Polygonflavanol A, a novel flavonostilbene glycoside from the roots of Polygonum multiflorum

Article abstract of DOI:10.1016/j.phytol.2012.08.007

Polygonflavanol A (1), a novel flavonostilbene glycoside with an unusual coupling pattern, together with five known stilbene glycosides (2-6), was isolated from the roots of Polygonum multiflorum. The structures were elucidated on the basis of extensive spectroscopic analysis and chemical evidence. The absolute configuration of 1 was further determined by CD analysis. Most of the compounds could inhibit nitric oxide (NO) secretion of RAW264.7 cells in respond to lipopolysaccharide (LPS) in a dose dependent manner. Among them, compound 2 displayed the highest inhibitory activity.

Full text of DOI:10.1016/j.phytol.2012.08.007

Phytochemistry Letters 5 (2012) 756–760
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Polygonflavanol A, a novel flavonostilbene glycoside from the roots of Polygonum
multiflorum
Li-Li Chen a,b,1, Xiao-Jun Huang a,b,c,1, Man-Mei Li a,b, Guo-Min Ou a,b, Bing-Xin Zhao a,b, Min-Feng Chen a,b
a,b,**
,
Qing-Wen Zhang c, Ying Wang a,b, , Wen-Cai Ye
*
a Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, PR China
c State Key Laboratory of Quality Research in Chinese Medicine (University of Macau) and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Polygonflavanol A (1), a novel flavonostilbene glycoside with an unusual coupling pattern, together with
five known stilbene glycosides (2–6), was isolated from the roots of Polygonum multiflorum. The
structures were elucidated on the basis of extensive spectroscopic analysis and chemical evidence. The
absolute configuration of 1 was further determined by CD analysis. Most of the compounds could inhibit
nitric oxide (NO) secretion of RAW264.7 cells in respond to lipopolysaccharide (LPS) in a dose dependent
manner. Among them, compound 2 displayed the highest inhibitory activity.
Received 26 April 2012
Received in revised form 3 August 2012
Accepted 19 August 2012
Available online 30 August 2012
Keywords:
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Polygonum multiflorum
Flavanostilbene
Stilbene glycoside
Polygonflavanol A
Anti-inflammatory
1. Introduction
multiflorum, some of which have been reported to possess potent
anti-inflammoatory (Zhang et al., 2007), antioxidant (Chen et al.,
The plant Polygonum multiflorum Thunb. (Polygonaceae) is
widely cultivated in southern China. The root of this plant,
commonly known as ‘‘Heshouwu’’ in China, is a famous traditional
Chinese medicine, which has been widely used for the treatment of
hyperlipidemia, coronary heart disease, neurosis, and other
diseases (Wu, 2009). Recent pharmacological studies demonstrat-
ed that the crude extract of the roots of P. multiflorum showed
obvious anti-inflammoatory and anti-atherosclerotic effects
(Zhang et al., 2007), as well as the strongly suppressing oxidation
property (Lv et al., 2007) and the ability to clean the active oxygen
free radicals (Ryu et al., 2002) activities. Previous phytochemical
investigation of this plant had led to the isolation of a number of
compounds, such as anthraquinones, stilbene glycosides, flavo-
noids, and tannins (Zhang et al., 2008). Among them, stilbene
glycosides are the major and characteristic composition of P.
1999a,b), anti-HIV (Lin et al., 2010), and liver protection (Wang
et al., 2012) activities. As a part of our ongoing program to assess
the chemical and biological diversities of medicinal plants in
southern China, we carried out the phytochemical study on the
roots of P. multiflorum. A novel flavonostilbene glycoside, poly-
gonflavanol A (1), with an unusual coupling pattern between
flavanone and stilbene moiety, together with five known stilbene
glycosides (2–6) (Fig. 1) was isolated from the n-butanol soluble
fraction. The structure of compound 1 was elucidated by means of
HRESIMS, NMR, as well as CD analysis. Herein, we report the
isolation and structural elucidation of the new compound, as well
as the anti-inflammatory activity of the six isolated compounds.
