DSpace at VNU: Cytotoxic Activity of New Phenolic Compounds from Vietnamese Caesalpinia sappan

Biosci. Biotechnol. Biochem., 77 (12), 2378–2382, 2013

Cytotoxic Activity of New Phenolic Compounds
from Vietnamese Caesalpinia sappan
Tran Manh H UNG,1;2; y Nguyen Xuan H AI,1 Nguyen Trung N HAN,1 Ton That Q UANG,1
Tran Le Q UAN,1 To Dao C UONG,2 Nguyen Hai D ANG,3 and Nguyen Tien D AT3
1

Faculty of Chemistry, University of Science, Vietnam National University,
227 Nguyen Van Cu Street, District 5, HoChiMinh city, Vietnam
2
College of Pharmacy, Catholic University of Daegu, Kyeongbuk 712-702, Korea
3
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST),
18 Hoang Quoc Viet, Caugiay District, Hanoi, Vietnam
Received June 19, 2013; Accepted September 11, 2013; Online Publication, December 7, 2013
[doi:10.1271/bbb.130493]

Two new phenolic compounds, caesalpiniaphenols G–
H (1 and 2), were isolated from Vietnamese Caesalpinia
sappan heartwood. The chemical structures were established mainly by extensive spectroscopic studies and
chemical evidence. Compounds 1 and 2 showed potent
inhibitory activity against HL-60 cancer cell lines with
respective IC50 values of 16.7 and 22.5 g/mL. Treating
HL-60 cells with various concentrations of 1 resulted in
growth inhibition and the induction of apoptosis.
Key words:

Caesalpinia sappan; Leguminosae; caesalpiniaphenols; HL-60; apoptosis

Caesalpinia sappan L. (Leguminosae) is distributed

in Southeast Asia. It has been used as a herbal medicine
for treating inflammation and to improve blood circulation,1) as well as for its anti-influenza, anti-allergic and
neuroprotective activities.2–4) This plant is called Toˆ
Mo:^ c in Vietnam and is scattered in low mountainous
areas, being light-demanding and drought-tolerant. Toˆ
Mo:^ c has been used in traditional Vietnamese medicine
for treating menstrual and post-partum haematometra,
trauma blood static, dizziness and post-partum blood
loss. It has also been employed in the therapy of bloody
dysentery, enteralgia, intestinal haemorrhage and infectious diarrhea, and for cleansing wounds.5) Many reports
have shown that the main components in C. sappan are
phenolics with four structural sub-types: brazilin, chalcone, protosappanin and homoisoflavonoids.6–11) An
ethanolic extract of C. sappan has ameliorated hypercholesterolemia in C57BL/6 mice and suppressed the
inflammatory response in human umbilical vein endothelial cells (HUVECs) through an anti-oxidizing mechanism.7) The compounds with a sappanchalcone skeleton
have shown anti-inflammatory, anti-bacterial and antiinfluenza activities.8–13) Nguyen et al. have reported that
a methanolic extract and the active compounds from the
heartwood of C. sappan collected in Vietnam exhibited
significant XO inhibitory activity.14,15) However, limited
information is available concerning the cytotoxic activity principles of C. sappan origining in Vietnam. To
y

2

OH
HO 7

8

6
5

8a O
4a

4

4' OH

2'
1'

2
3
9

6'

OH

OH

O
1

3 3a O
11

1
10

HO 9

10b
10a

5
5a
6

6a
7
8

OH
2

Fig. 1. Chemical Structures of 1 and 2 from C. sappan.

further study the phytochemical and biological activity
available from this plant, fractionation of the ethyl
acetate-soluble fraction resulted in the isolation of two
new phenolic compounds (1 and 2). This study describes
the isolation and structural elucidation of these compounds, as well as an evaluation of their in vitro
cytotoxicity and apoptosis induction toward HL-60,
human premyelocytic leukemia cancer cell lines.

