Phytochemistry Letters 11 (2015) 316–319

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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Two new flavonols from flowers of Getonia floribunda Roxb.

Kultida Suttaratrungse, Patcharee Chanhan, Kwanjai Kanokmedhakul,
Panawan Moosophon *, Somdej Kanokmedhakul
Natural Products Research Unit, Department of Chemistry, and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University,
Khon Kaen 40002, Thailand

A R T I C L E I N F O A B S T R A C T

Article history: Phytochemical investigation of the EtOAc and MeOH extracts from flowers of Getonia floribunda Roxb., a
Received 17 October 2014 Thai herbal medicine, resulted in the isolation of two new flavonols, 40 -hydroxy-6,7,8,30 -tetramethoxy-
Received in revised form 1 February 2015 flavonol (1) and 40 -hydroxy-6,7,8-trimethoxyflavonol (2), along with a known pachypodol (3). Their
Accepted 4 February 2015
structures were elucidated by spectroscopic methods including UV, IR, 1D and 2D NMR techniques and
Available online 16 February 2015
MS analysis. Compound 1 showed cytotoxicity against the oral cavity cancer (KB) cell line with an IC50
value of 8.99  2.00 mg/ml.
Keywords:
ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Getonia floribunda Roxb.
Flavonol
Oral cavity cancer
Cytotoxicity

1. Introduction and antioxidant activities, and cytotoxicity against three cancer

Getonia floribunda Roxb. (Combretacea) is used in Asian
traditional medicine systems, including Ayurveda, Unani and folk
medicine (Ali et al., 2008). It is a woody climbing shrub and
flowering plant commonly found in many part of the south-east
Asia. It is known as ‘‘Kradaeng’’ in Thai, and a water decoction of
the stems and/or roots has been used in Thai traditional medicine
as a tonic to heal infertile women (Pathommapas et al., 2008–
2009). Previous phytochemical studies have revealed the presence
of various classes of bioactive constituents: calycopterin with
anthelmintic properties (Ratnagiriswaran et al., 1934), biflvonoids,
calycopterones exhibiting cytotoxicity against human cancer cell
lines (Mayer, 1999; Wall et al., 1994) and calyflorenones (Mayer,
2004), and macrocyclic lactones, combretastatins, with cytotoxici-
ty towards the small cell lung cancer cell line (Vongvanich et al.,
2005). In order to search for active flavonoids from flowers of G.
floribunda, further chemical investigation of this plant was
conducted, which led to the isolation of two new flavonols, 40 -
hydroxy-6,7,8,30 -tetramethoxyflavonol (1) and 40 -hydroxy-6,7,8-
trimethoxyflavonol (2) and a known flavonol, pachypodol (3) (Ali
et al., 2008; Sy and Brown, 1998) from EtOAc and MeOH extracts.
We report herein the isolation, structural elucidation and
biological evaluation including antiplasmodial, antimycobacterial
* Corresponding author. Tel.: +66 43 202222 41x12243; fax: +66 43 202373.
E-mail address: mpanaw@kku.ac.th (P. Moosophon).
cell lines of isolated compounds.
2. Results and discussion
Crude EtOAc and MeOH extracts were obtained from dried
flowers of G. floribunda. Chromatographic separation of these
extracts gave two new flavonols, 40 -hydroxy-6,7,8,30 -tetramethox-
yflavonol (1) and 40 -hydroxy-6,7,8-trimethoxyflavonol (2), and a
known pachypodol (3) (Fig. 1). Their structures were identified on
the basis of spectroscopic methods, mass spectrometric techniques
and comparison of the data with those reported in the literature.
Compound 1 was obtained as a pale yellow powder and had the
molecular formula of C19H18O8 (11 degrees of unsaturation) based
on a molecular ion peak at m/z 397.0983 [M+Na]+ in the HRESIMS.
The IR spectrum showed the presence of hydroxy (3419 cm1) and
unsaturated carbonyl (1657 cm1) functional groups. The UV
spectrum displayed absorption bands at lmax 226 and 350 nm. The
1
H and 13C NMR (Table 1) and DEPT spectroscopic data showed
19 carbon signals attributable to four methoxy (dC 56.3, 56.5,
60.3 and 61.0), four methine aromatic (dC 90.5, 111.1, 114.8 and
122.7), and eleven nonprotonated carbons (dC 106.7, 122.5, 132.5,
138.8, 146.6, 148.5, 152.4, 152.9, 156.1, 158.9 and 179.0). These
data allowed the formulation of a flavonoid skeleton which was
supported by the above IR and UV spectroscopic data (Plazonić
et al., 2009). The 1H NMR spectrum showed a singlet signal at dH
6.49 (H-5), which showed HMBC correlations (Fig. 2) with C-4, C-6,

http://dx.doi.org/10.1016/j.phytol.2015.02.004
1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
 K. Suttaratrungse et al. / Phytochemistry Letters 11 (2015) 316–319 317