2. Results and discussion
Compound 1 was isolated as brown oil with positive optical
rotation value ½a D19
ꢀ
: þ9:46 (c 0.01, MeOH). The HR-ESI-MS showed
a
quasi-molecular ion at m/z 717.1789 [M+Na]+ (calcd for
* Corresponding author at: Institute of Traditional Chinese Medicine & Natural
Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China.
** Corresponding author at: Institute of Traditional Chinese Medicine & Natural
Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China.
Tel.: +86 20 85220936; fax: +86 20 85221559.
C35H34O15Na: 717.1790), which is consistent with a molecular
formula of C35H34O15
.
The UV spectrum of showed the
1
absorptions maxima at 205 and 280 nm. The IR spectrum implied
the presence of hydroxyl group (3444 cmꢁ1) and aromatic ring
(1618, 1508 and 1461 cmꢁ1). The 1H NMR spectrum of 1 revealed
E-mail address: chywc@yahoo.com.cn (W.-C. Ye).
1
These authors contributed equally to this work.
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2012.08.007

L.-L. Chen et al. / Phytochemistry Letters 5 (2012) 756–760
757
Fig. 2. Key 1H–1H COSY and HMBC correlations of 1.
on the 3JH1,H2 coupling constant of the anomeric proton (J = 7.9 Hz).
The 13C NMR and DEPT-135 spectra of 1 revealed the presence of
thirty-five carbon signals, including twenty-four aromatic carbons,
Fig. 1. Chemical structures of compounds 1–6.
three oxy-methines and two alkyl-methines, as well as a
b-D-
signals for a para-disubstituted benzene ring [dH 7.08 (2H, d,
J = 8.5 Hz, H-1000 and H-1400) and 6.58 (2H, d, J = 8.5 Hz, H-1100 and
H-1300)], a 1,3,4-trisubstituted benzene ring [dH 6.89 (1H, s, H-20),
6.73 (1H, d, J = 8.5 Hz, H-60) and 6.71 (1H, d, J = 8.5 Hz, H-50)], two
tetrasubstituted benzene rings [dH 6.09 (1H, d, J = 2.6 Hz, H-400),
6.07 (1H, d, J = 2.6 Hz, H-600), 6.01 (1H, d, J = 2.2 Hz, H-8) and 5.93
(1H, d, J = 2.2 Hz, H-6)], and five methines [dH 5.03 (1H, d,
J = 10.4 Hz, H-800), 4.97 (1H, d, J = 3.8 Hz, H-2), 4.30 (1H, t,
J = 10.4 Hz, H-700), 4.18 (1H, dd, J = 7.0, 3.8 Hz, H-3) and 3.02 (1H,
dd, J = 11.3, 7.0 Hz, H-4)]. In addition, the 1H NMR spectrum of 1
displayed the signals due to an anomeric proton [dH 4.46 (1H, d,
J = 7.9 Hz, H-1000)] and a hydroxymethyl group [dH 3.79 (1H, d,
J = 12.0 Hz, H-6000a) and 3.72 (1H, dd, J = 12.0, 4.3 Hz, H-6000b)]. Acid
glucopyranosyl moiety. All above information suggested that 1
possessed a flavonone moiety (1a) and a stilbene unit (1b). The
1H–1H COSY, HSQC, and HMBC spectra of 1 allowed the full
assignment of all proton and carbon signals (Table 1).
