study,
the anticancer effect of hydrophilic and lipophilic
extracts of butterfly pea petal and seed on decreased
laryngeal cancer cell (HEp-2) viability was studied and
compared. The aim of this study was to provide the
bioactive phytochemical profiles in both butterfly pea
petals and seeds and also emphasise the role of those
bioactive compounds in anticancer activity.
Materials and methods
Chemicals and materials
High-performance liquid chromatography (HPLC)-
grade acetonitrile, acetic acid, methanol and hexane
were purchased from Fisher Chemicals (Fair Lawn,
NJ, USA). Acetone was purchased from Macron
(Charlotte, NC, USA). Ethyl acetate was purchased
from EM Science (Gibbstown, NJ, USA). Trimethylsi-
lyl imidazole (TMS), BCl
3
methanol and phenolics,
fatty acids and tocopherol standards were purchased
from Sigma Aldrich (St. Louis, MO, USA). Fresh but-
terfly pea seeds and petals (Clitoria ternatea) were
obtained from a local garden in Baton Rouge, LA,
USA. The human carcinoma HEp-2 cell line was pur-
chased from American Type Culture Collection
(ATCC, Manassas, VA, USA). Other reagents and
culture media containing foetal bovine serum (FBS),
antibiotic (penicillin–streptomycin), CellTiter-Blue,
dimethyl sulphoxide (DMSO) and phosphate-buffered
saline (PBS) were purchased from Invitrogen (Grand
Island, NY, USA).
Extraction of hydrophilic and lipophilic bioactive
compounds in butterfly pea seeds and petals
The freeze-dried butterfly pea seeds and petals were
ground by a coffee blender (Hamilton Beach, Southern
Pines, NC, USA). The hydrophilic compounds in the
petals or seeds (20 g) were extracted using 50 mL of
methanol at 60 °C for 20 min. After centrifugation,
the methanol layer was transferred to a clean tube.
Then, the solid residues were extracted repeatedly for
two more times at the same condition and using the
same procedure. The dried hydrophilic extract was
obtained by evaporating the collected methanol using
a vacuum centrifuge evaporator (Labconco, Kansas
City, MO, USA). The dried extracts were then
prepared for a stock solution (50 mg mL
1
in metha-
nol). For the lipophilic extract of butterfly pea petals
or seeds, 50 mL of a solvent mixture of ethyl acetate
and hexane (50:50; v:v) was used for the extraction
according to the same procedure used for obtaining
the hydrophilic extract. After the solvent extract was
dried, a stock solution of the lipophilic extract
(50 mg mL
1
in hexane) was prepared as well.
Identification and quantification of hydrophilic and
lipophilic phytochemicals and fatty acids
Phytochemical profiles of the hydrophilic extracts were
determined by a reverse-phase HPLC (2690; Waters,
Torrance, CA, USA) coupled with C18 column (id
250 94.60 mm, 5 lm; Phenomenex, Torrance, CA,
USA) and a diode array detector and the operation
condition for HPLC was as reported in the study of
Du et al. (2014). The concentrations of the phenolics
were calculated based on their corresponding standard
curves. Both the extracts were also subject to an LC-
MS analysis to identify the compounds without their
standards available. The LC-MS consisted of a ultra
performance liquid chromatography (UPLC) system
(Thermo Scientific Dionex UltiMate 3000, Waltham,
MA, USA) and a mass spectrometry (MS) (Q Exac-
tive
TM
Plus Hybrid Quadrupole-Orbitrap
TM
) that had
an electrospray ionisation source (ESI) in the positive
mode with a full MS scan from 150 to 2000 m/z. The
LC-MS separation was carried out using a reverse-
phase column (Acclaim
Ò
Mixed-Mode WAX-1,
150 92.1 mm, 5 lm). The mobile phase consisted of
solution A (1% formic acid solution) and solution B
(acetonitrile) at a constant flow rate of 0.2 mL min
1
with a gradient programme of 0–70% B at 0.0–
8.0 min, 70–100% B at 8.0–10.0 min, 100–0% B at
10–15 min. The MS parameters were set as follows:
electric potential of the ESI source, 3.0 kV; capillary
temperature, 300 °C; heater temperature, 200 °C. The
concentrations of ternatins and delphinidin derivative
were calculated by the standard curve of cyanidin
chloride in molar concentration and then converted to
mass unit (mg g
1
) in the sample based on their
molecular weights. Tocopherols in the samples were
determined by a normal-phase HPLC (1100 series;
Agilent, Santa Clara, CA, USA) with Supelcosil LC-Si
column (id 250 94.60 mm 5 lm, Supelco, Bellefonte,
PA, USA). The condition of the HPLC analysis was
the same as the method described in Jang & Xu
(2009).
