Fitoterapia 82 (2011) 489492

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

A new dimeric resveratrol from the roots of Shorea roxburghii

Winitra Patcharamun a, Jirapast Sichaem a, Pongpan Siripong b, Suttira Khumkratok c,
Jonkolnee Jong-aramruang d, Santi Tip-pyang a,
a
b
c
d

Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Natural Products Research Section, Research Division, National Cancer Institute, Bangkok 10400, Thailand
Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand
Department of Chemistry, Faculty of Science, Burapha University, Chonburi 20231, Thailand

a r t i c l e

i n f o

Article history:
Received 15 October 2010
Accepted in revised form 31 December 2010
Available online 14 January 2011
Keywords:
Dipterocarpaceae
Shorea roxburghii
Roxburghiol A
Cytotoxicity

a b s t r a c t
A new resveratrol dimer, roxburghiol A (1) together with eleven known compounds were isolated
from the roots of Shorea roxburghii. Their structures were identied on the basis of spectroscopic
evidence and physicochemical properties. All isolated compounds were evaluated for their
cytotoxicity (KB and HeLa cells). Compounds 8 and 9 showed potent cytotoxicity against both KB
and HeLa cell lines with IC50 values of 6.5, 8.5 and 8.7, 10.1 g/mL, respectively.
2011 Elsevier B.V. All rights reserved.

1. Introduction
The genus Shorea belongs to the Dipterocarpaceae family
and comprises 22 species [1] in Thailand. Shorea roxburghii
locally known as Pha-yom is a deciduous and medium to
large-sized tree widely distributed in many parts of Thailand. Its
bark has been used to treat dysentery, diarrhoea and cholera by
the local people of India [2]. There are no reports on
phytochemical investigation of this plant. Previous phytochemical study on the Shorea species revealed the presence of various
stilbenoids [3]. These stilbenoids, some of which show
interesting biological activities such as cytotoxic [4], antioxidant [5], antiplatelet aggregation [6] and cyclooxygenase
inhibitory activities [7]. Our phytochemical investigation of
this plant has resulted in the isolation of a new resveratrol
dimer, roxburghiol A (1) along with eleven known compounds
(212) (Fig. 1). This paper also describes the isolation and
structure elucidation of a new resveratrol dimer (1), and all

Corresponding author. Natural Products Research Unit, Department of

Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,
Thailand. Tel.: +66 2 218 7625; fax: +66 2 218 7598.
E-mail address: Santi.Ti@chula.ac.th (S. Tip-pyang).
0367-326X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.tote.2011.01.003

isolated compounds were tested for their cytotoxicity (KB and

HeLa cells).
2. Experimental
2.1. General
NMR spectra were recorded with a Varian model Mercury+
400 spectrometer operated at 400 MHz for 1H NMR and
100 MHz for 13C NMR and a Bruker 400 AVANCE spectrometer
at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Adsorbents
such as Sephadex LH-20 and silica gel (60 Merck cat. No. 7730,
7734 and 7749) were used for quick column chromatography,
preparative TLC, open column chromatography and centrifugal
thin layer chromatograph (Chromatotron), respectively. ESIMS
and HRESIMS data were obtained from a mass spectrometer
model VG TRIO 2000 and Micromass LCT and Bruker MICROTOF
models, respectively. Circular dichroism (CD) spectra were
recorded on a JASCOJ-715 spectropolarimeter. UVvisible
adsorption spectra were recorded on UV-2552PC UVVis
spectrometer (Shimadzu, Kyoto, Japan). Optical rotations
were measured on a Jasco P-1010 polarimeter. Melting points
were determined with FisherJohns Melting Point Apparatus.