4' OH OH OH OH
OMe 1 OMe OMe
B 3'
MeO 7 9 O 2 MeO O MeO O MeO O
1' R OMe OMe OMe
A C 2'
3
MeO 6 10 OH OMe MeO OH MeO OH
5 4
O OH O O O

1 : R = OMe 3 Fig. 2. Key HMBC (left) and NOESY (right) correlations of 1.

2:R =H

Fig. 1. Structures of compounds 1–3.

C-7, C-9 and C-10 suggesting the presence of a 1,2,3,4,5-
pentasubstitution on the benzene ring A. Ring A contained three
methoxy groups at C-6, C-7 and C-8, confirmed by HMBC
correlations from methoxy protons at dH 3.95 to C-6, methoxy
protons at dH 3.91 to C-7 and methoxy protons at dH 3.85 to C-8 as
well as NOESY correlation (Fig. 2) between H-5 and methoxy
protons at C-6. The 1H NMR spectrum of ring B exhibited proton
resonances at dH 7.03 (d, J = 8.4 Hz, H-50 ), 7.65 (dd, J = 8.4, 1.4 Hz,
H-60 ) and 7.69 (d, J = 1.4 Hz, H-20 ) as well as the HMBC correlations
(Fig. 2), consistent with a 1,3,4-trisubstituted benzene ring. A
methoxy group appearing at dH 3.98 showed an HMBC correlation
with C-30 (dC 146.6) and a NOESY correlation with H-20 , confirming
the location of the methoxy group at C-30 . The chemical shift of C-40
appeared at dC 148.5 revealing oxygenation at this carbon. The
connectivity of ring B to ring C was established by HMBC
correlations from both H-20 and H-60 to the oxygenated carbon
C-2. Thus, compound 1 was elucidated as a new flavonol, 40 -
hydroxy-6,7,8,30 -tetramethoxyflavonol.
Compound 2 was obtained as a pale yellow powder and
displayed a [M+Na]+ ion at m/z 367.0769 in the HRESIMS,
consistent with a molecular formula C18H16O7 (11 degrees of
unsaturation). The UV and IR spectra were similar to those of 1. The
1
H and 13C NMR spectral data (Table 1), and a DEPT experiment,
revealed that 2 had a flavonol structure with 18 carbon signals
indicating one less methoxy carbon than 1. The 1H NMR data for 2
was compatible with those of 1, except for the proton spin patterns
of ring B. Ring B of 2 showed two overlapped two-proton doublets
at dH 7.99 (J = 8.8 Hz, H-20 /60 ) and 6.93 (J = 8.8 Hz, H-30 /50 ) that
were consistent with a 1,4-disubstitution benzene ring. This was
confirmed by HMBC correlations from H-20 /60 to C-2, C-40 , C-60 /20
and from H-30 /50 to C-10 , C-40 , C-50 /30 as well as NOESY correlation
between H-20 /60 and H-30 /5. The chemical shifts of C-40 (dC 160.9)
helped establish the attachment of a hydroxy group at this
position. Therefore, compound 2 was defined as a new 40 -hydroxy-
6,7,8,-trimethoxyflavonol.
Compound 3 was elucidated as pachypodol by comparing its
spectroscopic data with those reported in the literature (Ali et al.,
2008; Sy and Brown, 1998). All isolated compounds were
evaluated for their biological activities. Flavonol 1 displayed
moderate cytotoxicity towards the KB cell line with IC50 value of
8.99  2.00 mg/ml, while compounds 2 and 3 showed no cytotoxicity.
The results indicate that the methoxy at C-30 and the hydroxy at C-3 of
1 play an important role in enhancing cytotoxicity against the KB
cancer cell line, compared to 2 and 3, respectively. However these
compounds were non-cytotoxicity against the MCF7 cell line and
were inactive for antimycobacterial, antiplasmodial and antioxidant
tests.
3. Experimental
3.1. General experimental procedures
UV spectra were recorded using an Agilent 8453 UV-visible
spectrophotometer. IR spectra were obtained using a Bruker
Tenser 27 spectrophotometer. NMR spectra were recorded in
CDCl3 and CD3OD, as solvents on a Varian Mercury Plus
400 spectrometer; the internal standards were referenced from
the residue of those solvents. HRESITOFMS data were obtained
using a Micromass Q-TOF-2 spectrometer. Column chromatogra-
phy (CC) was carried out on MERCK silica gel 60 (230–400 mesh)
and Sephadex LH-20. Preparative TLC was carried out on silica gel

