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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-Marenco1 &
Zhimin Xu1*
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
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 g1. 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.
doi:10.1111/ijfs.13158
© 2016 Institute of Food Science and Technology
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1861
difficulty in treating the cancer (Pienta et al., 2008). prepared for a stock solution (50 mg mL1 in metha-
Primary clinical treatments for laryngeal cancer are nol). For the lipophilic extract of butterfly pea petals
surgery, chemotherapy and radiotherapy. However, or seeds, 50 mL of a solvent mixture of ethyl acetate
they induce seriously adverse side effects or even result and hexane (50:50; v:v) was used for the extraction
in resistance to these therapies (Agostinis et al., 2011). according to the same procedure used for obtaining
Recently, much attention to cancer treatment has been the hydrophilic extract. After the solvent extract was
focused on some plant-derived compounds that have dried, a stock solution of the lipophilic extract
pharmacological functions on the tumour but have less (50 mg mL1 in hexane) was prepared as well.
side effects (Veerabadran et al., 2013). In this study,
the anticancer effect of hydrophilic and lipophilic
Identification and quantification of hydrophilic and
extracts of butterfly pea petal and seed on decreased
lipophilic phytochemicals and fatty acids
laryngeal cancer cell (HEp-2) viability was studied and
compared. The aim of this study was to provide the Phytochemical profiles of the hydrophilic extracts were
bioactive phytochemical profiles in both butterfly pea determined by a reverse-phase HPLC (2690; Waters,
petals and seeds and also emphasise the role of those Torrance, CA, USA) coupled with C18 column (id
bioactive compounds in anticancer activity. 250 9 4.60 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
Materials and methods
Du et al. (2014). The concentrations of the phenolics
were calculated based on their corresponding standard
Chemicals and materials
curves. Both the extracts were also subject to an LC-
High-performance liquid chromatography (HPLC)- MS analysis to identify the compounds without their
grade acetonitrile, acetic acid, methanol and hexane standards available. The LC-MS consisted of a ultra
were purchased from Fisher Chemicals (Fair Lawn, performance liquid chromatography (UPLC) system
NJ, USA). Acetone was purchased from Macron (Thermo Scientific Dionex UltiMate 3000, Waltham,
(Charlotte, NC, USA). Ethyl acetate was purchased MA, USA) and a mass spectrometry (MS) (Q Exac-
from EM Science (Gibbstown, NJ, USA). Trimethylsi- tiveTM Plus Hybrid Quadrupole-OrbitrapTM) that had
lyl imidazole (TMS), BCl3 methanol and phenolics, an electrospray ionisation source (ESI) in the positive
fatty acids and tocopherol standards were purchased mode with a full MS scan from 150 to 2000 m/z. The
from Sigma Aldrich (St. Louis, MO, USA). Fresh but- LC-MS separation was carried out using a reverse-
terfly pea seeds and petals (Clitoria ternatea) were phase column (AcclaimÒ Mixed-Mode WAX-1,
obtained from a local garden in Baton Rouge, LA, 150 9 2.1 mm, 5 lm). The mobile phase consisted of
USA. The human carcinoma HEp-2 cell line was pur- solution A (1% formic acid solution) and solution B
chased from American Type Culture Collection (acetonitrile) at a constant flow rate of 0.2 mL min1
(ATCC, Manassas, VA, USA). Other reagents and with a gradient programme of 0–70% B at 0.0–
culture media containing foetal bovine serum (FBS), 8.0 min, 70–100% B at 8.0–10.0 min, 100–0% B at
antibiotic (penicillin–streptomycin), CellTiter-Blue, 10–15 min. The MS parameters were set as follows:
dimethyl sulphoxide (DMSO) and phosphate-buffered electric potential of the ESI source, 3.0 kV; capillary
saline (PBS) were purchased from Invitrogen (Grand temperature, 300 °C; heater temperature, 200 °C. The
Island, NY, USA). concentrations of ternatins and delphinidin derivative
were calculated by the standard curve of cyanidin
chloride in molar concentration and then converted to
Extraction of hydrophilic and lipophilic bioactive
mass unit (mg g1) in the sample based on their
compounds in butterfly pea seeds and petals
molecular weights. Tocopherols in the samples were
The freeze-dried butterfly pea seeds and petals were determined by a normal-phase HPLC (1100 series;
ground by a coffee blender (Hamilton Beach, Southern Agilent, Santa Clara, CA, USA) with Supelcosil LC-Si
Pines, NC, USA). The hydrophilic compounds in the column (id 250 9 4.60 mm 5 lm, Supelco, Bellefonte,
petals or seeds (20 g) were extracted using 50 mL of PA, USA). The condition of the HPLC analysis was
methanol at 60 °C for 20 min. After centrifugation, the same as the method described in Jang & Xu
the methanol layer was transferred to a clean tube. (2009).
