Polyphenols from the bark of Rhus verniciflua and their biological evaluation on antitumor and anti-inflammatory activities

Article abstract of DOI:10.1016/j.phytochem.2013.05.005

Bioassay-guided fractionation and chemical investigation of the extract of Rhus verniciflua bark resulted in the identification of six polyphenols, rhusopolyphenols A-F (1-6), together with four known compounds including (2R,3S,10S)-7,8,9,13-tetrahydroxy-2-(3,4-dihydroxyphenyl)-2,3-trans-3,4-cis-2,3, 10-trihydrobenzopyrano[3,4-c]-2-benzopyran-1-one (7), peapolyphenol C (8), cilicione-b (9) and (αR)- α,3,4,2',4'-pentahydroxydihydrochalcone (10). The structures of these polyphenols were elucidated by spectroscopic analysis, including 1D and 2D NMR, and HR-ESIMS, and their absolute configurations were further confirmed by a combination of chemical methods and CD data analysis. All isolates were evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and compounds 4-6, 9 and 10 showed antiproliferative activity against the tested cells, with IC₅₀ values of 3.31-18.51 μM. On the basis of the expanded understanding that inflammation is a crucial cause of tumor progression, the anti-inflammatory activities of these compounds were determined by measuring nitric oxide (NO) levels in the medium of murine microglia BV-2 cells. Compounds 5 and 10 significantly inhibited NO production in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cells with IC ₅₀ values of 28.90 and 12.70 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2013.05.005

Phytochemistry 92 (2013) 113–121
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Polyphenols from the bark of Rhus verniciflua and their biological
evaluation on antitumor and anti-inflammatory activities
Ki Hyun Kim a, Eunjung Moon b, Sang Un Choi c, Sun Yeou Kim d, Kang Ro Lee e,
⇑
a Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
b Graduate School of East-West Medical Science, Kyung Hee University Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
c Korea Research Institute of Chemical Technology, Teajeon 305-600, Republic of Korea
d College of Pharmacy, Gachon University, #191 Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea
e Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay-guided fractionation and chemical investigation of the extract of Rhus verniciflua bark resulted
in the identification of six polyphenols, rhusopolyphenols A–F (1–6), together with four known
compounds including (2R,3S,10S)-7,8,9,13-tetrahydroxy-2-(3,4-dihydroxyphenyl)-2,3-trans-3,4-cis-2,3,
Received 13 November 2012
Received in revised form 27 February 2013
Accepted 8 May 2013
10-trihydrobenzopyrano[3,4-c]-2-benzopyran-1-one (7), peapolyphenol C (8), cilicione-b (9) and (
aR)-
Available online 8 June 2013
a
,3,4,20,40-pentahydroxydihydrochalcone (10). The structures of these polyphenols were elucidated by
spectroscopic analysis, including 1D and 2D NMR, and HR-ESIMS, and their absolute configurations were
further confirmed by a combination of chemical methods and CD data analysis. All isolates were evalu-
ated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2,
and HCT-15), and compounds 4–6, 9 and 10 showed antiproliferative activity against the tested cells,
Keywords:
Rhus verniciflua
Anacardiaceae
Polyphenols
Rhusopolyphenols
Antitumor
with IC50 values of 3.31–18.51 lM. On the basis of the expanded understanding that inflammation is a
crucial cause of tumor progression, the anti-inflammatory activities of these compounds were deter-
mined by measuring nitric oxide (NO) levels in the medium of murine microglia BV-2 cells. Compounds
5 and 10 significantly inhibited NO production in lipopolysaccharide (LPS)-stimulated murine microglia
Anti-inflammation
BV-2 cells with IC50 values of 28.90 and 12.70 lM, respectively.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
2007). In the course of this study to search for bioactive metabo-
lites from Korean medicinal sources, it was found that the EtOH ex-
tract of Rhus verniciflua bark exhibited considerable cytotoxicity
against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines during
the screening procedures employed.
Polyphenols constitute one of the most ubiquitous groups of
plant metabolites that are utilized as important components in
both human and animal diets (Bravo, 1998). For decades, plant
polyphenols have interested scientists because they are essential
to plant growth and reproduction and some can also provide plants
with resistance to pathogens and predators by acting as phytoalex-
ins (Bravo, 1998). Polyphenols exhibit a wide range of biological
effects as a consequence of their antioxidant properties such as
antithrombotic, anti-inflammatory, and antimicrobial effects
(Gerritsen et al., 1995; Muldoon and Kritchevsky, 1996; Chung
et al., 1998). Several studies have shown that polyphenols have
anticarcinogenic and antimutagenic effects by inactivating carcin-
ogens and inhibiting expression of mutant genes as well as by
inhibiting the activity of enzymes involved in activation of procar-
cinogens (Hour et al., 1999; Dai and Mumper, 2010; Coates et al.,
Medicinal plants have been used to treat various diseases since
ancient times. R. verniciflua (Anacardiaceae) has been used in tradi-
tional Korean medicine as a herbal therapy for treating abdominal
disorders, inflammatory diseases, and various cancers in South
Korea (Lee et al., 2010). The medical use of this plant has been lim-
ited because of its toxic allergen, urushiol, which causes severe
dermatitis. After boiling the plant with other foods such as chicken
and duck to alleviate toxicity, the cooked herb is eaten to acquire
its useful effects (Lee et al., 2010). In the studies of biological activ-
ities of this herb by a number of investigators, the extract of this
plant exhibited anticancer and antiproliferative effects in various
cancer cell lines (Lee et al., 2003, 2010; Kook et al., 2007; Son
et al., 2005; Samoszuk et al., 2005), which has also been confirmed
by our screening tests showing that the R. verniciflua EtOH extract
had excellent cytotoxic activity against the above-mentioned tu-
mor cells using a sulforhodamine B (SRB) bioassay. But, few reports
have emphasized the active constituents responsible for the
⇑
Corresponding author. Address: Natural Products Laboratory, School of Phar-
macy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon 440-746,
Republic of Korea. Tel.: +82 31 290 7710; fax: +82 31 290 7730.
E-mail address: krlee@skku.ac.kr (K.R. Lee).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.05.005

114
K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
antitumor effect in this plant. Therefore, in the present study, focus
was placed upon R. verniciflua constituents with antitumor activity,
which will be potentially helpful to develop novel drugs and func-
tional foods. Bioassay-guided fractionation and chemical investiga-
tion of the EtOH extract of the R. verniciflua bark resulted in
isolating and identifying six new polyphenols (1–6), together with
four known analogs (7–10). Herein, we describe the isolation and
structural elucidation of these isolates (1–10) and the biological
evaluation of antitumor and anti-inflammatory activities.
flux, and the extract was further extracted with EtOH to afford a
crude EtOH extract. The EtOH extract was successively fractionated
by octadecylsilyl (ODS) chromatography, and the resulting active
fractions were purified by ODS HPLC to yield six new polyphenols
(1–6, Fig. 1), and four known analogs (7–10).