The fatty acids in each lipophilic extract were esteri-
fied using 2 mL of BCl
3
(boron trichloride) methanol
after 200 lg of internal standard (C17:0) was added.
The reaction mixture was incubated at 60 °C for
30 min. Then, 1 mL of water and 1 mL of hexane
were added to the reaction solution and vortexed.
©2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1861
After centrifugation, the upper hexane layer was trans-
ferred to a clean test tube and mixed with anhydrous
Na
2
SO
4
to remove any moisture before it was trans-
ferred to a gas chromatography (GC) vial. The fatty
acids were determined by a GC equipped with an
flame ionisation detector (FID) and a Supelco SP2380
(30 m 90.25 mm) column. The GC condition was the
same as that used in the study of Yue et al. (2008).
The determination of phytosterols was based on the
study of Xu & Godber (1999). After the lipophilic
extract was mixed with an aliquot of hexane contain-
ing 20 lg cholesterol internal standard, it was dried
and then reacted with 200 lL of TMS and 50 lLof
acetonitrile at 65 °C for 30 min. The derived products
were extracted using 200 lL of hexane and analysed
by GC-MS. A Varian CP-3800 GC (Walnut Creek,
CA, USA) coupled with a Saturn 2200 mass spectrom-
eter and a DB-5 column (60 m 90.25 mm) (Supelco)
was used in the analysis. The initial oven temperature
was 200 °C. Then, it was ramped to 280 °C at a rate
of 10 °C min
1
and held at the final temperature for
62 min. Helium was the carrier gas at a constant flow
rate of 1.5 mL min
1
. The injection port temperature
and split ratio were 280 °C and 1:50, respectively. The
ratios of peak areas at different levels (5, 10, 20, 50
and 100 lg) of campesterol, stigmasterol and b-sitos-
terol to 20 lg of cholesterol internal standard were
used to set up the standard curves for quantifying the
phytosterols.
Determination of capability of decreasing human
carcinoma cell (HEp-2) viability
The human carcinoma HEp-2 cell line was used to
assess the potential of butterfly pea seed and petal
extracts in decreasing cancer cell viability. The cell line
was cultured in Dulbecco’s modified Eagle’s media
(DMEM), supplemented with 10% foetal bovine
serum (FBS) and 1% antibiotic (penicillin–strepto-
mycin), and grown in a 5% CO
2
atmosphere with
95% humidity at 37 °C for 24 h. Then, the cells were
harvested, counted (3 910
4
cells mL
1
) and trans-
ferred into a 96-well plate. The working solution of
each extract was prepared by dissolving the extract
with 0.2% DMSO in PBS culture media. The treat-
ment solutions were prepared based on a group of the
concentrations multiplied by the low and effective con-
centration of each type of extract that was obtained in
our preliminary study. The HEp-2 cells were incubated
with a series of concentrations of the hydrophilic
extract working solution (1.0, 0.5, 0.25, 0.12 and
0.06 mg mL
1
) or lipophilic extract working solution
(12.0, 9.0, 6.0, 3.0 and 1.5 mg mL
1
) for 96 h at
37 °C for a dose-dependent study. The cells only
mixed with the media and 0.2% DMSO were used as
the control group. For measuring the cell viability, the
media were discarded and replaced with 100 lLof
fresh media containing 20% CellTiter-Blue. After the
cells were stained for 4 h, the fluorescence intensity of
the media was read at excitation/emission wavelengths
of 570/615 nm using a FluoStar Optima microplate
reader (BMG, Ortenberg, Germany). The potential of
the extract in decreasing human carcinoma cell viabil-
ity at each concentration treatment was expressed by a
survival rate, which was the percentage of the intensity
of the treatment vs. the intensity of the control.