Then, the solid residues were extracted repeatedly for The fatty acids in each lipophilic extract were esteri-
two more times at the same condition and using the fied using 2 mL of BCl3 (boron trichloride) methanol
same procedure. The dried hydrophilic extract was after 200 lg of internal standard (C17:0) was added.
obtained by evaporating the collected methanol using The reaction mixture was incubated at 60 °C for
a vacuum centrifuge evaporator (Labconco, Kansas 30 min. Then, 1 mL of water and 1 mL of hexane
City, MO, USA). The dried extracts were then were added to the reaction solution and vortexed.
© 2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
1862 Butterfly pea extracts decrease carcinoma cells Y. Shen et al.
After centrifugation, the upper hexane layer was trans- media were discarded and replaced with 100 lL of
ferred to a clean test tube and mixed with anhydrous fresh media containing 20% CellTiter-Blue. After the
Na2SO4 to remove any moisture before it was trans- cells were stained for 4 h, the fluorescence intensity of
ferred to a gas chromatography (GC) vial. The fatty the media was read at excitation/emission wavelengths
acids were determined by a GC equipped with an of 570/615 nm using a FluoStar Optima microplate
flame ionisation detector (FID) and a Supelco SP2380 reader (BMG, Ortenberg, Germany). The potential of
(30 m 9 0.25 mm) column. The GC condition was the the extract in decreasing human carcinoma cell viabil-
same as that used in the study of Yue et al. (2008). ity at each concentration treatment was expressed by a
The determination of phytosterols was based on the survival rate, which was the percentage of the intensity
study of Xu & Godber (1999). After the lipophilic of the treatment vs. the intensity of the control.
extract was mixed with an aliquot of hexane contain-
ing 20 lg cholesterol internal standard, it was dried
Data analysis
and then reacted with 200 lL of TMS and 50 lL of
acetonitrile at 65 °C for 30 min. The derived products The determinations of hydrophilic and lipophilic phy-
were extracted using 200 lL of hexane and analysed tochemicals and fatty acids in each extract were
by GC-MS. A Varian CP-3800 GC (Walnut Creek, repeated in triplicate and expressed as means stan-
CA, USA) coupled with a Saturn 2200 mass spectrom- dard deviation. The significant differences between the
eter and a DB-5 column (60 m 9 0.25 mm) (Supelco) concentrations of each compound in the hydrophilic
was used in the analysis. The initial oven temperature or lipophilic extracts were determined by one-way
was 200 °C. Then, it was ramped to 280 °C at a rate ANOVA at P < 0.05 (SAS, 9.1.3, Cary, NY, USA). The
of 10 °C min1 and held at the final temperature for determination of the potential of each treatment con-
62 min. Helium was the carrier gas at a constant flow centration or control in decreasing the cell viability
rate of 1.5 mL min1. The injection port temperature was repeated five times and analysed by GraphPad
and split ratio were 280 °C and 1:50, respectively. The Prism (version 6.0; GraphPad Software Inc., La Jolla,
ratios of peak areas at different levels (5, 10, 20, 50 CA, USA). The differences in the potential in decreas-
and 100 lg) of campesterol, stigmasterol and b-sitos- ing the cell viability between the treatments and con-
terol to 20 lg of cholesterol internal standard were trol were analysed by two-way ANOVA at P < 0.05.
used to set up the standard curves for quantifying the
phytosterols.