Compound 1 was obtained as a colorless gum. The molecular
formula was established as C15H16O7, based on HR-ESIMS (m/z
331.0797 [M+Na]+, calcd. for C15H16NaO7, 331.0794), suggesting
eight degrees of unsaturation. The IR absorption spectrum sug-
gested the presence of phenyl (2947 and 1451 cmꢀ1) and hydroxyl
(3378 cmꢀ1) groups. The 1H and 13C NMR spectra (Table 1) were
very similar to those of peapolyphenol C (8) (Evidente et al.,
2010), indicating that both 1 and 8 shared the same carbon frame-
work. A minor difference between the two compounds was in the
substitution patterns of the two aromatic rings in 1, which were
identified as 2,4-dihydroxylated and 3,4-dihydroxylated aromatic
rings by analysis of the 1H–1H COSY, HMQC, and HMBC spectra
(Fig. 2). The 1H and 13C NMR spectra also showed signals typical
of a 1,2,3-propanetriol unit. In fact, the double doublet (J = 9.5
and 3.5 Hz), resonating at dH 3.89 and assigned to H-8, showed cor-
relations with two doublets (J = 9.5 Hz at dH 4.87 and J = 3.5 Hz at
dH 4.57) in the COSY system, which were attributed to H-7 and
H-9 of the 1,2,3-propanetriol chain (Fig. 2). The correlations ob-
served in the HMQC spectrum supported assignment of the signals
at dC 76.8 (C-7), 70.9 (C-8), and 66.4 (C-9) of the propanetriol chain.
The chemical shift of C-9 at dC 66.4 showed an upfield shift, com-
pared to those of other methine carbons at C-7 and C-8, which is
2. Results and discussion
Dried R. verniciflua bark in H2O was fermented by Aspergillus
oryzae for 35 h at 30 °C to augment diversified active constituents
and remove the toxic constituent urushiol, which is an oily organic
allergen found in plants of the family Anacardiaceae (Kim et al.,
2011). The fermented material was extracted with H2O under re-
3'
5
2
HO
OH
OH
R2
2'
1'
HO
OH
OH
OH
OH
6
OH
4
4'
5'
9
7
1
3
8
6'
OR1 OH
OH OH
1
2
8
R1 = H
R2 = OH
R2 = H
R2 = H
3
R1 = CH3
R1 = H
caused by the c
-effect due to the C-20 and C-7 substituents of the
hydroxyl group. The gross structure of 1 was established by HMBC
correlations of H-7/C-2, C-6, C-9; H-8/C-1, C-10; and H-9/C-20, C-60
(Fig. 2).
HO
OH
OH
OH
R2
HO
OH
OH
OH
R1
O
OH
O
OH
The absolute configuration of 1 was determined by chemical
methods and by CD data analysis. The configuration of bioactive
natural products provides essential information for total synthesis
and the molecular mode of action. Thus, characterization of the
configuration by X-ray analysis, synthetic methods, or spectro-
scopic techniques has been important. Unfortunately, this poly-
phenol 1, obtained as a gum in very small amounts, is resistant
to crystallization, and it is laborious to synthesize totally and apply
Mosher’s method for the determination of its absolute configura-
tion because of the number of chiral centers in 1. Thus, compound
1 was converted to flavanol (1a) using a Mitsunobu cyclization
(Scheme 1) (Krohn et al., 2009; Jew et al., 2000). Compound 1a
was identified as (ꢀ)-30,40,7-trihydroxyflavan-2,3-cis-3,4-trans-diol
4
5
9
R1 = H R2 = OH
R1 = H R2 = H
6
R
1 = OH R2 = OH
5'
2'
OH
4'
3'
6'
1'
1
O
8
2
HO
7
OH
3
4
HO
OH
OH
OH
OH
6
O
5
HO
7''
6''
O
5''
4''
1''
2''
O
HO
3''
10
OH
7
Fig. 1. Chemical structures of the isolated compounds (1–10).
Table 1
1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of rhusopolyphenols A–C (1–3) in CD3OD (d in ppm, J values in parentheses).a
No
Rhusopolyphenol A (1)
dH (J in Hz)
Rhusopolyphenol B (2)
dH (J in Hz)
Rhusopolyphenol C (3)
dH (J in Hz)
dC
dC
dC
1
2
3
4
5
6
7
8
130.6
114.6
145.0
145.2
114.7
119.4
76.8
129.9
128.7
114.6
157.1
114.6
128.7
76.8
129.7
114.5
144.8
145.2
114.5
119.5
81.3
6.88 d (2.0)
7.28 dd (8.5, 2.0)
6.82 dd (8.5, 2.0)
6.93 d (1.5)
6.77 br s
6.77 br s
4.87 d (9.5)
3.89 dd (9.5, 3.5)
4.57 d (3.5)
6.82 dd (8.5, 2.0)
7.28 dd (8.5, 2.0)
5.01 d (10.0)
3.98 dd (10.0, 3.0)
4.20 d (3.0)
6.79 br s
6.79 br s
4.60 d (10.0)
3.73 dd (10.0, 8.0)
4.65 d (8.0)
70.9
66.4
70.2
76.2
73.4
71.5
9
10
20
30
40
50
60
OMe
114.8
155.4
102.1
158.7
108.5
131.3
111.7
155.2
102.2
159.0
107.4
131.3
55.6
116.1
155.0
101.7
157.6
108.5
128.2
6.26 d (2.0)
6.29 d (2.0)
6.22 d (2.5)
6.38 dd (8.5, 2.0)
7.12 d (8.5)
6.40 dd (8.5, 2.0)
7.10 d (8.5)
3.47 s
6.44 dd (8.5, 2.5)
7.28 d (8.5)
a
The assignments were based on 1H-1H-COSY, HMQC and HMBC experiments.

K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
115
ꢀ17,400) with those of 1 ([h]236 ꢀ15,200, [h]274 ꢀ31,300), which
is reported for the first time here.
Compound 2 was isolated as a colorless gum. The molecular
formula was C16H18O6 based on HR-ESIMS (m/z 329.1003
HO
HO
OH
OH
OH
OH
HO
HO
OH
OH
OH
OH OH
OCH3OH
1
2
[M+Na]+, calcd. for C16H18NaO6, 329.1001), suggesting eight de-
grees of unsaturation. Similar to compound 1, compound 2
showed IR absorptions typical of phenyl and hydroxyl groups.