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W. Patcharamun et al. / Fitoterapia 82 (2011) 489492

HO

A1

1a

7a

HO

8a

9a

13a

OH

HO

4a

A2

B2
9b

10a

11a

13b

OH

HO

OH

HO

OH

8b

B1

OH

HO

4b

HO

HO

O
HO
O

HO

HO
O

OH

OH

OH

HO

HO

HO

HO

OH

1b

OH

HO

HO

OH

OH

OH GlcO

HO
OH

OH

OH

OH

OH
OH

OH

HO

10
OH

HO
O H

7a

HO

HO

8b
H

7b
HO

OH

OH

8d
H

7c
H

OH

7d
OH

OH
Glc

HO
OH HO

OH

OGlc

OH

7
H O

HO

OH

OH

11b

H-7a H-7b H-8b H-7c H-7d H-8d

11

12

8
9

Fig. 1. Structure of compounds 112 isolated from S. roxburghii.

IR data were obtained from a Nicolet 6700 FT-IR spectrometer

(Thermo Electron Corporation, Madison, WI, USA) equipped
with a mercurycadmiumtelluride (MCT) detector.
2.2. Plant material
The roots of S. roxburghii were collected in May 2008 from
Mahasarakham province, Thailand. A voucher specimen
(Khumkratok no. 02-09) was deposited by Ms. Suttira
Khumkratok, a botanist at the Walai Rukhavej Botanical
Research Institute, Mahasarakham University.
2.3. Extraction and isolation
The air-dried roots of S. roxburghii (2.0 kg) were successively extracted in a Soxhlet apparatus with CH2Cl2 and acetone
(each 5 L, 24 h). The acetone soluble part was evaporated under
vacuum to yield 105.4 g. The acetone extract was fractionated

by vacuum liquid chromatography (VLC) over silica gel (500 g),

using hexane, CH2Cl2, EtOAc and MeOH with increasing
polarity. Each fraction (75 mL) was monitored by TLC and
fractions with similar TLC patterns were collected to afford six
major fractions (AF). The VLC fraction B was chromatographed
on the silica gel column (250 g) using a stepwise gradient
system of CH2Cl2, EtOAc and MeOH as eluting solvents, to
provide four fractions (B1B4). Fraction B2 was subjected to
Sephadex LH-20 (150 g) using a stepwise gradient elution of
CH2Cl2 and MeOH to yield three fractions (B2-1B2-3). Fraction
B2-2 was further puried by preparative TLC on silica gel using
CH2Cl2/EtOAc/MeOH (9:0.5:0.5) as eluent (developed 2), to
afford 2 (5.2 mg) [8] and 3 (4.5 mg) [9]. Fraction B3 was
subjected to Chromatotron eluted with a mixture of CH2Cl2/
EtOAc/MeOH (8:1:1) to give 1 (10.2 mg), 4 (12.1 mg) [10], 5
(8.0 mg) [11] and 6 (5.5 mg) [12].
Similarly, the VLC fraction C was chromatographed on
silica gel column using a stepwise gradient elution of CH2Cl2

W. Patcharamun et al. / Fitoterapia 82 (2011) 489492

and MeOH to obtain ve fractions (C1C5). Fraction C2 was

chromatographed on silica gel column (200 g) using CH2Cl2/
EtOAc/MeOH (8:1:1, 7:2:1 and 6:2:2) as elution systems, to
obtain four fractions (C2-1C2-4). Fraction C2-1 yielded 7
(43.6 mg) [13]. Fraction C2-3 was puried by preparative TLC
using CH2Cl2/EtOAc/MeOH (7:2:1 and 7:1.5:1.5) as eluent, to
give 8 (25.5 mg) and 9 (8.9 mg) [14]. Fraction C3 was
chromatographed on silica gel column (200 g) using a
mixture of CH2Cl2/EtOAc/MeOH (7:2:1 and 6:2:2), and 100%
EtOAc as elution systems to obtain three fractions (C3-1C33). Fraction C3-2 was further puried by preparative TLC
using CH2Cl2/EtOAc/MeOH (8:1:1) as eluent (developed 2),
and yielded 10 (13.0 mg) [15], 11 (5.3 mg) [16] and 12
(32.0 mg) [17].
Roxburghiol A (1): brown amorphous powder; mp 234
236 C; []25D 66.0 (c 0.20, MeOH); UV (MeOH) max (log
): 315 (4.8), 279 (2.9) nm; IR bands (KBr): 3399, 2925, 2856,
1606, 1512, 1444, 1384, 1254, 1111, 830 cm-1; positive ion
HRESIMS m/z: [M + H]+ 453.1364 (calcd for C28H20O6,
453.1370); CD (c 20.0 M, MeOH) nm () 232 (28.8); 1H
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) are
shown in Table 1.