The 1H and 13C NMR signals assigned to 1a were very similar to
those of (ꢁ)-epi-catechin (Hwang et al., 2001), indicating that 1
possessed the same flavan-3-ol moiety. Moreover, a comparison of
the NMR data assigned to 1b with those of 2,3,5,40-tetrahydrox-
ystilbene 2-O-b-D-glucopyranoside (2) (Chen et al., 1999a,b)
suggested that they were very similar, except for the absence of
trans-1,2-disubstituted vinyl unit and the presence of two
methines in 1b. The 1H–1H COSY spectrum of 1 revealed the
presence of spin systems in bold as shown in Fig. 2. The HMBC
correlations between H-2 and C-4, between H-800 and C-4/C-1000, as
well as between H-700 and C-600 indicated that 1a and 1b were
hydrolysis of 1 afforded
D-glucose, which was identified by GC
analysis. The
b
-configuration of -glucose was determined based
D
Table 1
1H and 13C NMR spectral data of compound 1 (CD3OD, J in Hz).a,b
Position
dC
dH
Position
dC
dH
2
3
80.9
4.97 d (3.8)
100
136.8
139.1
151.7
103.3
155.9
106.1
40.5
–
71.9
41.3
4.18 dd (7.0, 3.8)
200
–
4
3.02 dd (11.3, 7.0)
300
–
5
157.1
96.8
–
400
6.09 d (2.6)
–
6
5.93 d (2.2)
500
7
158.9
96.4
–
600
6.07 d (2.6)
4.30 t (10.4)
5.03 d (10.4)
–
8
6.01 d (2.2)
700
8a
4a
10
20
30
40
50
60
156.9
102.0
131.4
115.7
146.0
146.0
116.0
120.1
–
800
85.8
–
900
132.0
130.6
115.5
158.0
107.7
75.4
–
1000, 1400
1100, 1300
1200
1000
7.08 d (8.5)
6.58 d (8.5)
–
6.89 s
–
–
4.46 d (7.9)
3.46
6.71 d (8.5)
6.73 d (8.5)
2000
3000
78.3
3.34
4000
70.8
3.43
5000
78.5
3.37
6000
62.3
3.79 d (12.0)
3.72 dd (12.0, 4.3)
a
Assignments were established by interpretation of the 1H–1H COSY, HSQC, and HMBC spectra.
Overlapped signals are reported without designating multiplicity.
b

758
L.-L. Chen et al. / Phytochemistry Letters 5 (2012) 756–760
connected via C-4–C-700 bond. Furthermore, according to the
molecular formula information, as well as the obvious downfield
chemical shift value of C-800 (
dC 85.8) and upfield one of the C-5 (dC
157.1), the remaining oxygen atom was assigned to bridge C-5 and
C-800 to form a dihydropyran moiety (Fig. 2). The linkage position of
the
b-D-glucopyranosyl moiety was confirmed by the HMBC
correlation between H-1000 and C-200.
The relative configuration of 1 was deduced by the vicinal
coupling constants and the ROESY experiment (Fig. 3). The
coupling constant (J = 3.8 Hz) between H-2 and H-3 indicated
that the two protons were on the same side of the molecule plane
(assigned as
b
-orientation), which was identical to that of (ꢁ)-epi-
catechin (Hwang et al., 2001). On the contrary, the greater coupling
constant (J = 7.0 Hz) between H-3 and H-4 indicated that they were
in the opposite orientation (Friebolin, 1993). Thus, H-4 was
Fig. 4. Calculated and experimental CD spectra of 1.
assigned to be
a-orientation. Moreover, the large coupling
constant (J = 10.4 Hz) between H-700 and H-800 suggested the
opposite orientation of these two protons. The ROESY correlations
between H-3 and H-700 as well as between H-4 and H-800 suggested
flavonone and a stilbene through one of the three ring system
(rings A–C). It is noteworthy that 1 is the first example of
flavonostilbene whose flavanone moiety is coupled with the
stilbene moiety through both rings A and C. In addition, all the
known flavonostilbenes exist naturally as aglycone form, and 1
represents the first example of flavonostilbene glycoside.
that the orientations of H-700 and H-800 were
b and a, respectively.
The negative Cotton effects at max 286.7 and 245.8 nm in the CD
l
spectrum indicated the presence of 2R and 3R in 1 (Slade et al.,
2005). Thus, the absolute configurations of all chiral centers of the
aglycone part in 1 were determined to be 2R, 3R, 4S, 700R, and 800S,
respectively.