The 1H and 13C NMR spectra (Table 1) were similar to those of
1, indicating that both 1 and 2 shared the same carbon frame-
work with a 1,2,3-propanetriol unit. The major differences were
the presence of a methoxyl group (dH 3.47) and different aromatic
substitution patterns in 2, which were identified as 2,4-dihydrox-
ylated and 4-hydroxylated aromatic rings by analysis of the
1H–1H COSY, HMQC, and HMBC spectra (Fig. 2). The location of
the methoxyl group was deduced by a downfield shift of C-9 in
the 13C NMR spectrum of 2 compared to that of 1 and confirmed
by the HMBC correlation from OMe (dH 3.47) to C-9 (dC 76.2)
(Fig. 2). Full assignments of the 1H and 13C NMR spectra (Table 1)
were carried out by analysis of the COSY, HMQC, and HMBC data.
The absolute configuration of 2 was the same as that of 1 by com-
paring its coupling constants, specific rotation, and CD data ([h]237
ꢀ25,600, [h]283 ꢀ27,700) with those of 1 ([h]236 ꢀ15,200, [h]274
OH
OH
OH
OH
OH
OH
OH
O
OH
O
OH
4
6
Fig. 2. Key 1H–1H COSY (—) and HMBC (?) correlations of 1, 2, 4, and 6.
by its 1H NMR, specific rotation, and MS data. Standard SN2-type
Mitsunobu reaction conditions were employed to obtain the enan-
tioselective flavonol which was confirmed to be the expected 2,3-
cis-3,4-trans-configuration in the 1H NMR spectrum of 1a (Krohn
et al., 2009). Remarkably, the theoretically possible formation of
a five-membered ring was not observed during the conditions used
(Krohn et al., 2009). The CD spectrum of 1a ([h]240 ꢀ11,500, [h]285
ꢀ17,800) established the absolute configuration to be 2R,3R,4S, as
shown in Scheme 1 (Ferreira et al., 2004). Thus, the absolute con-
figuration of 1 was assigned 7S,8S,9S based on the absolute config-
uration of 1a because a change in the configuration at C-2 and C-3
in 1a occurs during the SN2-type Mitsunobu reaction (Krohn et al.,
2009; Jew et al., 2000). On the basis of these considerations, com-
pound 1 was established as shown in Fig. 1, for which the trivial
name rhusopolyphenol A is proposed. In addition, the absolute
configuration of peapolyphenol C (8) was determined to be identi-
cal to 1 by comparing the 1H and 13C NMR spectroscopic data, the
coupling constants, and its CD spectrum ([h]239 ꢀ19,500, [h]281
ꢀ31,300). Thus, compound
2 was determined as shown in
Fig. 1, named rhusopolyphenol B.
Compound 3 was obtained as a colorless gum. Its HR-ESIMS
data showed an ion peak at m/z 331.0790 [M+Na]+ (calcd. for C15-
H16NaO7, 331.0794) for the molecular formula C15H16O7 with eight
degrees of unsaturation. The 1H and 13C NMR spectra (Table 1)
were remarkably similar to those of 1, suggesting that both 1
and 3 belonged to the same carbon framework with a 1,2,3-pro-
panetriol unit. The only differences between them were the chem-
ical shifts and splitting patterns of C-7 [dH 4.60 (d, J = 10.0 Hz); dC
OH
OH
OH
OH
HO
OH
OH
OH
OH
HO
OH
HO
O
(S)
(R)
(R)
(S)
OH
OH
(S)
(S)
(S)
(S)
(S)
OH
OH OH
OH
OH
1
1a
1
OH
OH
OH
OH
HO
OH
OH
OH
OH
HO
OH
HO
O
(S)
(R)
OH
OH
(R)
(S)
(S)
(R)
(R)
(S)
(R)
OH
OH OH
OH
OH
3
3a
3
OH
OH
OH
OH
HO
OH
OH
OH
OH
HO
OH
(S)
HO
O
(S)
(R)
(S)
OH
OH
(S)
(S)
OH
4a
O
OH
O
O
4
4
OH
OH
OH
OH
HO
OH
O
OH
OH
HO
OH
HO
O
(R)
(S)
OH
(R)
OH
O
O
6
6a
6
Scheme 1. Reagents and conditions: DEAD, Ph3P, THF, 3 h.

116
K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
Table 2
1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of rhusopolyphenols D–F (4–6) in CD3OD (d in ppm, J values in parentheses).a
No
Rhusopolyphenol D (4)
dH (J in Hz)
Rhusopolyphenol E (5)
dH (J in Hz)
Rhusopolyphenol F (6)
dH (J in Hz)
dC
dC
dC
1
2
3
4
5
6
7
8
128.7
114.5
144.9
145.6
114.6
119.5
84.2
128.1
128.9
114.7
157.7
114.7
128.9
84.0
128.9
114.6
145.2
146.0
115.2
119.1
84.6
7.00 d (2.0)
7.35 dd (8.5, 2.0)
6.82 dd (8.5, 2.0)
6.56 d (2.0)
6.82 d (8.0)
6.82 dd (8.5, 2.0)
7.35 dd (8.5, 2.0)
4.99 d (12.0)
6.74 d (8.0)
6.41 dd (8.0, 2.0)
5.79 dd (13.0, 1.0)
2.66 dd (14.0, 1.0)3.33 overlapped
6.88 dd (8.0, 2.0)
4.95 d (11.5)
4.50 d (11.5)
73.1
4.51 d (12.0)
73.1
51.3
9
193.0
112.0
163.6
102.3
165.4
110.7
128.7
193.1
112.0
163.7
102.3
165.4
110.7
128.6
192.5
113.9
163.8
102.4
165.4
110.5
128.8
10
20
30
40
50
60
6.35 d (2.0)
6.32 d (2.0)
6.26 d (2.0)
6.54 dd (8.5, 2.0)
7.73 d (8.5)
6.52 dd (8.5, 2.0)
7.72 d (8.5)
6.48 dd (8.5, 2.0)
7.66 d (8.5)
a
The assignments were based on 1H-1H-COSY, HMQC and HMBC experiments.
81.3], C-8 [dH 3.73 (dd, J = 10.0, 8.0 Hz); dC 73.4], and C-9 [dH 4.65
(d, J = 8.0 Hz); dC 71.5] in 3 compared to those of corresponding
carbons in 1, suggesting that they had different configurations at
the 1,2,3-propanetriol unit. Similarly, as described for 1, the abso-
lute configuration of 3 was determined using Mitsunobu cycliza-
tion (Scheme 1) (Krohn et al., 2009; Jew et al., 2000), which
afforded (ꢀ)-30,40,7-trihydroxyflavan-2,3-cis-3,4-cis-diol (3a). This
was identified by its 1H NMR, specific rotation, and MS data, and
its absolute configuration was assigned as 2R,3R,4R by means of
the CD data of 3a ([h]242 ꢀ48,500, [h]286 ꢀ34,800) (Ferreira et al.,
2004). Thus, the absolute configuration of 3 was elucidated to be
7S,8S,9R, as described for 1. On the basis of the evidence, the struc-
ture of compound 3 was assigned as shown in Fig. 1, named rhuso-
polyphenol C.