Materials and Methods
General experimental procedures. Optical rotation was measured
with a Jasco DIP 370 digital polarimeter. UV spectra were obtained in
MeOH by using a Thermo 9423AQA2200E UV spectrometer, and IR

spectra were obtained with a Bruker Equinox 55 FT-IR spectrometer.
The nuclear magnetic resonance (NMR) spectra were obtained with a
Varian Unity Inova 400 MHz spectrometer. Silica gel (Merck, 63–
200 mm particle size) and RP-18 silica gel (Merck, 75 mm particle size)
were used for column chromatography. TLC was carried out by using
Merck silica gel 60 F254 and RP-18 F254 plates. HPLC was performed
with a Waters 600 Controller system equipped with a UV detector and
a YMC Pak ODS-A column (20 Â 250 mm, 5 mm particle size, YMC
Co., Japan), and HPLC solvents were obtained from Burdick &
Jackson, USA.
Plant materials. The heartwood of Caesalpinia sappan L. was
collected in An Giang province of Vietnam in September 2010.
Professor Tran Cong Luan at Vietnam National Institute of Medicinal
Material botanically authenticated the plant, where voucher specimen
number TCL-00120 describing the plant is deposited.
Extraction and isolation. C. sappan (1.5 kg) was extracted three
times (3 h  5 L) with refluxing methanol. After the solvent had been
removed under reduced pressure, the residue was suspended in H2 O
and then successively partitioned with n-hexane, EtOAc, and n-BuOH.

To whom correspondence should be addressed. Tel: +84-8-3832-4457; Fax: +84-8-3835-0096; e-mail:
Abbreviations: C. sappan, Caesalpinia sappan; NMR, nuclear magnetic resonance; HL-60, human promyelocytic leukemia; HeLa, human cervical
adenocarcinoma; MCF-7, human breast adenocarcinoma; LLC, Lewis lung carcinoma cells; ATCC, American Type Culture Collection.


Cytotoxic Activity of Phenolic Compounds from C. sappan
The EtOAc-soluble fraction (16 g) was chromatographed in a silica gel
column, using a stepwise gradient of CHCl3 :MeOH (80:1 to 0:1), to
yield sixteen sub-fractions (E.1–E.16) according to their TLC profiles.
Fraction E10 (1.12 g) was subjected to reversed-phase (ODS-A)

column chromatography, eluting with MeOH/H2 O (from 3:1 to 1:0,
2 L for each step) to afford six sub-fractions (E.10-1–E.10-6). Further
purification of E10-5 (127 mg) by a semi-preparative Waters HPLC
system [using an isocratic solvent system of 30% MeOH in
H2 O þ 0:1% trifluoroacetic acid at a flow rate of 5 mL/min over
90 min; UV detection at 210 nm; a YMC Pak ODS-A column
(20 Â 250 mm, 5 mm particle size] resulted in the isolation of
compound 1 (12.7 mg; tR ¼ 26:5 min). Further purification of E10-6
(68.5 mg) by the semi-preparative Waters HPLC system [using an
isocratic solvent system of 25% MeOH in H2 O þ 0:1% trifluoroacetic
acid at a flow rate of 5 mL/min over 90 min; UV detection at 210 nm; a
YMC Pak ODS-A column (20 Â 250 mm, 5 mm particle size] resulted
in the isolation of compound 2 (5.7 mg; tR ¼ 32:5 min).
Caesalpiniaphenol G (1): yellowish needles (MeOH); mp 176–
178  C; IR max (KBr) cmÀ1 : 3464 (hydroxy group), 1725 (carbonyl
group), 1440 (aromatic absorption); 1 H-NMR (400 MHz, methanol-d4 )
and 13 C-NMR (100 MHz, methanol-d4 ): see Table 1 for the spectroscopic data; HR-EI-MS m=z: 316.0596 [M]þ , calcd. for C16 H12 O7 ,
316.0595.
Caesalpiniaphenol H (2): colorless amorphous powder; ½Š25
D
À165.2 (c 0.05, MeOH); IR max (KBr) cmÀ1 : 3380 (hydroxy group),
1452 (aromatic absorption); UV (MeOH) max (log ") nm: 225 (4.33),
260 (3.75); 1 H-NMR (400 MHz, methanol-d4 ) and 13 C-NMR
(100 MHz, methanol-d4 ): see Table 2 for the spectroscopic data; HREI-MS m=z: 242.0968 [M]þ , calcd. for C15 H14 O3 , 242.0969.
Cell lines and culture. HL-60 (human promyelocytic leukemia),
HeLa (human cervical adenocarcinoma), MCF-7 (human breast
adenocarcinoma) and LLC (Lewis lung carcinoma) cells were obtained
from the American Type Culture Collection (ATCC). The cells were
maintained in RPMI or IMDM (Gibco BRL, NY, USA) with 10% fetal
bovine serum (FBS) supplemented with 2% penicillin and 100 mg/mL