2.4. Cytotoxicity test

Cytotoxicity of the isolated compounds (112) was
evaluated using the MTT assay method [18]. The cell lines
used in this experiment were HeLa (human cervical carcinoma) and KB (human epidermoid carcinoma) and adriamycin
was used as the standard substance.

Table 1
1
H, 13C and HMBC NMR data of compound 1 in acetone-d6.
Position

H (mult, J in Hz)

HMBC

1a
2a, 6a
3a, 5a
4a-OH
7a
8a
9a
10a
11a-OH
12a
13a-OH
14a
1b
2b, 6b
3b, 5b
4b-OH
7b
8b
9b
10b
11b
12b
13b-OH
14b

133.7
126.9
115.4
157.4
85.0
53.2
134.0
115.0 a
157.0
101.3 b
158.9
101.2 b
135.9
127.6
115.0 a
156.6
141.8
127.1
145.2
122.0
157.7
95.6
156.9
105.0

7.28 (2 H, d, J = 8.4 Hz)

6.81 (2 H, d, J = 8.4 Hz)
8.50 (1 H, s)
6.38 (1 H, d, J = 4.4 Hz)
4.05 (1 H, d, J = 4.4 Hz)

7.39 (1 H, s)
6.23 (1 H, d, J = 2.0 Hz)
8.55 (1 H, s)
6.73 (1 H, d, J = 2.0 Hz)

7.23 (2 H, d, J = 8.4 Hz)

6.75 (2 H, d, J = 8.4 Hz)
8.38 c (1 H, s)

7.09 (1 H, br s)

6.25 (1 H, d, J = 1.6 Hz)

8.38 c(1 H, s)
6.37 (1 H, d, J = 1.6 Hz)

C-4a, C-7a
C-1a
C-3a, 5a
C-2a, 6a, C-11b
C-10a, C-9b

C-10a, 12a
C-10a, C-14a
C-12a, C-14a
C-8a, C-10a, C-12a

C-4b, C-7b
C-1b
C-3b, 5b

C-10a, C-1b, C-10b, 14b

C-10b, 14b
C-12b, C-14b
C-10b

a
b
c

Signals were overlapped.

Signals were overlapped.
Signals were overlapped.