Data analysis
The determinations of hydrophilic and lipophilic phy-
tochemicals and fatty acids in each extract were
repeated in triplicate and expressed as means stan-
dard deviation. The significant differences between the
concentrations of each compound in the hydrophilic
or lipophilic extracts were determined by one-way
ANOVA at P<0.05 (SAS, 9.1.3, Cary, NY, USA). The
determination of the potential of each treatment con-
centration or control in decreasing the cell viability
was repeated five times and analysed by GraphPad
Prism (version 6.0; GraphPad Software Inc., La Jolla,
CA, USA). The differences in the potential in decreas-
ing the cell viability between the treatments and con-
trol were analysed by two-way ANOVA at P<0.05.
Results and discussions
Hydrophilic and lipophilic phytochemicals and fatty acids
in butterfly pea seeds and petals
The yields of the HBS and HBP extracts were 3.66
and 4.05%, respectively. The chromatograms of the
hydrophilic phenolics in HBS and HBP are shown in
Figs 1 and 2, respectively. In addition to the high con-
centration of ascorbic acid [1.32 0.02 mg g
1
fresh
weight (FW)], sinapic acid was the dominant phenolic
compound among the fifteen major hydrophilic pheno-
lics at a concentration of 1.01 0.07 mg g
1
FW, fol-
lowed by epicatechin and gallic acid in the seed
(Table 1). Compared with the contents in rapeseeds
(0.09–0.59 mg g
1
FW) and camelina seeds
(0.39 mg g
1
FW) (Ni
ciforovi
c & Abramovi
c, 2014),
the sinapic acid content in butterfly pea seeds was
much higher than each of them (Table 1). It was
reported that sinapic acid could help suppress the
expression of proinflammatory mediators via NF-jB
inactivation in regulating inflammatory status and
immune response (Yun et al., 2008). Epicatechin, in
the butterfly pea seeds, which was reported to exhibit
immunoregulatory, antihypertensive effects as well,
was ten times higher (0.56 mg g
1
FW, Table 1) than
that reported in the garden pea seeds (Pisum sativum)
(0.05 mg g
1
) (Ferraro et al., 2014; Litterio et al.,
©2016 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al.1862
2015). Protocatechuic, p-coumaric, rutin and two
hydroxycinnamic acid derivatives in the butterfly pea
seeds were all above 0.30 mg g
1
FW, while kaemp-
ferol, apigenin, caffeic, syringic, ferulic, rosmarinic and
cinnamic acids were in a range of 0.04 to 0.22 mg g
1
FW (Table 1). Compared with the rutin content in edi-
ble Amaranthus seeds, which was between 0.0711 and
0.0775 mg g
1
dry weight (DW) (Li et al., 2015), the
rutin content in butterfly pea seeds was much higher
and reached 0.31 mg g
1
FW (Table 1). Although
mung bean, radish, broccoli and sunflower seeds con-
tain gallic, protocatechuic, caffeic, p-coumaric, ferulic,
chlorogenic, sinapic acid, quercetin and kaempferol
(Paja
zket al., 2014), each of their levels was signifi-
cantly lower than that in the butterfly pea seeds.
The unique phytochemicals in butterfly pea petals
are ternatins, a group of polyacylated delphinidin
derivatives (Sasaki et al., 2013). It was reported that
ternatin A1-A3, B1-B4, C1-C5 and D1-D3 consist of
delphinidin 3, 30,5
0-triglucoside attached with malonic
acid, glucose, p-coumaric acid or caffeic acid (Kazuma
et al., 2003). In this study, ternatin C2 and D2 in the
butterfly pea petals were observed at higher concentra-
tions of 1.81 0.09 and 1.45 0.07 mg g
1
FW,
respectively. The concentrations of ternatin A1, B3,
D3 and B2 were in a range of 0.32 to 0.51 mg g
1
FW
(Table 2). The chemical structures of the six types of
ternatins are elucidated in Fig. 3. Two delphinidin
derivatives and cyanidin-3-sophoroside were also iden-
tified and responsible for the blue colour of the petals
together with ternatins. In general, kaempferol 3-neo-
hesperidoside, quercetin 3-(2G-rhamnosylrutinoside)
and rutin were the major flavanol glycoside com-
pounds in the petals, while ellagic acid was the only
phenolic acid identified in butterfly pea petals
(Table 2).