of streptomycin at 37  C in a humidified atmosphere containing 5%
CO2 .
Cytotoxic activity assay. The cancer cell lines were maintained in
RPMI 1640 or IMDM that included L-glutamine (Gibco) with 10%
FBS (Gibco) and 2% penicillin-streptomycin (Gibco). The cells were
cultured at 37  C in a 5% CO2 incubator. The cytotoxic activity was
measured by using a modified MTT assay.16) Viable cells were seeded
in the growth medium into 96-well microtiter plates (1 Â 104 cells/
well) and then incubated at 37  C in a 5% CO2 incubator. The test
sample was dissolved in DMSO and adjusted to a final sample
concentration ranging from 5 to 100 mg/mL by diluting with the
growth medium. Each sample was prepared in triplicate, the final
DMSO concentration being adjusted to <0.1%. After standing for 24 h,
the test sample was added to each well. The same volume of the
medium with 0.1% DMSO was added to the control wells. The MTT
reagent was added to the each well (a 5 mg/mL final concentration)
48 h after the test sample had been added and, 4 h later, the plate was
centrifuged for 5 min at 1500 rpm, the medium was removed, and the
resulting formazan crystals were dissolved with DMSO. The optical
density (O.D) was measured at 570 nm with a Titertek microplate
reader (Multiskan MCC/340, MKII Microplate Reader). The IC50
value is defined as the concentration of a sample which reduced the
absorbance by 50% relative to the vehicle-treated control.
DNA gel electrophoresis. DNA from the cells was purified by using
an Apoptotic DNA Ladder kit (Roche, Germany), and DNA bands
were photographed under ultraviolet illumination.16) HL-60 cells
(5 Â 105 cell/mL) were treated with the indicated concentration of
the test compound for 24 h. After the supernatant had been removed by
centrifugation (1500 rpm at 4  C), the cells were washed with 1 mL of
PBS and then precipitated by centrifugation at 3000 rpm for 10 min at

4  C. DNA from the cells was purified by using the Apoptotic DNA
Ladder kit (Roche, Germany), and then separated on 1% (w/v) agarose
gel containing 2.5 mL of 10 mg/mL ethidium bromide in a TBE buffer
[0.045 M Tris–borate at pH 8.0, 0.001 M EDTA].

2379

Caspase-3 activity. The Caspase-3 enzyme activity was measured
by proteolytic cleavage of the fluorogenic substrate, Ac-DEVD-AFC,
by counting with a Twinkle LB970 microplate fluorometer fluorescence plate reader (Berthold Technologies, Germany). HeLa cells
(1 Â 105 cell/well) were treated with the active compound at concentrations of 5, 12.5, 25, 50 and 100 mg/mL. After incubating for 24 h,
the cells were harvested and washed with cold PBS. The pellets were
lyzed with 15 mL of a lysis buffer [10 mM Tris–HCL at pH 8.0, 10 mM
EDTA, 0.5% Triton X-100] at room temperature for 10 min, and then
placed on ice; 100 mL of an assay buffer [100 mM Hepes at pH 7.5,
10 mM dithiothreitol, 10% (w/v) sucrose, 0.1% (v/v) Chaps, 0.1%
(v/v) BSA] and 10 mL of a substrate solutin (200 mM substrate in the
assay buffer) were finally added. After incubating at 37  C for 1 h, the
fluorescence was measured with excitation at 370 nm and emission at
505 nm.16)

Results
Structural elucidation of 1 and 2
Compound 1 was obtained as yellowish needles with
the molecular formula C16 H12 O7 , as determined by HREI-MS at m=z 316.0596 [M]þ . The IR spectrum of 1
suggested the presence of a hydroxy group (3464 cmÀ1 ),
a carbonyl group (1725 cmÀ1 ), and an aromatic absorption (1440 cmÀ1 ). The 1 H-NMR spectrum showed the
typical splitting pattern for a 7-hydroxy-chromen-type
homoisoflavonoid with H-2 resonances occurring as a
singlet signal at H 5.28 (2H, s).1) A singlet proton

