Phenanthrenes, 9,10-dihydrophenanthrenes, bibenzyls with their derivatives, and malate or tartrate benzyl ester glucosides from tubers of Cremastra appendiculata

Article abstract of DOI:10.1016/j.phytochem.2013.06.001

Eleven previously unknown compounds and 23 known compounds including 20 phenanthrene or 910- dihydrophenanthrene derivatives five bibenzyls, seven malate or tartrate benzyl ester glucosides, adenosine and gastrodin were isolated from tubers of Cremastra a

Full text of DOI:10.1016/j.phytochem.2013.06.001

Phytochemistry 94 (2013) 268–276
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenanthrenes, 9,10-dihydrophenanthrenes, bibenzyls
with their derivatives, and malate or tartrate benzyl ester glucosides
from tubers of Cremastra appendiculata
a
a
a
a
Yang Wang a,b, Shu-Hong Guan a, , Yu-Hui Meng , Yi-Bei Zhang , Chun-Ru Cheng , Yang-Yang Shi ,
⇑
Rui-Hong Feng a, Feng Zeng a, Zhi-Yuan Wu a, Jing-Xian Zhang a,b, Min Yang a, Xuan Liu a, Qing Li b,
Xiao-Hui Chen b, Kai-Shun Bi b, De-An Guo a,
⇑
a Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, 501 Haike Road, ZhangJiang Hi-Tech Park, Shanghai 201203, People’s Republic of China
b School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven previously unknown compounds and 23 known compounds, including 20 phenanthrene or 9,10-
dihydrophenanthrene derivatives, five bibenzyls, seven malate or tartrate benzyl ester glucosides, aden-
osine and gastrodin were isolated from tubers of Cremastra appendiculata. Among the obtained com-
pounds, two are the first isolated dimers with one phenanthrene or bibenzyl unit connected to C-3 of
2,3,4,5-tetrahydro-phenanthro[2,1-b]furan moiety. In addition, 33 of these compounds were evaluated
in vitro for their cytotoxic activity against two cancer cell lines. Among the compounds examined, one
compound showed moderate cytotoxic activity, while five showed weak cytotoxic activity against the
A549 cell line.
Received 19 November 2012
Received in revised form 3 June 2013
Available online 29 June 2013
Keywords:
Cremastra appendiculata
Orchidaceae
Bibenzyl
Phenanthrene
Ó 2013 Elsevier Ltd. All rights reserved.
9,10-Dihydrophenanthrene
2,3,4,5-Tetrahydro-phenanthro[2,1-b]furan
Malate benzyl ester glucoside
Tartrate benzyl ester glucoside
1. Introduction
as the authentic medicines. Mixed use of the title herbal medicine
and its adulterants will negatively influence the therapeutic effect
The tubers of orchidaceous plants, Cremastra appendiculata (D.
Don) Makino, together with Pleione bulbocodioides (Franch.) Rolfe
and Pleione yunnanensis Rolfe, namely ‘‘Shan-Ci-Gu’’ in traditional
Chinese medicine, are used for treatment of various cancers and
bacterial infections (Xie et al., 1996). In this regard, Orchidaceae
plants can biosynthesis various aromatic compounds, such as
bibenzyls, phenanthrenes, 9,10-dihydrophenanthrenes, 9,10-
dihydrophenanthrofurans, and tartrate benzyl ester glucosides
(Bai et al., 1998; Majumder et al., 1999; Xue et al., 2006; Yang
et al., 2007; Zi et al., 2008). Some of these compounds were reported
to possess various pharmacological activities (Hahn et al., 2012;
Kim et al., 2007; Matsuda et al., 2004; Shim et al., 2004; Toh,
1994; Xue et al., 2006; Yang et al., 2007; Yu et al., 2009). Because
C. appendiculata availability is far less than market demand result-
ing in a very high price of the tubers, however, cheaper adulterants
such as the tubers of Tulipa edulis (Miq.) Baker are sold in the market
though, and could cause even more serious problems, such as hepa-
tic injury and inherent toxicity. Therefore, some researchers have
investigated rapid propagation of the tubers of C. appendiculata
(Mao et al., 2007; Zhang et al., 2010). Although the technique has
not been commercialized so far, it is still a promising improved cul-
tivation method. However, the main and active components of the
herbal medicines may also change with alteration of the environ-
ment (Qi et al., 2006). Thus, it is important to define the chemotax-
onomic markers of the tubers of C. appendiculata in order to help
control its quality.
Previous chemical investigation of the title plant mainly fo-
cused on the low-polarity compounds (Dong et al., 2007; Ikeda
et al., 2005; Li et al., 2008; Liu et al., 2008; Shim et al., 2004; Xia
et al., 2005; Xue et al., 2005, 2006; Zhang et al., 2011), while the
constituents of the high-polarity fraction were not investigated
to the same extent. In this paper, the isolation and structural elu-
cidation of 11 previously unreported compounds (Figs. 1 and 2),
including two 2-isobutylmalate benzyl ester glucosides (2, 3),
two 2-benzylmalate benzyl ester glucosides (5, 6), three glycosidic
⇑
Corresponding authors. Tel.: +86 21 50271516; fax: +86 21 50272223.
E-mail addresses: sh_guan@mail.shcnc.ac.cn (S.-H. Guan), daguo@mail.shcn-
c.ac.cn (D.-A. Guo).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.06.001