Results and discussions
Determination of capability of decreasing human Hydrophilic and lipophilic phytochemicals and fatty acids
carcinoma cell (HEp-2) viability in butterfly pea seeds and petals
The human carcinoma HEp-2 cell line was used to The yields of the HBS and HBP extracts were 3.66
assess the potential of butterfly pea seed and petal and 4.05%, respectively. The chromatograms of the
extracts in decreasing cancer cell viability. The cell line hydrophilic phenolics in HBS and HBP are shown in
was cultured in Dulbecco’s modified Eagle’s media Figs 1 and 2, respectively. In addition to the high con-
(DMEM), supplemented with 10% foetal bovine centration of ascorbic acid [1.32 0.02 mg g1 fresh
serum (FBS) and 1% antibiotic (penicillin–strepto- weight (FW)], sinapic acid was the dominant phenolic
mycin), and grown in a 5% CO2 atmosphere with compound among the fifteen major hydrophilic pheno-
95% humidity at 37 °C for 24 h. Then, the cells were lics at a concentration of 1.01 0.07 mg g1 FW, fol-
harvested, counted (3 9 104 cells mL1) and trans- lowed by epicatechin and gallic acid in the seed
ferred into a 96-well plate. The working solution of (Table 1). Compared with the contents in rapeseeds
each extract was prepared by dissolving the extract (0.09–0.59 mg g1 FW) and camelina seeds
with 0.2% DMSO in PBS culture media. The treat- (0.39 mg g1 FW) (Niciforovic & Abramovic, 2014),
ment solutions were prepared based on a group of the the sinapic acid content in butterfly pea seeds was
concentrations multiplied by the low and effective con- much higher than each of them (Table 1). It was
centration of each type of extract that was obtained in reported that sinapic acid could help suppress the
our preliminary study. The HEp-2 cells were incubated expression of proinflammatory mediators via NF-jB
with a series of concentrations of the hydrophilic inactivation in regulating inflammatory status and
extract working solution (1.0, 0.5, 0.25, 0.12 and immune response (Yun et al., 2008). Epicatechin, in
0.06 mg mL1) or lipophilic extract working solution the butterfly pea seeds, which was reported to exhibit
(12.0, 9.0, 6.0, 3.0 and 1.5 mg mL1) for 96 h at immunoregulatory, antihypertensive effects as well,
37 °C for a dose-dependent study. The cells only was ten times higher (0.56 mg g1 FW, Table 1) than
mixed with the media and 0.2% DMSO were used as that reported in the garden pea seeds (Pisum sativum)
the control group. For measuring the cell viability, the (0.05 mg g1) (Ferraro et al., 2014; Litterio et al.,
International Journal of Food Science and Technology 2016 © 2016 Institute of Food Science and Technology
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1863
7 8
1.00 1 2
0.90
0.80
0.70 11
9
10
0.60 4
AU
0.50
Figure 1 Chromatogram of the hydrophilic
5
butterfly pea seed extract. 1 – vitamin C; 2 – 0.40
gallic acid; 3 – protocatechuic acid; 4 – epi- 3
0.30 12