resonance at H 7.69 (1H, s, H-9) in the 1 H-NMR
spectrum and two carbon signals at C 123.7 (C-3) and
134.4 (C-9) in the 13 C-NMR spectrum, which were
assigned to a trisubstituted double bond (–CH(3)=C(9)–),
were present (Table 1). In addition, the proposed Egeometry of the double bond was supported by
characteristic 4 J-coupling of the methylene protons at
H 5.28 (2H, s) at C-2 and the vinylic proton at C-9, and
accounted for the unusual chemical shift of the vinylic
proton H 7.69 (1H, s, H-9) appearing in a higher field.17)
A typical ABX spin system at H 7.97 (1H, d, J ¼
8:8 Hz, H-5), 6.71 (1H, dd, J ¼ 2:4, 8.8 Hz, H-6) and
6.55 (1H, d, J ¼ 2:4 Hz, H-8) accounted for the protons
of ring A (Table 1). The presence of one singlet signal at
H 6.97 (1H, s) accounted for the aromatic proton at H-20
of ring B (Fig. 1). Correlation in the NOESY spectrum
between the H-20 and the H-2 proton resonances enabled
placement of this aromatic at C-20 . The 13 C-NMR
spectrum of 1 contained 16 carbon signals consistent
Table 1. NMR Spectroscopic Data for Compound 1 (in methanol-d4 )
Position
2
3
4
4a
5
6
7
8
8a
9

10
20
30
40
50
60

1

H (400 MHz)

5.28 (2H, s)

7.97 (1H, d, J ¼ 8:8 Hz)
6.71 (1H, dd, J ¼ 2:4, 8.8 Hz)
6.55 (1H, d, J ¼ 2:4 Hz)
7.69 (1H, s)
6.97 (1H, s)

13

C (100 MHz)
68.1
123.7
184.4
119.0
108.0
108.7
164.9
98.8

161.7
134.4
120.5
113.6
163.2
146.4
145.2
150.3


2380

T. M. HUNG et al.
O
OH

HO

Table 3. Cytotoxic Activity of Compounds 1 and 2 from C. sappan

OH

O

IC50 value (mg/mL)a

Compound
OH
O

HO

OH
COSY
HMBC

1

OH
2

Fig. 2. Selected COSY and HMBC Correlations for 1 and 2.

Table 2. NMR Spectroscopic Data for Compound 2 (in methanol-d4 )
1

Position
1
2
3
3a
5
5a
6
6a
7
8
9
10
10a

10b
11

H (400 MHz)

6.84 (d, J ¼ 10:0 Hz)
6.42 (brd, J ¼ 10:0 Hz)
5.54 (brs)
4.39
4.24
2.46
2.24
2.09

(d, J ¼ 11:2 Hz)
(dd, J ¼ 3:0, 11.2 Hz)
(m)
(d, J ¼ 12:8 Hz)
(dd, J ¼ 3:0, 12.8 Hz)

6.41 (s)

6.64 (s)

2.95 (d, J ¼ 17:2 Hz)
3.27 (dd, J ¼ 5:6, 17.2 Hz)

13

1

2
Adriamycinb

HL-60

HeLa

MCF-7

LLC

16:7 Æ 2:2
22:5 Æ 5:1
3:0 Æ 0:4

28:1 Æ 3:6
39:2 Æ 2:0
4:9 Æ 0:2

>100
>100
2:4 Æ 0:2

>100
42:5 Æ 5:1
2:8 Æ 0:3

a
The inhibitory effect is presented as giving 50% inhibition (IC50 ) relative to
the vehicle control. These data represent the average values of three repeated

experiments (mean Æ SD).
b
Positive control.

C (100 MHz)
108.1
129.7
152.9
182.0
78.9
33.6
35.7
126.2
113.9
144.8
146.6
117.1
128.9
44.3
30.3

with the chromen-4-one skeleton with ten quaternary
carbons, the chemical shifts of these quaternary carbons
indicating that six of them were oxygen-bearing carbons
with one carbonyl functionality evident at C 184.4
(C-4) (Table 1). By comparison, it was found that the
1
H- and 13 C-NMR spectroscopic data for 1 were closely
related to those of sappanone A12) and 30 -deoxysappanone A,12) except for the additional hydroxyl groups at
C-30 , C-50 and C-60 of ring B. The complete NMR