The yields of the lipophilic extracts, LBS and LBP,
were 5.28 and 0.80%, respectively. Lipophilic phytos-
terols are chemically characterised as triterpenes and
considered to be one of the structural components of
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Min
10.00 20.00 30.00 40.00 50.00 60.00 70.00
1
2
3
4
5
78
9
10
11
12
13
14
15
16
6
Figure 1 Chromatogram of the hydrophilic
butterfly pea seed extract. 1 –vitamin C; 2 –
gallic acid; 3 –protocatechuic acid; 4 –epi-
catechin; 5 –caffeic acid; 6 –syringic acid;
7–sinapic acid; 8 –hydroxycinnamic acid
derivatives; 9 –p-coumaric acid; 10 –
hydroxycinnamic acid derivatives; 11 –rutin;
12 –ferulic acid; 13 –rosmarinic acid; 14 –
cinnamic acid; 15 –kaempferol; 16 –api-
genin.
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Min
10.00 20.00 30.00 40.00 50.00 60.00 70.00
1
2 4
5
6
7
8
9
10 12
13
11
3
Figure 2 Chromatogram of the hydrophilic
butterfly pea petal extract. 1 –cyanidin-
3-sophoroside; 2 –delphinidin derivative; 3 –
ternatin A1; 4 –ternatin B3; 5 –ternatin
D3; 6 –ellagic acid; 7 –rutin; 8 –delphini-
din derivative; 9 –kaempferol-3-neohesperi-
doside; 10 –quercetin-3-(2G-
rhamnosylrutinoside); 11 –ternatin B2;
12 –ternatin C2; 13 –ternatin D2.
©2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1863
plant cell membranes (Moreau et al., 2002). Similar to
the function of cholesterol in animal cells, free phytos-
terols serve to stabilise phospholipid bilayers in the
plant cells (Moreau et al., 2002). Although several
studies investigated phytosterols in Clitoria ternatea
species, most of them only focused on leaves, roots or
petals (Kapoor & Purohit, 2013). In this study, b-sitos-
terol (40.17 3.73 mg/100 g FW) in the butterfly pea
seed extract LBS was significantly higher than that of
the petal extract LBP (6.77 0.19 mg/100 g FW)
(Table 3). It was reported that the level in butterfly
pea roots and shoots was between 6 and 9 mg/100 g
(Kapoor & Purohit, 2013). Also, the seed extract LBS
contained campesterol at a level of 8.07 0.22 mg/
100 g FW, which was several times higher than that of
the petal extract LBP (1.24 0.02 mg/100 g FW)
(Table 3) and some vegetable seeds such as pepper
seeds (4.23–5.41 mg/100 g FW) and tomato seeds
(1.08–6.56 mg/100 g FW) (Silva et al., 2013; Ancos
et al., 2015). However, the levels of stigmasterol in
butterfly pea seeds and petals were similar
(7.95 0.63 and 6.70 0.83 mg/100 g FW, respec-
tively) (Table 3) and approximately five and eight
times higher than in berryfruit (0.50–1.60 mg/100 g
FW) and pepper seeds (0.63–0.93 mg/100 g FW) (Silva
et al., 2013; Salvador et al., 2015). Phytosterols were
confirmed to possess hypocholesterolaemic function
and reduce the risks of benign prostatic hyperplasia,
cardiovascular diseases, colon and breast cancer devel-
opment, as well as immunological effects in macro-
phages (Hamedi et al., 2014).
As for the tocol contents, the butterfly pea seeds
had abundant c-tocopherol (5.44 0.30 mg/100 g
FW) compared with the grape seeds (14.1–
30.2 mg kg
1
) and Jatropha curcas seeds
(33.9 mg kg
1
) reported in the studies of Sabir et al.