catechin; 5 – caffeic acid; 6 – syringic acid; 14
0.20 16
7 – sinapic acid; 8 – hydroxycinnamic acid
6 13 15
derivatives; 9 – p-coumaric acid; 10 – 0.10
hydroxycinnamic acid derivatives; 11 – rutin;
0.00
12 – ferulic acid; 13 – rosmarinic acid; 14 –
cinnamic acid; 15 – kaempferol; 16 – api- 10.00 20.00 30.00 40.00 50.00 60.00 70.00
genin. Min
0.14
9
0.12
11
0.10
0.08
6 8
AU
10 12
0.06 7
Figure 2 Chromatogram of the hydrophilic
butterfly pea petal extract. 1 – cyanidin- 0.04 13
3-sophoroside; 2 – delphinidin derivative; 3 – 5
2 4
ternatin A1; 4 – ternatin B3; 5 – ternatin 0.02 1
D3; 6 – ellagic acid; 7 – rutin; 8 – delphini- 3
din derivative; 9 – kaempferol-3-neohesperi- 0.00
doside; 10 – quercetin-3-(2G-
rhamnosylrutinoside); 11 – ternatin B2; 10.00 20.00 30.00 40.00 50.00 60.00 70.00
12 – ternatin C2; 13 – ternatin D2. Min
2015). Protocatechuic, p-coumaric, rutin and two acid, glucose, p-coumaric acid or caffeic acid (Kazuma
hydroxycinnamic acid derivatives in the butterfly pea et al., 2003). In this study, ternatin C2 and D2 in the
seeds were all above 0.30 mg g1 FW, while kaemp- butterfly pea petals were observed at higher concentra-
ferol, apigenin, caffeic, syringic, ferulic, rosmarinic and tions of 1.81 0.09 and 1.45 0.07 mg g1 FW,
cinnamic acids were in a range of 0.04 to 0.22 mg g1 respectively. The concentrations of ternatin A1, B3,
FW (Table 1). Compared with the rutin content in edi- D3 and B2 were in a range of 0.32 to 0.51 mg g1 FW
ble Amaranthus seeds, which was between 0.0711 and (Table 2). The chemical structures of the six types of
0.0775 mg g1 dry weight (DW) (Li et al., 2015), the ternatins are elucidated in Fig. 3. Two delphinidin
rutin content in butterfly pea seeds was much higher derivatives and cyanidin-3-sophoroside were also iden-
and reached 0.31 mg g1 FW (Table 1). Although tified and responsible for the blue colour of the petals
mung bean, radish, broccoli and sunflower seeds con- together with ternatins. In general, kaempferol 3-neo-
tain gallic, protocatechuic, caffeic, p-coumaric, ferulic, hesperidoside, quercetin 3-(2G-rhamnosylrutinoside)
chlorogenic, sinapic acid, quercetin and kaempferol and rutin were the major flavanol glycoside com-
(Pajaz k et al., 2014), each of their levels was signifi- pounds in the petals, while ellagic acid was the only
cantly lower than that in the butterfly pea seeds. phenolic acid identified in butterfly pea petals
The unique phytochemicals in butterfly pea petals (Table 2).
are ternatins, a group of polyacylated delphinidin The yields of the lipophilic extracts, LBS and LBP,
derivatives (Sasaki et al., 2013). It was reported that were 5.28 and 0.80%, respectively. Lipophilic phytos-
ternatin A1-A3, B1-B4, C1-C5 and D1-D3 consist of terols are chemically characterised as triterpenes and
delphinidin 3, 30 , 50 -triglucoside attached with malonic considered to be one of the structural components of