Compound 4 was isolated as a colorless gum. The molecular for-
mula was C15H14O7 with nine degrees of unsaturation by analysis
of the molecular ion peak at m/z 329.0645 [M+Na]+ (calcd. for C15-
H14NaO7, 329.0637) in the HR-ESIMS in combination with 1H and
13C NMR spectra. The IR absorption implied the presence of car-
bonyl (1713 cmꢀ1), phenyl (2947 and 1451 cmꢀ1), and hydroxyl
(3382 cmꢀ1) groups. The 1H and 13C NMR spectra (Table 2) were
very similar to those of cilicione-b (9) (Ahmed and Al-Howiriny,
2007), suggesting that compound 4 belonged to the same chalcone
derivative as 9. The only difference was the substitution pattern of
the aromatic ring in 4, which was confirmed by analyzing the
1H–1H COSY, HMQC, and HMBC spectra (Fig. 2). The gross structure
of 4 was elucidated by analyzing the 2D NMR spectra (COSY, HMQC,
and HMBC). The trend for larger couplings in anti-orientated pro-
tons (J = 8–9 Hz) compared to their syn-isomers (J = 2–3 Hz) has
been well established in closely related acyclic polyol derivatives
(Wiesler and Nakanishi, 1990), which allowed us to demonstrate
that the relative configuration of 4 was the 7,8-erythro form. Next,
as described for 1, compound 4 was cyclizated by Mitsunobu reac-
tion to afford compound 4a for determining its absolute configura-
tion (Scheme 1) (Krohn et al., 2009; Jew et al., 2000). Compound 4a
was identified as (ꢀ)-fustin by its 1H NMR, specific rotation, and
MS data, and the absolute configuration was established as 2R,3S
by the CD data ([h]307 ꢀ14,600, [h]340 +8500) (Van Rensburg
et al., 1997). Similar to 1, the absolute configuration of 4 was
7S,8S based on the absolute configuration of 4a. Thus, the structure
of compound 4 was elucidated as shown in Fig. 1, named rhuso-
polyphenol D. Additionally, the absolute configuration of cilici-
one-b (9) was identical to 4 by comparing its 1H and 13C NMR
spectroscopic data, the coupling constants, and the CD data
([h]245 +12,600, [h]276 +17,100, [h]290 +13,700) with those of 4
([h]246 +16,500, [h]276 +14,300, [h]290 +12,800), which is suggested
for the first time in this study.
The HR-ESIMS data (m/z 313.0690 [M+Na]+, calcd. for C15H14-
NaO6, 313.0688) of 5 indicated that this molecule possessed the
molecular formula C15H14O6 with nine degrees of unsaturation.
The 1H and 13C NMR spectroscopic data (Table 2) showed high sim-
ilarity to those of 4, except for the different substitution pattern of
the aromatic ring in 5, which was determined by analyzing the
1H–1H COSY, HMQC, HMBC spectra. The remaining structural fea-
tures of 5 were confirmed based on comprehensive 2D NMR stud-
ies (COSY, HMQC, and HMBC spectra). The absolute configuration
of 5 was the same as that of 4 by comparing its coupling constants,
specific rotation, and CD data ([h]246 +10,600, [h]275 +15,900, [h]291
+18,200) with those of 4 ([h]246 +16,500, [h]276 +14,300, [h]290
+12,800). Consequently, compound 5 was established as shown
in Fig. 1, named rhusopolyphenol E.
Compound 6 was isolated as a colorless gum. The HR-ESIMS of
6 displayed a molecular ion peak [M+Na]+ at m/z 313.0693 (calcd.
for C15H14NaO6, 313.0688), consistent with the molecular formula
of C15H14O6 with nine of unsaturation. The IR spectrum exhibited
absorptions of carbonyl (1705 cmꢀ1), phenyl (2945 and
1499 cmꢀ1), and hydroxyl (3398 cmꢀ1) groups. Inspection of the
1H and 13C NMR spectroscopic data (Table 2) established high
similarity to those of (aR)-a
,3,4,20,40-pentahydroxydihydrochal-
cone (10) (Alvarez and Delgado, 1999), except for the chemical
shifts of C-7 [dH 5.79 (dd, J = 13.0, 1.0 Hz); dC 84.6] and C-8 [dH
2.66 (dd, J = 14.0, 1.0 Hz), 3.33 (overlapped); dC 51.3] in 6 com-
pared to those [dH 2.70 (1H, dd, J = 16.5, 3.0 Hz, H-7a), 2.99 (1H,
dd, J = 16.5, 13.0 Hz, H-7b); dC 43.8 (C-7) and dH 5.30 (1H, dd,
J = 13.0, 3.0 Hz, H-8); dC 79.8 (C-8)] of corresponding parts in
10. The differences were confirmed by analyzing the 1H–1H COSY
and HMQC spectra, and the HMBC correlations of H-7/C-2, C-6, C-
9 and H-8/C-1, C-10 allowed us to assign an aliphatic carbinol at
C-7, a methylene at C-8, and a ketone at C-9 (Fig. 2). The gross
structure of 6 was unambiguously established by analyzing the
2D NMR data (COSY, HMQC, and HMBC). Compound 6 was
cyclizated using a Mitsunobu reaction to identify its absolute
configuration (Scheme 1) (Krohn et al., 2009; Jew et al., 2000).
(ꢀ)-Butin (6a) was synthesized in the reaction, which was identi-
fied by its 1H NMR, specific rotation, MS, and CD data ([h]292
ꢀ15,200) (Chokchaisiri et al., 2009). Thus, the absolute configura-
tion of 6 was 7R based on the absolute configuration of the
synthetic compound, (ꢀ)-butin. On the basis of the evidence,
the structure of compound 6 was determined as shown in
Fig. 1, named rhusopolyphenol F.