The five known stilbene glycosides were identified as (E)-
2,3,5,40-tetrahydroxystilbene-2-O-
b
-
D
-glucoside (2) (Chen et al.,
1999a,b), (E)-2,3,5,40-tetrahydroxystilbene-2-O-(60-O-acetyl)-
glucoside (3) (Chen et al., 2000a), (E)-2,3,5,40-tetrahydroxystil-
bene-2,3-di-O- -glucoside (4) (Zhou et al., 1994), (E)-2,3,5,
40-tetrahydroxystilbene-2-O-(600-O-
-glucoside)- -glucoside
(5) (Chen et al., 2000b), and (Z)-2,3,5,40-tetrahydroxystilbene-2-O-
-glucoside (6) (Xu et al., 2006), respectively, by comparison of
b-D-
The absolute configuration of 1 was further confirmed by the
quantum chemical CD calculation method (Li et al., 2010). By using
MMFF94S force field, a conformational analysis was performed.
The resulted global minima conformers were found and used as the
basis for the B3LYP/6-311++G(2d,p)//B3LYP/6-31G(d) calculations
to finally provide the theoretically predicted CD spectrum with
PCM model in CH3CN solvent (Fig. 4) by using Gaussian 09
software. The calculated CD curve of 1 revealed a good agreement
with the measured spectrum. Thus, the absolute configuration of 1
was further confirmed as 2R, 3R, 4S, 700R, and 800S.
The term flavonostilbene was first applied by Shirataki and co-
workers in 1991 to describe the coupling of flavanones and
stilbenes. Up to now, about twenty-three flavonostilbenes and one
nor-flavonostilbene had been isolated from natural sources (Ayers
et al., 2008; Iliya et al., 2003; Linuma et al., 1995, 1994, 1991;
Shirataki et al., 1991; Steynberg et al., 1987; Wada et al., 2007). All
skeletons of the reported flavonostilbenes were formed by a
b
-D
a
-D
b-D
b
-D
their spectroscopic data with those reported in the literature.
The anti-inflammatory activities of compounds 1–6 were
investigated. The results showed that compounds 1–3, 5 and 6
could inhibit NO secretion of RAW264.7 cells in respond to LPS in a
dose dependent manner. Compound 2 displayed the highest
inhibitory activity with NO concentration value of 0.89 ꢂ 0.06 mM.
However, all the compounds showed weak inhibitory activity against
the proliferation of cells.
3. Materials and methods
3.1. General experimental procedures
Optical rotation values were measured on a JASCO P-1020
polarimeter with a 0.01 dm length cell. UV spectra were recorded
on a JASCO V-550 UV/VIS spectrophotometer. IR spectra were
measured on a JASCO FT/IR-480 plus Fourier transform infrared
spectrometer with KBr pellets. CD spectra were obtained on a
JASCO J-810 spectropolarimeter at room temperature. 1D and 2D
NMR spectra were recorded on a Bruker AV-400 spectrometer with
TMS as internal standard. ESI-MS data were obtained on a Finnigan
LCQ advantage spectrometer. HR-ESI-MS was conducted on an
Agilent 6210 LC/MSD TOF mass spectrometer. GC–MS analyses
were performed on a Shimadzu GCMS-QP2010 plus gas chromato-
graph-mass spectrometer. Column chromatographies were carried
out on macroporous resin Diaion HP-20 (Mitsubishi Chemical
Corporation), silica gel (200–300 mesh; Qingdao Marine Chemical
˚
Group Co. Ltd.), ODS (50
mm, 120 A; YMC), or Sephadex LH-20
(Pharmacia Biotech AB). TLC analyses were performed on pre-
coated silica gel GF254 plates (Yantai Chemical Industrial Institute,
Yantai, China). Analytical high-performance liquid chromatogra-
phy (HPLC) was carried out on a Agilent chromatograph equipped
with a 1260 pump and a 1260 Dual
Cosmosil 5C18-MS-II reversed-phase column (4.6 mm ꢃ 250 mm,
m, Nacalai Tesque, Kyoto, Japan). Preparative HPLC was carried
out on an Agilent chromatograph equipped with a 1260 pump and
l absorbance detector with a
5
m
Fig. 3. Key ROESY correlations of 1.