assignment and connectivity of 1 were further determined by analyzing the COSY, HMQC and HMBC
spectroscopic data (Fig. 2). Based on the this analysis,
the structure of compound 1 was elucidated as (E)-3(2,3,4,5-tetrahydroxybenzylidene)-2,3-dihydro-7-hydroxychromen-4-one, trivially named caesalpiniaphenol G.
Compound 2 was obtained as a colorless amorphous
powder with the molecular formula C15 H14 O3 , as
established by a molecular ion peak [M]þ at m=z
242.0968 in the HR-EI-MS data. IR absorptions at
3380 cmÀ1 and 1452 cmÀ1 respectively implied the
presence of hydroxyl and aromatic absorptions. In the
1
H-NMR spectrum, two aromatic proton singlet signals
at H 6.41 (1H, s, H-7) and 6.64 (1H, s, H-10) were
observed. The resonances were ascribed at H 4.39
(d, J ¼ 11:2 Hz, H-5ax), 4.24 (dd, J ¼ 3:0, 11.2 Hz,
H-5eq), 2.46 (1H, m, H-5a), 2.24 (d, J ¼ 12:8 Hz,
H-6ax), 2.09 (dd, J ¼ 3:0, 12.8 Hz, H-6eq), 2.95
(d, J ¼ 17:2 Hz, H-11ax) and 3.27 (dd, J ¼ 5:6,
17.2 Hz, H-11eq), respectively accounting for the presence of one oxygenated methylene, a methine proton,
and two methylene groups. In the 13 C-NMR (DEPT)
spectra (Table 2), 15 signals were recognized, viz.
6 Â C, 6 Â CH, 3 Â CH2 , suggesting that the structural
skeleton of 2 should be close to that of 10,11-

Fig. 3. Induction of DNA Fragmentation by 1 in HL-60 Cells in
Vitro.
HL-60 cells were treated with 1 for 24 h (5, 10, 20 and 50 mg/mL).
Total genomic DNA was extracted and resolved on 1% agarose gel.
Apoptotic DNA fragmentation was visualized by ethidium bromide
staining. M, size marker; À, mature cell; þ, positive control treated
with 2.5 mg/mL of camptothecin.

dihydroxydracaenone C,18) except that the signals for a
cyclohexa-2,5-dienone group at C-4a and 11a of 10,11dihydroxydracaenone were absent in 2. Instead, a set of
olefinic proton signals at H 6.84 (d, J ¼ 10:0 Hz, H-1),
6.42 (brd, J ¼ 10:0 Hz, H-2) and 5.54 (brs, H-3) in the
1
H-NMR spectrum and three carbon signals at C 108.1
(C-1), 129.7 (C-2) and 152.9 (C-3) in the 13 C-NMR
spectrum, which are characteristic of a cyclopenta-1,3diene, were present (Table 2). The 1 H–1 H COSY
spectrum, coupled with the HMQC analysis, allowed
the establishment of three H-atom spin systems H-1/
H-2/H-3, corresponding to structural fragments of C-1/
C-2/C-3 (Fig. 2). The HMBC correlations of H-1, H-2
and H-3 with C-3a and C-10b indicated the direct
linkage of cyclopenta-1,3-diene located at C-3a and
C-10b. The stereochemistry of 2 was deduced from the
negative optical rotation data, ½Š25
D À165.2 (c 0.05,
MeOH), compared with that of 10,11-dihydroxydracae18)
This evidence
none C (½Š25
D À411.3, c 0.10, MeOH).
was used to deduce the stereochemistry of 2 as shown
in Fig. 1, with the trivial name of caesalpiniaphenol H.
Cytotoxic effects
Cancer cells were seeded in 96-well plates, then
incubated for 4 h, and were treated with the caesalpiniaphenol G (1) and caesalpiniaphenol H (2) at various
concentrations (5, 10, 20, 50 and 100 mg/mL). The
inhibitory process was assessed by using an MTT
assay.16) As shown in Table 3, both of 1 and 2 showed

effective inhibition against HL-60 and HeLa cells with
the IC50 values of 16:7 Æ 2:2, 22:5 Æ 5:1, 28:1 Æ 3:6
and 39:2 Æ 2:0 mg/mL, respectively. Caesalpiniaphenol
H (2) also exhibited significant inhibition against LLC
cancer cells with the IC50 value as 42:5 Æ 5:1 mg/mL.