K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
117
Table 3
the SRB bioassay (Skehan et al., 1990). Compounds 4–6, 9, and 10
showed antiproliferative activity against the tested cells with IC50
Cytotoxicity of compounds 4–6, 9, and 10 against four cultured human cancer cell
lines in the SRB bioassay.
values of 3.31–18.51
(IC50 > 30.0 M) (Table 3). Compound 10 exhibited the most potent
antiproliferative activity against A549, SK-OV-3, SK-MEL-2, and
HCT-15 cells, with IC50 values of 3.31, 4.28, 7.70, and 8.81 M,
lM, but the other compounds were inactive
Compound
IC50
A549
17.05 1.21b 17.41 1.71
(l
M)a
l
SK-OV-3
SK-MEL-2
HCT-15
l
4
5
6
13.18 1.19
4.38 0.49
9.74 0.13
10.13 1.25
7.70 1.18
8.54 0.83
12.91 1.43
11.65 2.66
17.82 3.23
8.81 0.79
respectively. Although more structurally related compounds need
to be tested to confirm this hypothesis, it appears that the presence
of a ketone group at C-9 in these molecules is essential for mani-
festing the activity in such polyphenols as compounds 4–6, 9,
and 10 showed the inhibitory activity against cell proliferation in
the tested lines, whereas the other compounds 1–3, 7, and 8 were
inactive. The lipophilicity may modulate the in vitro cytotoxicity of
compounds 1–10. Compounds 4–6, 9, and 10, which were more
lipophilic than inactive compounds 1–3, 7, and 8 (estimated on
the basis of HPLC retention time and TLC Rf value), showed higher
cytotoxicities (Table 3). This result is in agreement with other re-
ports of increases in cytotoxicity with the absence or with the
decreasing number of hydroxyl groups (Bao et al., 2005; Sun
et al., 2003). Higher lipophilicity may lead to facilitated penetration
through the lipophilic plasma membrane of the tumor cells, and
subsequently, to higher cytotoxicity (Beekman et al., 1997). The
change in the hydroxyl group position present in the active com-
pounds, leads to important changes in cytotoxicity. Compound 4,
without a hydroxyl group at C-60, was twice as potent against the
HCT-15 cell line with respect to that of 9, and compound 5, without
a hydroxyl group at C-3, was three times more potent compared to
4 against the SK-MEL-2 cell line. In particular, the change in hydro-
xyl group location at positions C-7 and C-8 affected the significant
changes in the cytotoxicity of 6 and 10, and the presence of a hy-
droxyl group at C-8 in 10 led to an increase in activity against all
the tested cell lines in comparison to that of 6. The change in the
hydroxyl group position in molecules results in alteration of the
reactive position which may be associated with the pro-oxidant ef-
fect influencing inhibition of cell viability, induction of apoptosis
and necrosis, and cell cycle arrest in cancer cells through electron
transfer reactions leading to moderate formation of ROS (Sanchez-
Tena et al., 2012). The alteration of pro-oxidant effect may cause
different potency in cytotoxicity of the active compounds.
16.42 0.31
18.51 1.80
18.57 1.40
3.31 0.68
12.10 0.90
10.32 0.97
12.38 1.54
4.28 0.13
9
10
Doxorubicinc 0.016 0.005 0.027 0.003 0.036 0.001 1.073 0.067
a
50% inhibitory concentration; the concentration of compound that caused a 50%
inhibition in cell growth.
b
Data are expressed as mean SEM of three independent experiments.
Doxorubicin as a positive control.
c
Compound 7, (2R,3S,10S)-7,8,9,13-tetrahydroxy-2-(3,4-dihy-
droxyphenyl)-2,3-trans-3,4-cis-2,3,10-trihydrobenzopyrano[3,4-c]-
2-benzopyran-1-one was previously isolated from the heartwood
of Peltophorum africanum (Bam et al., 1990). But, its 13C NMR
spectroscopic data has not been reported, though that of its hexa-
methyl ether was published (Bam et al., 1990). Full NMR data
assignments of 7 were thus performed by analyzing the 2D NMR
data (including DEPT, HMQC, HMBC, and NOESY). The absolute
configuration was identified by coupling constants (J2,3 = 2.5 Hz,
J3,4 = 3.0 Hz) and its CD spectrum showing the positive Cotton
effect in the 225–240 nm consistent with those of the reported cor-
responding compounds (Bam et al., 1990). To the best of our
knowledge, the 13C NMR spectroscopic data is reported in this
study for the first time.
The other known compounds were identified as peapolyphenol
C (8) (Evidente et al., 2010), cilicione-b (9) (Ahmed and Al-howiriny,
2007), and
(Alvarez and Delgado, 1999) by comparing their spectroscopic data
with values reported previously. These known compounds 7–10
were isolated from this plant for the first time.
The fermentation or hot water extraction might change some
constitutions and structures of naturally occurring compounds. In
order to identify whether the isolated compounds 1–10 were gen-
uine natural compounds or artifacts, R. verniciflua barks were ex-
tracted with 50% EtOH and 80% MeOH in H2O at room
temperature to obtain crude EtOH and MeOH extracts, respec-
tively. HPLC analyses of the extracts indicated that the chemical
profiles are quite similar to each other. The peaks of all compounds
except for 9 were observed in the HPLC chromatogram of the EtOH
extract, which was also confirmed by LC-MS analyses. The peak
corresponding to 9 could be identified in the HPLC chromatogram
of the MeOH extract. This evidence proves that the compounds 1–
10 are genuine natural products.
(aR)-a
,3,4,20,40-pentahydroxydihydrochalcone (10)
Inflammatory gene expression is often negatively correlated
with cancer stage and prognosis (Chang et al., 2004; Galon et al.,
2006; Wang et al., 2006). Moreover, non-steroidal anti-inflamma-
tory drugs show preventive effects against cancer (Ulrich et al.,
2006). Therefore, cancer and inflammation are related by epidemi-
ology, histopathology, and inflammatory profiles (Rakoff-Nahoum,
2006). As inflammation plays a crucial role in tumor progression
(Coussens and Werb, 2002), the anti-inflammatory activities of
polyphenols 1–10 isolated from R. verniciflua in lipopolysaccharide
(LPS)-activated BV-2 cells were evaluated by measuring the nitric
oxide (NO) levels produced. BV-2 has both the phenotypic and
functional properties of reactive microglia cells (Blasi et al., 1990)
and is activated following stimulation by various agents such as
To evaluate the isolated compounds 1–10 as cytotoxic agents,
evaluation of their antiproliferative activities was performed
against the A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines using
Table 4
Inhibitory effect of compounds 1–10 on nitric oxide (NO) production in LPS-activated BV-2 cells.
Compound
IC50
(
l
M)a
Cell viability (%)b
Compound
IC50
(l
M)a
Cell viability (%)b
1
2
3
4
5
6
91.02
31.29
52.40
42.51
28.90
84.05
94.1 3.5⁄
100.0 5.1
92.8 2.8⁄
97.4 4.2
7
8
9
>500
104.3 3.1
96.8 6.9
96.7 2.6
96.8 4.2
101.3 4.5
75.24
74.51
12.70
18.29
10
102.5 5.3
NMMAc
90.5 4.9⁄
Results are averages of three independent experiments, and data are expressed as mean SD (⁄p-value < 0.05).
a
IC50 value of each compound was defined as the concentration (
l
M) that caused 50% inhibition of NO production in LPS-activated BV-2 cells.
b
c
Cell viability after treatment with 20
NMMA was the positive control.
lM of each compound is expressed as a percentage (%) of the LPS only treatment group.