Y. Wang et al. / Phytochemistry 94 (2013) 268–276
269
O
1
7'
R1
2
2
4
1'
O
OH
HO
HO
HO
OH
O
O
O
O
HO
6
O
O
HO
HO
1''''
OH
7
OH
2 R1= OMe
1
4
3 R1= OEt
O
O
1
H
OH
O
7'
3
OMe
HO
OMe
1'
O
O
7''
HO
OH
OH
OH
4'
O
O
O
HO
HO
O
1''''
1''
HO
HO
OH
OH
OH
OH
5
1'''''
O
O
HO
OH
O
1
7'
2
6
O
O
1'
O
O
1'''
HO
O
O
HO
O
1''
HO
1''''
OH
OH
OH
O
O
HO
OH
O
R1 R2
HO
OH
O
O
HO
HO
O
OH
7 R1=R2=H
8 R1=H R2=OH
9
9
HO
HO
HO
1
8a
10a
1
O
O
HO
1'
1'
OH
HO
O
OH
HO
O
4a
OH
OH
MeO
10
OMe
9
HO
HO
1
O
OH
OH
1''
1'
O
HO
HO
O
O
OH
OH
MeO
11
Fig. 1. Structures of compounds 1–11.
9,10-dihydrophenanthrenes (9–11), one biphenanthrene (27), and
three 2,3,4,5-tetrahydro-phenanthro[2,1-b]furan derivatives (29,
32, 33) from the ethyl acetate-soluble and water-soluble fractions
of C. appendiculata are described. The in vitro cytotoxicities of com-
pounds 1–33 are also evaluated against two human cancer cell
lines.
compound gastrodin (1) (Baek et al., 1999) because part of the
1H and 13C NMR spectra of 2 were similar to those reported for
1. Moreover, compared with the carbon signals of 1, the C-70 in 2
was shifted upfield to dC 67.9, which implied an esterification prod-
uct of gastrodin (1). This deduction was supported by the corre-
sponding HSQC and HMBC correlations. It was elucidated that, in
comparison to 1, 2 was esterified by 4-methyl-2-isobutylmalate
moiety at C-70.
2. Results and discussion
In order to determine the absolute configuration of 2, it was
subjected to alkaline hydrolysis with this affording (2R)-2-hydro-
xy-2-(2-methylpropyl) butanedioic acid (Kizu et al., 1999; Yin
Compound 2 was obtained as a colorless gum. Its IR spectrum
showed the presence of hydroxyl (3427 cmꢀ1), carbonyl
(1736 cmꢀ1), and aromatic ring (1514 cmꢀ1) functional groups,
and its molecular formula was determined from HRESIMS as
et al., 2010). Accordingly, structure 2 was elucidated as 1-(4-b-
glucopyranosyloxybenzyl) 4-methyl (2R)-2-isobutylmalate.
D-
Compound 3 was obtained as a colorless gum, with a molecular
C22H32O11
.
Compound
2
was derived from the analogous
formula of C23H34O11 determined by HRESIMS. The 1H and 13C

270
Y. Wang et al. / Phytochemistry 94 (2013) 268–276
R1
R1
12 R1=H R2=OH R3=OMe
13 R1=H R2=R3=OMe
HO
HO
R2
R2
14 R1=CH2C6H4OH R2=OMe R3=OH
R3
R3
15 R1=H R2=OH R3=OMe
16 R1=H R2=R3=OMe
17 R1=CH2C6H4OH R2=OH R3=OMe
18 R1=R3=H R2=OH R4=OMe
19 R1=R3=H R2=R4=OH
R4
R3
OH
20 R1=H R2=OMe R3=CH2C6H4OH R4=OH
21 R1=CH2C6H4OH R2=OH R3=H R4=OMe
22 R1=CH2C6H4OH R2=OMe R3=H R4=OH
R1
R2
HO
HO
HO
OMe
OH
OMe
OMe
OH
OH
OH
HO
OH HO
HO
OH
MeO
23
MeO
MeO
25
24
8a
10a
1
HO
OH
HO
OH
HO
OH
4a
MeO
OH
OMe
OH
MeO
OH
1'
HO
HO
HO
MeO
MeO
MeO
27
28
26
MeO
2'
OH
MeO
O
OH
OH
OMe
OH
O
1''
O
3
2
4
3a
O
11a
1
3b
O
5
trans-2,3
5a
11
HO
OMe
31
O
6
9a
9
10
OMe
O
OMe
HO
7
HO
8
29
MeO
30
3'''
MeO
5''
NH2
3''
3''
N
N
HO
HO
OH
N
HO
HO
OH
OH
OMe
OH
3'
1''
1''
3
1'''
11''
7''
N
O
α'
α
4
1'
1'
3
2
2
OMe
OH
1
O
O
1
OH
trans-2,3
OH
trans-2,3
MeO
33
MeO
34
32
Fig. 2. Structures of compounds 12–34.
NMR spectroscopic data (Table 1) of 3 were very similar to those of
2. The only difference was the absence of the O-methyl moiety, this
being replaced by an O-ethyl moiety in 3, showing corresponding
signals at dH 1.19 (3H, t, J = 7.2 Hz) and dH 4.05 (2H, q, J = 7.2 Hz)