(2012) and Corzo-Valladares et al. (2012), respectively.
However, c-tocopherol in the butterfly pea petals was
twenty times lower than that in the seeds (Table 3).
The levels of a-tocopherol in butterfly pea seeds and
petals were similar (0.17 0.06 and 0.20 0.06 mg/
100 g FW, respectively) (Table 3). These tocols have
been evidenced for protecting cell membrane against
reactive lipid radicals and for the prevention of
atherosclerosis and carcinogenesis (Yang et al., 2013).
For the fatty acids profile, both the butterfly pea
seeds and petals had palmitic acid (C16:0), stearic acid
(C18:0), petroselinic acid (C18:1), linoleic acid (C18:2),
arachidic acid (C20:0) and behenic acid (C22:0)
(Table 3). Among them, linoleic acid was the most
abundant fatty acid and had 8.73 0.61 and
4.72 0.51 mg g
1
FW in the butterfly pea seeds and
petals, respectively. It is well known that linoleic acid
is an essential fatty acid and required for assisting nor-
mal biological activities in the brain and heart (Blan-
chard et al., 2013). The palmitic, stearic and
petroselinic acids in the seeds and petals were all above
1.0 mg g
1
(Table 3). Different from the results of a
previous study (Mukherjee et al., 2008), arachidic and
behenic acids were first time observed in the butterfly
pea by this study (Table 3). Furthermore, phytanic
acid was found in the butterfly pea petals and might
be derived from a microbial breakdown of chlorophyll
to release phytol followed by the further oxidation to
phytanic acid (Jansen & Wanders, 2006). In the study
Table 1 Hydrophilic compounds identified in the butterfly pea
seeds
Peak No. Compounds
Concentration
(mg g
1
FW)
1 Ascorbic acid 1.32 0.02
2 Gallic acid 0.42 0.00
3 Protocatechuic acid 0.34 0.01
4 Epicatechin 0.56 0.03
5 Caffeic acid 0.22 0.01
6 Syringic acid 0.14 0.01
7 Sinapic acid 1.01 0.07
8 Hydroxycinnamic
acid derivative 1
0.57 0.19
9p-Coumaric acid 0.30 0.01
10 Hydroxycinnamic
acid derivative 2
0.44 0.02
11 Rutin 0.31 0.01
12 Ferulic acid 0.15 0.00
13 Rosmarinic acid 0.05 0.00
14 Cinnamic acid 0.08 0.00
15 Kaempferol 0.04 0.00
16 Apigenin 0.09 0.00
Table 2 Hydrophilic compounds identified in the butterfly pea
petals
Peak No. Compounds
Concentration
(mg g
1
FW)
1 Cyanidin-3-sophoroside 0.31 0.02
2 Delphinidin derivative 0.28 0.01
3 Ternatin A1 0.51 0.03
4 Ternatin B3 0.50 0.03
5 Ternatin D3 0.54 0.01
6 Ellagic acid 0.21 0.01
7 Rutin 0.89 0.04
8 Delphinidin derivative 2.13 0.16
9 Kaempferol
3-neohesperidoside
1.76 0.05
10 Quercetin
3-(2G-rhamnosylrutinoside)
0.37 0.01
11 Ternatin B2 0.32 0.01
12 Ternatin C2 1.81 0.09
13 Ternatin D2 1.45 0.07
©2016 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al.1864
of Jansen & Wanders (2006), phytanic acid was
involved in several mechanisms for regulating triglyc-
erides/cholesterol status in the skeletal muscles.