118
K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
LPS (Dheen et al., 2007). Activated BV-2 cells exhibit various
inflammatory responses including production of the proinflamma-
tory factor NO (Hanisch, 2002). It has been known that polyphenols
exhibit anti-inflammatory properties (Muldoon and Kritchevsky,
1996). In this study, compounds 5 and 10 significantly inhibited
chromatography (TLC). Spots were detected on TLC under UV light
or by heating after spraying with anisaldehyde-sulfuric acid.
4.2. Plant material
NO levels (IC50 values < 30
cell toxicity (Table 4). In particular, compound 10 exhibited the
highest inhibitory activity with an IC50 of 12.70 M, which was
more potent than that of NG-monomethyl-
-arginine ( -NMMA),
an inducible NO synthase inhibitor (Reif and McCreedy, 1995).
Interestingly, the presence of a ketone group at C-9 in these poly-
phenols played an important role in the anti-inflammatory proper-
ties as well as cytotoxic activity.
l
M) in LPS-stimulated BV-2 without
R. verniciflua bark was collected in Hoengseong, Gangwon-do,
Korea, in March 2010. The plant material was identified by one
of the authors (K.R. Lee). A voucher specimen (SKKU-RV 2010-3)
was deposited at the Farm Corporation, Dongyi’s Farms, Boryung,
Chungcheongnam-do, Korea.
l
L
L
4.3. Extraction and isolation
Air-dried and pulverized plant material (1.3 kg) was soaked in
distilled H2O (10 L), thus begin heated until reflux began and held
for 1 h with the material in boiling water then being cooled. The
material in the cooled water was then fermented at 30 °C for 35 h
to augment the active constituents and remove toxic components
by A. oryzae. The fermented material was extracted with H2O twice
(each 20 L ꢁ 3 h) under conditions of reflux and the whole filtered.
The filtrate was evaporated in vacuo to obtain a crude extract
(120 g), which was further extracted with EtOH to afford a crude
EtOH extract (60 g). The EtOH extract (10 g) was subjected to C18 re-
verse-phased silica gel CC, eluted with a gradient of MeOH–H2O
(2:3 ? 1:0) to yield six fractions (fractions A–F). Each fraction
was evaluated for cytotoxic activity against A549, SK-OV-3, SK-
MEL-2, and HCT-15 human tumor cell lines using the SRB bioassay,
and a bioactivity-guided fractionation method was used for the iso-
lation work. Fractions B and C showed weak cytotoxicity, whereas
fractions D and E exhibited significant cytotoxicity against the
tested cell lines. The active fraction B (3.5 g) was applied to
Sephadex LH-20 and eluted with MeOH–H2O (4:1) to give five sub-
fractions (B1–B5). Fraction B5 (450 mg) was further purified by
semi-preparative HPLC using a solvent system of MeOH–H2O
(2:3) at a flow rate of 2.0 mL/min (Econosil RP-18 column;
3. Concluding remarks
This study dealt with the isolation and biological activity of 10
polyphenols including the structural elucidation of six new poly-
phenols, rhusopolyphenols A–F (1–6) from R. verniciflua bark
through bioactivity-guided isolation techniques. It was confirmed
that the polyphenols represented a portion of the molecules
with cytotoxic and anti-inflammatory activities. Although all
compounds were not active, compounds 4–6, 9, and 10 can be
considered highly cytotoxic agents (IC50 values < 20 lM) in A549,
SK-OV-3, SK-MEL-2, and HCT-15 cell lines, whereas compounds 5
and 10 exhibited an anti-inflammatory effect by inhibiting NO lev-
els (IC50 values < 30 lM) in LPS-stimulated BV-2 cells. Taking into
account that compounds 5 and 10 showed higher antitumor and
anti-inflammatory activities, it is suggested that they might be
useful as bioactive molecules for regulating various cancers and
inflammation-related diseases and their structures can be
considered as new lead molecules for the design of analogs with
therapeutic potential. These results also demonstrate that the use
of R. verniciflua as Korean traditional medicine to treat inflamma-
tory diseases and various cancers is reasonable, because the
polyphenols were isolated with cytotoxic and anti-inflammatory
effects as a main component.
250 ꢁ 10 mm; 10
lm particle size; Shodex refractive index detec-
tor) to afford 7 (4 mg). The active fraction C (550 mg) was subjected
to Sephadex LH-20 chromatography, eluted with MeOH–H2O (4:1)
to yield five subfractions (C1–C5). Fraction C4 (130 mg) was puri-
fied by semi-preparative HPLC, as described above, using a solvent
system of MeOH–H2O (9:11) to provide 9 (15 mg). The active frac-
tion D (560 mg) was applied to a silica gel column eluted with
CHCl3–MeOH (17:1) to give six subfractions (D1–D6). Fraction D3
(185 mg) was purified by semi-preparative HPLC, as described
above, using a solvent system of MeOH–H2O (9:11) to obtain com-
pounds 2 (5 mg), 5 (10 mg), and 8 (7 mg). Fraction D4 (50 mg) was
purified by semi-preparative HPLC, as described above, using a sol-
vent system of MeOH–H2O (1:1) to afford 10 (18 mg). Finally, active
fraction E (1.9 g) was subjected to silica gel CC eluted with CHCl3–
MeOH (15:1) to furnish five subfractions (E1–E5). Fraction E3
(350 mg) was purified by semi-preparative HPLC, using a solvent
system of MeOH–H2O (3:2) to give 4 (4 mg). Fraction E5 (800 mg)
was subjected to C18 reverse-phased silica gel CC and eluted with
MeOH–H2O (2:3 ? 4:1, gradient system) to yield two subfractions
(E51 and E52). Fraction E51 (150 mg) was purified by semi-
preparative HPLC, using a solvent system of MeOH–H2O (3:7) to
obtain compounds 1 (4 mg) and 3 (3 mg). Fraction E52 (110 mg)
was purified by semi-preparative HPLC, using a solvent system of
MeOH–H2O (3:2) to yield 6 (5 mg).
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a Jasco P-1020 polarimeter
(Jasco, Easton, MD, USA). IR spectra were recorded on a Bruker IFS-
66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany). Circular
dichroism (CD) spectra were measured on a Jasco J-715 spectropo-
larimeter (Jasco, Easton, MD, USA). Ultraviolet (UV) spectra were
recorded with a Shimadzu UV-1601 UV–Visible spectrophotometer
(Shimadzu, Tokyo, Japan). Electron spray ionization (ESI) and high-
resolution (HR)-ESI mass spectra were acquired on a SI-2/LCQ
DecaXP Liquid chromatography (LC)-mass spectrometer (Thermo
Scientific, West Palm Beach, PL, USA). Nuclear magnetic resonance
(NMR) spectra were obtained using a Varian UNITY INOVA 500
NMR spectrometer (Varian, Palo Alto, CA, USA) operating at
500 MHz (1H) and 125 MHz (13C), with chemical shifts given in
ppm (d). Preparative high performance liquid chromatography
(HPLC) used a Gilson 306 pump (Gilson, Middleton, WI, USA) with
a Shodex refractive index detector (Shodex, New York, NY, USA).