491

3. Results and discussion

Roxburghiol A (1) was obtained as an optically active brown
amorphous powder. The molecular formula of C28H20O6 was
deduced from the HRESIMS ion at m/z 453.1364 [M+ H]+
(calcd for C28H20O6, 453.1370) and NMR data.
The 1H NMR spectrum exhibited the signals of two sets of
ortho-coupled protons on the para-substituted phenyl moieties
(rings A1 and B1) at 7.28 (2 H, d, J = 8.4 Hz, H-2a, 6a)/6.81
(2 H, d, J = 8.4 Hz, H-3a, 5a) and 7.23 (2 H, d, J = 8.4 Hz, H-2b,
6b)/6.75 (2 H, d, J = 8.4 Hz, H-3b, 5b), and two sets of metacoupled protons on tetrasubstituted benzene rings (rings A2
and B2) at 6.23 (1 H, d, J = 2.0 Hz, H-12a)/6.73 (1 H, d,
J = 2.0 Hz, H-14a) and 6.25 (1 H, d, J = 1.6 Hz, H-12b)/6.37
(1 H, d, J = 1.6 Hz, H-14b). A set of mutually coupled aliphatic
methine protons at 6.38 (1 H, d, J = 4.4 Hz, H-7a)/4.05 (1 H, d,
J = 4.4 Hz, H-8a), an olenic methine proton at 7.09 (1 H, br s,
H-8b), and ve phenolic hydroxyl protons at 8.50 (1 H, br s,
OH-4a), 7.39 (1 H, br s, OH-11a), 8.55 (1 H, br s, OH-13a) and
8.38 (2 H, br s, OH-4b, 13b) were also observed in the spectrum.
The molecular formula (C28H20O6) and NMR (1H and 13C)
spectral data revealed that 1 was composed of two resveratrol
units. The correlations of all protons to the respective carbons
were claried with the help of HMQC and HMBC spectrum. The
HMBC correlations between H-7a/C-2a(6a), H-7a/C-11b, H-8a/
C-10a, H-8a/C-9b, H-12a/C-10a, H-14a/C-10a, H-8b/C-10a, H8b/C-1b, H-8b/C-10b, H-8b/C-14b and H-2b(6b)/C-7b revealed
the connectivities between C-1a/C-7a, C-8a/C-9a, C-1b/C-7b
and C-8b/C-9b (Fig. 2). The signals at H-7a ( 6.38) and H-8a (
4.05) with their HMQC correlated at C-7a ( 85.0) and C-8a (
53.2) showed the characteristics of a resveratrol derivative
containing a 1,2-diaryl-dihydrobenzofuran moiety, that the
conformation of this 1,2-diaryl-dihydrobenzofuran was
assigned to be trans-oriented by NOE experiments.
The absolute congurations at C-7 and C-8 of the
dihydrobenzofuran skeleton of 1 were assigned based on
the circular dichroism (CD) spectroscopic evidence. The CD
spectra of 1 exhibited the Cotton signal at 232 nm ( 28.8
(c 20.0 M, MeOH)); the sign and wavelength maxima are
consistent with similar with CD spectra of 4 at 236 nm (

HO

O
HO

OH

OH

OH

1
Fig. 2. The key HMBC correlations of compound 1. Arrows indicate
correlation between hydrogen (point of origin) and carbon (arrowhead).

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W. Patcharamun et al. / Fitoterapia 82 (2011) 489492

24.8 (c 32.8 M, MeOH)), suggesting that two chiral centers

of the dihydrobenzofuran skeleton of 1 have same absolute
congurations as 4 which have been assigned as absolute R
congurations [19]. Thus, this new dimeric resveratrol was
given the name as roxburghiol A (1).
The eleven known compounds, melanoxylin A (2), caragaphenol A (3), (-)--viniferin (4), hopeahainanphenol (5),
vitisinol G (6), vaticanol A (7), (-)-hopeaphenol (8), isohopeaphenol (9), apigenin 7-O-arabinoside (10), trans-piceid (11)
and trans-3,5,4-trihydroxyresveratrol 2-C-glucoside (12) were
established on the basis of their spectroscopic data as well as
comparison with the previous literature data.
All isolated compounds were evaluated for their cytotoxicity towards HeLa and KB cells.
Compounds 8 and 9 showed potent cytotoxicity against
both KB and HeLa cells with IC50 values of 6.5, 8.5 and 8.7,
10.1 g/mL, respectively. In addition, other compounds were
inactive to both cells (IC50 N 50 g/mL).

Acknowledgment
The authors are grateful to the Center for Petroleum,
Petrochemical and Advanced Materials, Chulalongkorn University and the National Research University Project of CHE and the
Ratchadaphiseksomphot Endowment Fund (FW001A) (to ST)
for partially supporting this project. We also thank Assist. Prof.
Tetsuro Ito, Gifu Pharmaceutical University, Japan, for his

invaluable comments on the structure elucidation of the

compounds in this work.
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