Capabilities of hydrophilic and lipophilic butterfly pea
seed and petal extracts in decreasing carcinoma cell
(HEp-2) viability
The media with different concentrations of the four
extracts HBS, HBP, LBS and LBP were prepared,
respectively, and used to treat HEp-2 cells. HBS was
the most effective extract against the survival of
HEp-2 cells which rapidly decreased from 100.0 to
7.2% as the level of HBS increased from 0 to
0.25 mg mL
1
(Fig. 4). However, the survival rates of
HEp-2 in HBP treatment still remained over 90% in
the same concentration range (Fig. 4). As the concen-
tration of HBP increased from 0.25 to 0.50 mg mL
1
,
the survival rate of HEp-2 then rapidly reduced to
17.2% (Fig. 4). Both HBP and HBS could decrease
95% of the HEp-2 cell viability after the concentration
was increased to 1.0 mg mL
1
(Fig. 4). On the other
hand, the decreasing effect was observed in LBS and
LBP treatment only after their concentrations were
increased to 1.5 mg mL
1
. There was no significant
difference between the two treatments with their
concentrations lower than 6 mg mL
1
(Fig. 4). At a
concentration of 9 mg mL
1
, LBS exhibited approxi-
mately 2.5 times higher capability than LBP in
decreasing HEp-2 cell growth. In general, compared
with the lipophilic extracts of seeds and petals, the
hydrophilic extracts had much higher capability in
decreasing carcinoma viability (Fig. 4).
Generally, Krebs cycle is the primary metabolic
pathway for providing ATP for normal cell growth
through the involvement of mitochondria (Wen et al.,
2013). Different from the normal cells, however, can-
cer cells inevitably rely on metabolic reprogramming
and undergo glycolytic pathway to carry out a rapid
energy generation and macromolecular synthesis
because of mitochondria dysfunction (Suh et al.,
2013). The high glycolytic fluxes in cancer cells fur-
ther induce apoptosis in the neighbourhood normal
cells, block immune system and induce tissue inva-
sion by tumours (Suh et al., 2013). Thus, the inhibi-
tion of glycolysis is a biochemical way for decreasing
the cancer cell viability with the minimal residual sys-
temic toxicity and can be used in designing therapeu-
tic strategies. Because the butterfly pea seeds
contained abundant phenolics, they might act individ-
ually or synergistically to deactivate the key enzyme
involved in glycolytic metabolism in the cancer cells
C1
B3
D3 D2
B2
A1
Figure 3 Chemical structures of ternatin
A1, B2, B3, C2, D2 and D3.
Table 3 Lipophilic compounds identified in the butterfly pea seeds
and petals
Compounds Seeds Petals
Fatty Acid
(mg g
1
FW)
Palmitic acid
(C16:0)
3.61 0.13b 2.13 0.18a
Stearic acid
(C18:0)
2.85 0.15b 1.99 0.16a
Petroselinic
acid
(C18:1n6c)
1.55 0.10b 1.01 0.04a
Linoleic acid
(C18:2n6c)
8.73 0.61b 4.72 0.51a
Arachidic acid
(C20:0)
0.46 0.03b 0.36 0.01a
Behenic acid
(C22:0)
0.41 0.02b 0.30 0.03a
Phytanic acid N.D. 0.81 0.06a
Phytosterol
(mg/100 g FW)
Campesterol 8.07 0.22b 1.24 0.02a
Stigmasterol 7.95 0.63a 6.70 0.83a
b-Sitosterol 40.17 3.73b 6.77 0.19a
Sitostanol 5.10 0.05b 1.20 0.03a
Tocols
(mg/100 g FW)
a-Tocopherol 0.17 0.06a 0.20 0.01a
c-Tocopherol 5.44 0.30b 0.24 0.02a
N.D., not detected
Concentrations in each row with different letters are statistically differ-
ent at p<0.05.
©2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1865
and decrease their viability. It has been reported that
the noncovalent interactions between cellular proteins
and phenolics could prevent the cancer cell prolifera-
tion (Aslan et al., 2015). Phenolics such as rutin,
quercetin, kaempferol, catechin, p-coumaric, sinapic,
ferulic, syringic, caffeic and gallic acids found in the
butterfly pea seeds have also been proved to affect
important control point enzyme (pyruvate kinase
isoenzyme M2) or attack the glucose transporters
(GLUT) in glycolytic pathway to regulate the cancer
cell production (Aslan et al., 2015). Those phyto-
chemicals could also act as apoptosis-inducing factors
and could be released into the cytosol and translo-
cated to the nucleus to cleave DNA (Lee et al.,
2010). Moreover, the other mechanisms of cell cycle
regulation modulation, invasiveness and angiogenesis
suppression would be involved to decrease the HEp2
cancer cell viability (Lee et al., 2010). Thus, it may
be the reason that HBS exhibited the highest effi-
ciency among the four types of extracts in decreasing
HEp-2 cell viability.