Column chromatography (CC) was performed with silica gel 60
(Merck, 70–230 mesh and 230–400 mesh) and RP-C18 silica gel
(Merck, 230–400 mesh). The packing material for molecular sieve
CC was Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Merck pre-
coated silica gel F254 plates and reversed-phase (RP)-18 F254s plates
(Merck, Darmstadt, Germany) were used for thin-layer
4.3.1. Rhusopolyphenol A (1)
Ccolorless gum; ½a D25
ꢀ23.8 (c 0.18, MeOH); UV (MeOH) kmax nm
ꢂ
(log
e
): 218 (3.9), 275 (2.6); CD (MeOH): [h]236 ꢀ15,200, [h]274
ꢀ31,300; IR (KBr) mmax cmꢀ1: 3378, 2947, 2834, 2503, 1661,
1451, 1120, 1030; for 1H (500 MHz) and 13C (125 MHz) NMR spec-
troscopic data, see Table 1; ESIMS (positive-ion mode) m/z 331

K.H. Kim et al. / Phytochemistry 92 (2013) 113–121
119
[M+Na]+; HR-ESIMS (positive-ion mode) m/z 331.0797 [M+Na]+
(calcd. for C15H16NaO7, 331.0794).
4.4. General procedure for Mitsunobu reaction
To solution of individual polyphenols 1, 3, 4, and
a
6
4.3.2. Rhusopolyphenol B (2)
(0.005 mmol) in anhydrous THF (0.05 ml) was added Ph3P (2 mg,
0.008 mmol). Each mixture was stirred for 15 min, and DEAD
(Diethyl azodicarboxylate) (1.5 ll, 0.008 mmol) was added drop-
Colorless gum; ½a D25
ꢀ19.1 (c 0.20, MeOH); UV (MeOH) kmax nm
ꢂ
(log
e
): 218 (3.9), 275 (2.5); CD (MeOH): [h]237 ꢀ25,600, [h]283
ꢀ27,700; IR (KBr) mmax cmꢀ1: 3383, 2948, 2836, 2504, 1661,
1452, 1120, 1031; for 1H (500 MHz) and 13C (125 MHz) NMR spec-
troscopic data, see Table 1; ESIMS (positive-ion mode) m/z 329
[M+Na]+; HR-ESIMS (positive-ion mode) m/z 329.1003 [M+Na]+
(calcd. for C16H18NaO6, 329.1001).
wise to the reaction mixture. After stirring for 3 h at room temper-
ature (reaction monitored by TLC), each reaction mixture was
diluted with EtOAc (5 ml) and washed with H2O (5 ml) and brine
(4 ml). Each organic phase was then dried (MgSO4), filtered, and
concentrated under reduced pressure to produce a brown residue.
Each residue was purified with a silica gel Waters Sep-Pak Vac 6 cc
(n-Hexane–EtOAc, 3:1) (Water, Milford, MA, USA) to give the cy-
clized products 1a, 3a, 4a, and 6a as amorphous solids,
respectively.
4.3.3. Rhusopolyphenol C (3)
Colorless gum; ½a D25
ꢂ
+34.2 (c 0.13, MeOH); UV (MeOH) kmax nm
): 218 (3.9), 275 (2.6); CD (MeOH): [h]250 ꢀ11,200, [h]291
(log
e
+2500; IR (KBr) mmax cmꢀ1: 3419, 2947, 2835, 2506, 1644, 1449,
1058, 1033; for 1H (500 MHz) and 13C (125 MHz) NMR spectro-
scopic data, see Table 1; ESIMS (positive-ion mode) m/z 331
[M+Na]+; HR-ESIMS (positive-ion mode) m/z 331.0790 [M+Na]+
(calcd. for C15H16NaO7, 331.0794).
4.4.1. (ꢀ)-30,40,7-Trihydroxyflavan-2,3-cis-3,4-trans-diol (1a)
Amorphous solid; 75% yield; ½a D25
ꢀ7.4 (c 0.05, MeOH); CD
ꢂ
(MeOH): [h]240 ꢀ11,500, [h]285 ꢀ17,800; IR (KBr) mmax cmꢀ1
:
3407, 2912, 1585, 1483, 1310, 1241, 1030; 1H NMR (500 MHz,
CD3OD): d 6.97 (1H, d, J = 2.0 Hz, H-20), 6.85 (1H, d, J = 8.5 Hz,
H-5), 6.79 (1H, d, J = 8.5, 2.0 Hz, H-60), 6.75 (1H, dd, J = 8.5 Hz,
H-50), 6.45 (1H, d, J = 2.0 Hz, H-8), 6.22 (1H, dd, J = 8.5, 2.0 Hz,
H-6), 5.04 (1H, d, J = 2.5 Hz, H-2), 4.56 (1H, d, J = 6.0 Hz, H-4),
4.45 (1H, dd, J = 6.0, 2.5 Hz, H-3); ESIMS (positive-ion mode) m/
z 313 [M+Na]+.
4.3.4. Rhusopolyphenol D (4)
Colorless gum; ½a D25
ꢂ
+12.7 (c 0.20, MeOH); UV (MeOH) kmax nm
(log e): 213 (4.1), 285 (3.2); CD (MeOH): [h]246 +16,500, [h]276
+14,300, [h]290 +12,800; IR (KBr) mmax cmꢀ1: 3382, 2947, 2836,
2502, 1713, 1451, 1120, 1030; for 1H (500 MHz) and 13C
(125 MHz) NMR spectroscopic data, see Table 2; ESIMS (positive-
ion mode) m/z 329 [M+Na]+; HR-ESIMS (positive-ion mode) m/z
329.0645 [M+Na]+ (calcd. for C15H14NaO7, 329.0637).