Table 1
1
H and 13C NMR (400 and 100 MHz) and HMBC data of compounds 1 and 2 (d in ppm, J in Hz).

Position 1a 2b

dC dH HMBC dC dH HMBC

2 156.1 157.9
3 152.9 152.8
4 179.0 179.6
5 90.5 6.49 s 4, 6, 7, 9, 10 91.4 6.61 s 4, 6, 7, 9, 10
6 158.9 159.6
7 132.5 132.7
8 138.8 139.0
9 152.4 153.2
10 106.7 106.9
10 122.5 121.9
20 111.1 7.69 d (1.4) 2, 10 , 30 , 40 , 60 130.9 7.99 d (8.8) 2, 40 , 60
30 146.6 116.2 6.93 d (8.8) 10 , 40 , 50
40 148.5 160.9
50 114.8 7.03 d (8.4) 10 , 30 , 40 , 60 116.2 6.93 d (8.8) 10 , 30 , 40
60 122.7 7.65 dd (8.4, 1.4) 2, 20 , 40 130.9 7.99 d (8.8) 2, 20 , 40
6-OMe 56.5 3.95 s 6 56.8 3.95 s 6
7-OMe 61.0 3.91 s 7 61.2 3.86 s 7
8-OMe 60.3 3.85 s 8 60.5 3.77 s 8
30 -OMe 56.3 3.98 s 30 , 40
a
Recorded in CDCl3.
b
Recorded in a mixture of CD3OD and CDCl3.
318 K. Suttaratrungse et al. / Phytochemistry Letters 11 (2015) 316–319
PF254. TLC was performed on precoated MERCK silica gel 60 PF254;
spots were visualized under UV light (254 and 366 nm) and further
by spraying with anisaldehyde and then heating until charred.
3.2. Plant material
The flowers of G. floribunda were collected from Phu Dong Ee-
Pia forest, Ubonratana district, Khon Kaen, Thailand, in March
2012. The plant was identified by Professor James F. Maxwell,
Chiang Mai University, Thailand. A voucher specimen (SRITUBTIM
42) was deposited at the Udon Thani Rajabhat Universtiy
Herbarium, Udon Thani, Thailand.
3.3. Extraction and isolation
Air-dried flowers of G. floribunda (150 g) were ground and
extracted successively with EtOAc (3  1.5 l) and MeOH (3  1.5 l).
Removal of solvents from each extract under reduced pressure
gave crude EtOAc (3.93 g) and MeOH (5.37 g) extracts, respective-
ly. The EtOAc extract was subjected to silica gel flash column
chromatography (FCC), eluted with a gradient system of EtOAc/
hexane (0–100%) and MeOH/EtOAc (5–100%) to give 10 fractions as
E1–E10. Fraction E8 (0.6477 g) was separated by CC on silica gel,
eluted with CH2Cl2/hexane (40%), to give 5 subfractions, E8.1-E8.5.
Subfraction E8.3 (322.8 mg) was chromatographed on silica gel
column, eluted with EtOAc/hexane (50%), to give 7 subfractions,
E8.3.1-E8.3.7. Then subfraction E8.3.1 (69.4 mg) was purified by
Sephadex LH-20 column using MeOH as solvent to give a pale
yellow powder of compound 1 (26.9 mg). Purification of subfrac-
tion E8.4 (251.2 mg) by silica gel CC and using MeOH/CH2Cl2 (99%)
as eluent gave a pale yellow powder of compound 2 (21.3 g).
Fraction E6 (651.70 mg) was purified by silica gel CC, eluted with
acetone/hexane (10%), to afford a yellow solid of pachypodol (3)
(2.80 mg). The MeOH extract was subjected to FCC on silica gel,
eluted with gradient solvent system of MeOH/EtOAc, to give
19 fractions, M1–M19. Fraction M4 (111.0 mg) was purified by
preparative TLC using EtOAc/CH2Cl2 (2%) as developing solvent to
afford an additional amount of 2 (17.5 mg). Fraction M5 (19.7 mg)
was separated by preparative TLC using EtOAc/CH2Cl2 (50%) as
eluent and further purified by preparative TLC using n-BuOH/
CH2Cl2 (99%) as developing solvent to obtain an additional amount
of 1 (4.3 mg).
3.3.1. 40 -hydroxy-6,7,8,30 -tetramethoxyflavonol (1)
Pale yellow powder; UV (MeOH) lmax (log e) 226 (4.31), 350