L.-L. Chen et al. / Phytochemistry Letters 5 (2012) 756–760
759
a UV/VIS-1260 detector, with a Cosmosil 5 C18-MS-II reversed-
3.5. Bioassay of anti-inflammation activity
phase column (20 mm ꢃ 250 mm, 5
mm, Nacalai Tesque, Kyoto,
Japan).
In vitro anti-inflammation activity was evaluated by determin-
ing the nitrite concentration in the medium and the proliferation of
RAW264.7 cells. Briefly, the RAW264.7 cells (4 ꢃ 104 cells) were
cultured in the medium with different concentration of samples (0,
3.2. Plant material
The roots of P. multiflorum were collected in Deqing county,
Guangdong province of China, in October of 2010 and authenti-
cated by Prof. Guang-Xiong Zhou (College of Pharmacy, Jinan
University). A voucher specimen (No. 20101003) was deposited in
the Institute of Traditional Chinese Medicine & Natural Products,
Jinan University, Guangzhou, PR China.
12.5, 25, 50, 100
One hundred microliters of each supernatant was mixed with
100 L of Griess reagent and incubated at room temperature for
15 min. The absorbance was measured at 540 nm with
microplate reader (Thermo, USA). The cell proliferation was
determined in fresh medium by MTT method.
mM) for 48 h in the presence of LPS (200 ng/mL).
m
a
3.3. Extraction and isolation
Acknowledgments
The air-dried roots of P. multiflorum (14.5 kg) were pulverized
and extracted with 95% (v/v) EtOH for two times (2 ꢃ 25 L, 2 h each
time) under reflux. The solution was concentrated under vacuum
to yield a crude extract (1200 g). The suspension of the extract in
water was partitioned successively with petroleum ether (b.p. 60–
90 8C), ethyl acetate, and n-butanol, respectively. After removing
the solvent, the n-butanol soluble fraction (144 g) was re-dissolved
in water and subjected to macrosporous resin HP-20 column and
eluted successively with water, 30% EtOH, 50% EtOH, 70% EtOH,
and 95% EtOH. The 30% EtOH eluate (121 g) was subjected to silica
gel column and eluted with gradient mixtures of CHCl3–MeOH
(90:10 ! 0:100, v/v) to afford ten fractions (Fr.1–Fr.10). Fr.4 (5.6 g)
was separated by preparative HPLC on a reversed-phase C18
column using MeOH–H2O (35:65) to give compounds 2 (2 g), 3
(90 mg), and 6 (7 mg), respectively. Fr.6 (7.5 g) was separated by
ODS column with MeOH–H2O (10:90 ! 100:0) as eluent to give
nine subfractions (Fr.6a–Fr.6i). Fr.6e (1.5 g) was further separated
by ODS column using MeOH–H2O (15:85 ! 45:55) as eluent to
obtain compound 4 (16 mg) and a mixture. The mixture (1.1 g) was
reseparated by Sephadex LH-20 column (MeOH) and preparative
HPLC to yield compounds 1 (10 mg) and 5 (14 mg), respectively.
This work was supported financially by the Program for
Changjiang Scholars and Innovative Research Team in the
University (No. IRT0965), the Joint Fund of NSFC-Guangdong
Province (U0932004), the Team Project of Natural Science
Foundation of Guangdong Province (No. 8351063201000003),
and the Science and Technology Development Fund of Macau
Special Administrative Region (No. 013/2008/A1).
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.2012.08.007
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Brown oil. ½a 1D9
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