Cytotoxic Activity of Phenolic Compounds from C. sappan

2381

Fig. 4. Increase of Caspase-3 Activity by 1 in HL-60 Cells in Vitro.
After 12 h, 24 h and 48 h of incubation with the 1-treated HL-60 cells, the cell lysates were incubated for 1 h at 37  C with the caspase-3
substrate (Ac-DEVD-AFC). The fluorescence intensity of the cell lysates was measured to determine the caspase-3 activity. The blank group was
used as 0.1% DMSO-treated cells. Camptothecin (2.5 mg/mL) was used as a positive control. Data are presented as the mean Æ SD of the results
from three independent experiments.

However, in the case of MCF-7 cell lines, both of two
compounds showed very weak inhibitory activity with
the IC50 values over 100 mg/mL (Table 3).
Apoptosis effects
To evaluate whether the growth inhibition of HL-60
cells by caesalpiniaphenol G (1) was mediated through
the apoptotic process, we performed a DNA laddering
assay.16) HL-60 cells (5 Â 105 cell/mL) were treated
with 5, 10, 20 and 50 mg/mL of 1 for 24 h, and the DNA
bands were photographed under ultraviolet illumination.
The chromosomal DNA of the treated cells showed
internucleosomal DNA fragmentation consisting of
multiples of approximately 180–200 base pairs in the

agarose gel electrophoresis (Fig. 3). Caspase-3 is a
cytosolic protein that normally exists as a 32 kDa
inactive precursor, and is proteolytically cleaved into
a heterodimer when the cell undergoes apoptosis. The
activity of caspase-3 was measured by the proteolytic
cleavage of Ac-Asp-Glu-Val-Asp-8-amino-4-trifluoromethylcoumarin in the HL-60 cells for 12, 24, and 48 h
(Av-DEVD-AFC)17) after treating with compound 1 (5,
10, 20, 50 and 100 mg/mL). The result was the caspase-3
activity being increased 1–5-fold in a dose- and timedependent manner when compared to the control
(Fig. 4).

proliferator-activated receptor
(PPAR
).21) The toxic
effects of brazilein have been evaluated in terms of the
cell viability, induction of apoptosis, and activity of
caspase-3 in BCC cells.22) Brazilin has also shown dosedependent inhibition of the cell proliferation and induction of apoptosis in glioma cells by increasing the
ratio of cleaved poly-(ADP)-ribose polymerase and
decreasing the expression of caspase-3 and caspase7.21) This in now the first report on the chemical
constituents of Vietnamese C. sappan and its cytotoxic
activity. Treatment with one of the isolated compounds,
caesalpiniaphenol G (1), induced apoptosis, thereby
inhibiting the growth of HL-60 cancer cells. The
activation of caspase-3 is required for several typical
hallmarks of apoptosis and is indispensable for apoptotic
chromatin condensation and DNA fragmentation in
the HL-60 cell type examined. The apoptotic bodies
induced by DNA fragmentation were clearly observed
in HL-60 cells treated with caesalpiniaphenol G via
electrophoresis. Apoptosis is an important way to

maintain cellular homeostasis between cell division and
cell death. The induction of apoptosis in cancer cells is
therefore one of the most useful strategies for anticancer
drug development. According to these results, it is
suggested that the active constituents from Vietnamese
C. sappan may be an important source for developing
anti-cancer drugs.

Discussion
Acknowledgments
The heartwood of C. sappan L. is a common remedy
in traditional medicine and possesses diverse biological
activities. Among the compounds isolated from C.
sappan, isoliquiritigenin 20 -methyl ether has inhibited
the growth of oral cancer cells via a pathway involving
MAP kinases, NF-B, and Nrf2.19) Sappanchalcone,
a flavonoid, has suppressed oral cancer cell growth
and induced apoptosis through the activation of p53dependent mitochondrial, p38, ERK, JNK, and NF-B
signaling.20) In another report, brazilein, the main
phenolic compound from C. sappan, has exhibited an
antioxidative effect, inhibited the intracellular lipid
accumulation during adipocyte differentiation in 3T3L1 cells, and suppressed the induction of peroxisome

This study was funded by Vietnam National
Foundation for Science and Technology Development
(NAFOSTED) under grant number 106.05-2011.52.

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