4.4.2. (ꢀ)-30,40,7-Trihydroxyflavan-2,3-cis-3,4-cis-diol (3a)
Amorphous solid; 80% yield; ½a D25
ꢀ17.3 (c 0.05, MeOH); CD
ꢂ
(MeOH): [h]242 ꢀ48,500, [h]286 ꢀ34,800; IR (KBr) mmax cmꢀ1
:
4.3.5. Rhusopolyphenol E (5)
3427, 2930, 1610, 1501, 1338, 1241, 1030; 1H NMR (500 MHz,
CD3OD): d 7.01 (1H, d, J = 2.0 Hz, H-20), 6.88 (1H, d, J = 8.0 Hz,
H-5), 6.82 (1H, d, J = 8.5, 2.0 Hz, H-60), 6.77 (1H, dd, J = 8.5 Hz,
H-50), 6.45 (1H, d, J = 2.0 Hz, H-8), 6.21 (1H, dd, J = 8.0, 2.0 Hz,
H-6), 4.98 (1H, d, J = 1.0 Hz, H-2), 4.75 (1H, d, J = 4.0 Hz, H-4),
4.24 (1H, dd, J = 4.0, 1.0 Hz, H-3); ESIMS (positive-ion mode) m/
z 313 [M+Na]+.
Colorless gum; ½a D25
ꢂ
+10.8 (c 0.45, MeOH); UV (MeOH) kmax nm
(log e): 214 (4.1), 286 (3.0); CD (MeOH): [h]246 +10,600, [h]275
+15,900, [h]291 +18,200; IR (KBr) mmax cmꢀ1: 3382, 2947, 2833,
2502, 1712, 1451, 1115, 1031; for 1H (500 MHz) and 13C
(125 MHz) NMR spectroscopic data, see Table 2; ESIMS (positive-
ion mode) m/z 313 [M+Na]+; HR-ESIMS (positive-ion mode) m/z
313.0690 [M+Na]+ (calcd. for C15H14NaO6, 313.0688).
4.4.3. (ꢀ)-Fustin (4a)
4.3.6. Rhusopolyphenol F (6)
Amorphous solid; 75% yield; ½a D25
ꢀ11.4 (c 0.05, MeOH); CD
ꢂ
Amorphous solid; ½a D25
ꢂ
+19.4 (c 0.25, MeOH); UV (MeOH) kmax
nm (log e): 214 (4.1), 284 (3.4), 320 (3.3); CD (MeOH): [h]245
(MeOH): [h]307 ꢀ14,600, [h]340 +8500; IR (KBr) mmax cmꢀ1: 3415,
2932, 1665, 1500, 1398, 1245, 1030; 1H NMR (500 MHz, CD3OD):
d 7.68 (1H, d, J = 8.5 Hz, H-5), 6.87 (1H, d, J = 2.0 Hz, H-20), 6.77
(1H, d, J = 8.0, 2.0 Hz, H-60), 6.73 (1H, dd, J = 8.0 Hz, H-50), 6.45
(1H, dd, J = 8.5, 2.0 Hz, H-6), 6.30 (1H, d, J = 2.0 Hz, H-8), 5.05
(1H, d, J = 6.0 Hz, H-2), 4.45 (1H, dd, J = 6.0 Hz, H-3); ESIMS (posi-
tive-ion mode) m/z 311 [M+Na]+.
+6500, [h]280 +12,100, [h]293 +8900; IR (KBr) mmax cmꢀ1: 3398,
2945, 2832, 2501, 1705, 1499, 1120, 1030; for 1H (500 MHz) and
13C (125 MHz) NMR spectroscopic data, see Table 2; ESIMS (posi-
tive-ion mode) m/z 313 [M+Na]+; HR-ESIMS (positive-ion mode)
m/z 313.0693 [M+Na]+ (calcd. for C15H14NaO6, 313.0688).
4.3.7. (2R,3S,10S)-7,8,9,13-Tetrahydroxy-2-(3,4-dihydroxyphenyl)-
2,3-trans-3,4-cis-2,3,10-trihydrobenzopyrano[3,4-c]-2-benzopyran-
1-one (7)
4.4.4. (ꢀ)-Butin (6a)
Amorphous solid; 78% yield; ½a D25
ꢀ32.6 (c 0.05, MeOH); CD
ꢂ
(MeOH): [h]292 ꢀ15,200; IR (KBr) mmax cmꢀ1: 3454, 2930, 1662,
1580, 1502, 1361, 1345, 1285, 1170, 1030; 1H NMR (500 MHz, CD3-
OD): d 7.68 (1H, d, J = 8.0 Hz, H-5), 6.85 (1H, d, J = 2.0 Hz, H-20), 6.76
(1H, dd, J = 8.0 Hz, H-50), 6.71 (1H, d, J = 8.0, 1.5 Hz, H-60), 6.42 (1H,
dd, J = 8.5, 2.0 Hz, H-6), 6.29 (1H, d, J = 2.0 Hz, H-8), 5.21 (1H, dd,
J = 13.0, 3.0 Hz, H-2), 2.91 (1H, dd, J = 17.0, 13.0 Hz, H-3a), 2.65
(1H, dd, J = 17.0, 3.0 Hz, H-3b); ESIMS (positive-ion mode) m/z
295 [M+Na]+.
Amorphous solid; ½a D25
ꢀ47.4 (c 0.15, MeOH); UV (MeOH) kmax
ꢂ
nm (log e): 214 (4.0), 276 (3.6); CD (MeOH): [h]225 +59,500, [h]230
+65,300, [h]270 ꢀ5200; IR (KBr) mmax cmꢀ1: 3347, 2925, 1712,
1515, 1493, 1210, 1118, 1030; 1H NMR (500 MHz, CD3OD): d
7.07 (1H, s, H-200), 6.80 (1H, d, J = 2.0 Hz, H-20), 6.79 (1H, d,
J = 8.5 Hz, H-50), 6.69 (1H, d, J = 8.5 Hz, H-5), 6.67 (1H, dd, J = 8.5,
2.0 Hz, H-60), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.27 (1H, dd, J = 8.5,
2.0 Hz, H-6), 5.54 (1H, d, J = 2.5 Hz, H-2), 4.91 (1H, dd, J = 3.0,
2.5 Hz, H-3), 4.05 (1H, d, J = 3.0 Hz, H-4); 13C NMR (125 MHz, CD3-
OD): d 166.5 (C-700), 157.2 (C-7), 153.3 (C-8a), 145.2 (C-30), 144.8
(C-40, C-300), 142.5 (C-500), 139.8 (C-400), 130.1 (C-5), 129.5 (C-10),
121.8 (C-600), 116.4 (C-60), 115.2 (C-50), 113.7 (C-100), 112.1 (C-20),
110.8 (C-5a), 108.2 (C-6, C-200), 101.8 (C-8), 77.2 (C-3), 76.8 (C-2),
26.1 (C-4); HR-ESIMS (positive-ion mode) m/z 447.0689 [M+Na]+
(calcd. for C22H16NaO9, 447.0692).
4.5. Cell cultures
All tumor cell cultures were maintained using RPMI1640 cell
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streptomycin. Human tumor cell lines such as A549 (non-small cell
lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2

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This work was supported by the Postdoctoral Research Program
of Sungkyunkwan University (2012). We thank Drs. E.J. Bang, S.G.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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