(4.39) nm; IR (KBr) nmax 3419, 2939, 1657, 1591, 1515, 1461, 1354,
1273, 1218 cm1; 1H and 13C NMR data see Table 1; HRESIMS m/z
397.0983 [M+Na]+ (calcd. for C19H18O8Na, 397.0899)
3.3.2. 40 -hydroxy-6,7,8-trimethoxyflavonol (2)
Pale yellow powder; UV (MeOH) lmax (log e) 227 (3.15), 341
(3.43) nm; IR (KBr) nmax 3144, 2925, 2853, 1733, 1647,
1595,1462,1437, 1355 cm1; 1H and 13C NMR data see Table 1;
HRESIMS m/z 367.0769 [M+Na]+ (calcd. for C18H16O7Na,
367.0794).
3.4. Antimalarial assay
Antimalarial activity was evaluated in vitro against the parasite
Plasmodium falciparum (K1, multidrug resistant strain) using the
method of Trager and Jensen (1967). The in vitro antimalarial
activity was quantified using the microculture radioisotope
technique based on the method described by Desjardins et al.
(1979). The inhibitory concentration (IC50) represents the concen-
tration that caused a 50% reduction in parasite growth as indicated
by the in vitro uptake of [3H]-hypoxanthine by P. falciparum. The
standard compound dihydroartemisinin exhibited an IC50 value of
2.0 nM and the maximum final concentration of the tested sample
for antimalarial activity was 10 mg/ml.
3.5. Antimycobacterial assay
Antimycobacterial activity was assessed against Mycobacterium
tuberculosis H37Ra using the Microplate Alamar Blue Assay
(MABA) according to Collins and Franzblau (1997). The standard
drug isoniazid showed MIC value of 0.047 mg/ml and the highest
test concentration for anti TB was 50 mg/ml.
3.6. Antioxidant assay
Antioxidant activity was determined by the scavenging effect
on DPPH radicals. This method was adapted from that of Bonina
et al. (2000). Basically, the isolated compounds were added into
methanol solution of DPPH. After thorough mixing, the solutions
were kept in the dark for 30 min. Thereafter, the absorbency of the
samples was measured using a spectrophotometer (UV-2450,
Shimadzu) at 517 nm. Each sample was duplicated in the test, and
the values were averaged. For the determination of IC50, each of the
purified compounds was made into eight different concentrations
for DPPH tests. Standard gallic acid showed an IC50 value of 7.0 mg/
ml and the maximum final concentration of the tested sample was
200 mg/ml.
3.7. Cytotoxicity assay
Cytotoxicity assays using human oral cavity cancer (KB), human
breast cancer (MCF7), and human small cell lung cancer (NCI-
H187) cell lines were performed employing the colorimetric
method described by Skehan et al. (1990). The reference substance,
doxorubicin, showed cytotoxicity against the KB, MCF7 and NCI-
H187 with IC50 values of 0.633  0.352, 8.74 and 0.099 mg/ml,
respectively. The maximum final concentration of tested sample for
cytotoxicity against cancer cell lines was 50 mg/ml.
Acknowledgements
We thank the Center of Excellence for Innovation in Chemistry
(PERCH-CIC), Office of the Higher Education Commission, Ministry
of Education, for partial support. We are indebted to the Bioassay
Research Facility of the National Centre for Genetic Engineering
and Biotechnology via the Bioresource Research Network (BRN) for
bioactivity tests.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2015.02.004.
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