Furthermore, different anthocyanins such as ter-
natins and cyanidin glycoside in HBP could be the
compounds for contributing to its anticancer effect as
well. The anthocyanins extracted from black raspber-
ries were found to counteract cancer cell motility
through the disruption of an essential mediator
cyclooxygenase-2 (COX-2) in tumorigenesis (Wang &
Stoner, 2008). However, Dai et al. (2009) suggested
that anthocyanin extract alone could less contribute to
anticancer ability, but may act additively or synergisti-
cally with other active components in the inhibition of
cancer cell growth. It may be the reason that HBS had
greater anticancer potential than HBP.
Tocopherols and phytosterols were the primary
compounds in LBP and LBS. As reported by Kannap-
pan et al. (2012), the anticancer actions of c-toco-
pherol involved in death receptor 5 (DR5) protein
upregulation could further stimulate tumour necrosis
and restrict its proliferation. Of the four phytosterols,
b-sitosterol has been evidenced as the most effective
one in decreasing the growth of cancer cells via the
activation of certain enzymes, which in turn induce
cellular apoptosis (Bradford & Awad, 2007). Woyengo
et al. (2009) suggested that b-sitosterol and campes-
terol could alleviate the cancer development by reduc-
ing the production of carcinogens in biological
metabolism. The levels of b-sitosterol and campesterol
in LBS were approximately seven and eight times
higher than those in LBP, respectively. Thus, it may
explain why LBS had better performance than LBP in
decreasing HEp-2 cell growth in the concentrations
ranging from 5 to 12 mg mL
1
.
As discussed above, certain phenolics, tocopherols
and phytosterols could demonstrate the decreasing effi-
ciency by targeting specific enzymes or interfering with
the metabolic pathway of cancer cells without affecting
other nontumorigenic counterparts and cells. Similar
results showed that normal human epidermal ker-
atinocytes and astrocytes were able to survive when
exposed to flavonoids or green tea leaves rich in
polyphenols, while they elicited the death of tumour
cells (Hsu et al., 2003; Das et al., 2010). Therefore, the
butterfly pea seed or petal extracts, especially the
hydrophilic seed extract, have the potential in reducing
the risks of cancer. Although butterfly pea seeds and
flowers have been used as tea or a source of edible
food ingredient for a long time, the possible toxicity
level of the extracts on noncarcinogenic cells should be
considered and evaluated.
Conclusions
The concentration and profile of phenolics, toco-
pherols, phytosterols and fatty acids in butterfly pea
seeds and petals were determined. Anticancer effect of
butterfly pea seed and petal extracts (HBP, HBS, LBP
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
Concentration of the extracts (mg mL–1)
Concentration of the extracts (mg mL–1)
HBS
HBP
Survival rate (%)
0
20
40
60
80
100
Survival rate (%)
0 5 10 15
LBS
LBP
Figure 4 The survival rates of HEp2 cells treated by different con-
centrations of the hydrophilic (HBS and HBP) and lipophilic (LBS
and LBP) extracts of butterfly pea seeds and petals.
©2016 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2016
Butterfly pea extracts decrease carcinoma cells Y. Shen et al.1866
and LBS) was evaluated using HEp-2 carcinoma cell
line. The results indicated that HBS had the highest
capability in decreasing the survival of HEp-2 cells.
Both HBS and HBP exhibited greater performance
than LBS and LBP in decreasing the cell viability. In
general, the hydrophilic extracts of butterfly pea
seed and petal possess a variety of antioxidant phyto-
chemicals and the potential in decreasing cancer cell
viability.
Acknowledgments
We thank Dr. Graca Vicente (Department of Chem-
istry, Louisiana State University) who kindly provided
the facility for the cell culture experiment.
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