© 2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
1864 Butterfly pea extracts decrease carcinoma cells Y. Shen et al.
Table 1 Hydrophilic compounds identified in the butterfly pea Table 2 Hydrophilic compounds identified in the butterfly pea
seeds petals
Concentration Concentration
Peak No. Compounds (mg g1 FW) Peak No. Compounds (mg g1 FW)
International Journal of Food Science and Technology 2016 © 2016 Institute of Food Science and Technology
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1865
D3 D2
A1
C1
B3
Figure 3 Chemical structures of ternatin B2
Table 3 Lipophilic compounds identified in the butterfly pea seeds HEp-2 cells which rapidly decreased from 100.0 to
and petals 7.2% as the level of HBS increased from 0 to
0.25 mg mL1 (Fig. 4). However, the survival rates of
Compounds Seeds Petals HEp-2 in HBP treatment still remained over 90% in
Fatty Acid Palmitic acid 3.61 0.13b 2.13 0.18a the same concentration range (Fig. 4). As the concen-
(mg g1 FW) (C16:0) tration of HBP increased from 0.25 to 0.50 mg mL1,
Stearic acid 2.85 0.15b 1.99 0.16a the survival rate of HEp-2 then rapidly reduced to
(C18:0) 17.2% (Fig. 4). Both HBP and HBS could decrease
Petroselinic 1.55 0.10b 1.01 0.04a 95% of the HEp-2 cell viability after the concentration
acid was increased to 1.0 mg mL1 (Fig. 4). On the other
(C18:1n6c) hand, the decreasing effect was observed in LBS and
Linoleic acid 8.73 0.61b 4.72 0.51a
LBP treatment only after their concentrations were
(C18:2n6c)
increased to 1.5 mg mL1. There was no significant
Arachidic acid 0.46 0.03b 0.36 0.01a
(C20:0)
difference between the two treatments with their
Behenic acid 0.41 0.02b 0.30 0.03a concentrations lower than 6 mg mL1 (Fig. 4). At a
(C22:0) concentration of 9 mg mL1, LBS exhibited approxi-
Phytanic acid N.D. 0.81 0.06a mately 2.5 times higher capability than LBP in
Phytosterol Campesterol 8.07 0.22b 1.24 0.02a decreasing HEp-2 cell growth. In general, compared
(mg/100 g FW) Stigmasterol 7.95 0.63a 6.70 0.83a with the lipophilic extracts of seeds and petals, the
b-Sitosterol 40.17 3.73b 6.77 0.19a hydrophilic extracts had much higher capability in
Sitostanol 5.10 0.05b 1.20 0.03a decreasing carcinoma viability (Fig. 4).
Tocols a-Tocopherol 0.17 0.06a 0.20 0.01a
Generally, Krebs cycle is the primary metabolic
(mg/100 g FW) c-Tocopherol 5.44 0.30b 0.24 0.02a
pathway for providing ATP for normal cell growth
N.D., not detected through the involvement of mitochondria (Wen et al.,
Concentrations in each row with different letters are statistically differ- 2013). Different from the normal cells, however, can-
ent at p< 0.05. cer cells inevitably rely on metabolic reprogramming
and undergo glycolytic pathway to carry out a rapid
energy generation and macromolecular synthesis
of Jansen & Wanders (2006), phytanic acid was because of mitochondria dysfunction (Suh et al.,
involved in several mechanisms for regulating triglyc- 2013). The high glycolytic fluxes in cancer cells fur-
erides/cholesterol status in the skeletal muscles. 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-
Capabilities of hydrophilic and lipophilic butterfly pea
tion of glycolysis is a biochemical way for decreasing
seed and petal extracts in decreasing carcinoma cell
the cancer cell viability with the minimal residual sys-
(HEp-2) viability
temic toxicity and can be used in designing therapeu-
The media with different concentrations of the four tic strategies. Because the butterfly pea seeds
extracts HBS, HBP, LBS and LBP were prepared, contained abundant phenolics, they might act individ-
respectively, and used to treat HEp-2 cells. HBS was ually or synergistically to deactivate the key enzyme
the most effective extract against the survival of involved in glycolytic metabolism in the cancer cells
© 2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
1866 Butterfly pea extracts decrease carcinoma cells Y. Shen et al.
International Journal of Food Science and Technology 2016 © 2016 Institute of Food Science and Technology
Butterfly pea extracts decrease carcinoma cells Y. Shen et al. 1867
and LBS) was evaluated using HEp-2 carcinoma cell Hsu, S., Bollag, W.B., Lewis, J. et al. (2003). Green tea polyphenols
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