Y. Wang et al. / Phytochemistry 94 (2013) 268–276
271
Table 1
1H and 13C NMR spectroscopic data of compounds 2, 3, 5, 6.a
Pos.
2b
3c
5d
6
dc
dH (J in Hz)
dc
dH (J in Hz)
dc
dH (J in Hz)
dc
dH (J in Hz)
1
2
3
176.0
76.6
45.8
175.8
76.3
45.8
175.2
77.1
44.1
175.2
77.1
44.5
2.62 (d, 15.7)
2.91 (d, 15.7)
2.60 (d, 15.6)
2.90 (d, 15.6)
2.62 (d, 15.5)
2.97 (d, 15.5)
2.64 (d, 16.0)
3.01 (d, 16.0)
4
5
172.3
49.3
171.6
48.9
172.3
171.6
1.57 (dd, 5.6, 14.0)
1.68 (mf)
1.57 (d, 5.6, 14.0)
1.67 (mf)
6
25.0
1.72 (mf)
24.8
1.71 (mf)
7
24.7
23.9
130.8
131.3
0.91 (3H, d, 6.5)
0.80 (3H, d, 6.5)
24.4
23.7
130.6
131.0
0.92 (3H, d, 6.5)
0.80 (3H, d, 6.5)
8
10
130.8
131.5
(or 131.4)
117.7
159.3
68.0
130.7
131.5
20,60
7.34 (2H, d, 8.6)
7.10 (2H, d, 8.6)
7.34 (2H, d, 8.7)
7.10 (2H, d, 8.7)
7.28 (2H, d, 8.5)
7.09 (2H, d, 8.5)
5.07 (2H, s)
7.19 (2H, d, 9.0)
7.07 (2H, d, 9.0)
4.94 (2H, s)
30,50
40
70
117.7
159.2
67.9
117.4
159.0
67.6
117.7
159.2
68.0
5.15 (d, 11.6)
5.10 (d, 11.6)
5.15 (d, 12.0)
5.10 (d, 12.0)
100
200,600
136.6
131.5
(or 131.4)
129.1
127.9
46.4
136.6
131.3
7.10 (2H, dd, 4.0, 7.5)
7.11 (2H, dd, 6.5, 3.0)
300,500
400
7.21 (2H, mf)
7.20 (mf)
3.01 (d, 13.5)a
2.93 (d, 13.5)b
129.1
127.9
46.4
7.22 (2H, mf)
7.20 (mf)
3.00 (d, 14.0)
2.92 (d, 14.0)
700
1000
130.7
131.0
117.7
159.1
67.2
2000,6000
3000,5000
4000
7.25 (2H, d, 9.0)
7.06 (2H, d, 9.0)
7000
4.99 (2H, s)
4.91 (d, 7.5)
10000
(100000
102.2
74.8
77.9
71.3
4.92 (d, 7.2)
3.47 (mf)
3.44 (mf)
3.41 (mf)
102.0
74.6
77.7.
71.0
4.90 (d, 7.2e)
3.45 (mf)
102.2
74.9
78.0
71.3
4.93 (d, 7.5)
3.46 (mf)
3.46 (mf)
3.40 (mf)
102.3
)
)
)
)
20000
30000
40000
74.9
77.9
71.3
3.47 (mf)
3.47 (mf)
3.41 (mf)
(200000
(300000
(400000
3.45 (mf)
3.40 (mf)
50000 (500000
60000
(600000
)
78.1
62.4
3.44 (mf)
3.89 (dd, 1.8, 12.0)
3.70 (dd, 5.2, 12.0)
77.9
62.2
3.41 (mf)
3.89 (dd, 2.0, 12.0)
3.70 (dd, 5.2, 12.0)
78.2
62.5
3.41 (mf)
3.90 (dd, 2.0, 12.0)
3.71 (dd, 5.0, 12.0)
78.1
62.5
3.45 (mf)
3.89 (mf)
3.70 (mf)
)
a
b
c
d
e
f
1H NMR data were measured in CD3OD at 400 MHz for 2 and 3, at 500 MHz for 5 and 6. 13C NMR data were measured at 125 MHz in CD3OD.
Data for methoxy: dc 52.2, dH 3.57 (3H, s).
Data for ethoxy: dc 61.5, dc 14.2; dH 4.05 (2H, q, J = 7.2 Hz), dH 1.19 (3H, t, J = 7.2 Hz).
Data for methoxy: dc 52.2, dH 3.57 (3H, s).
J were measured in DMSO-d6.
Overlapping signals.
in the 1H NMR spectrum. Other resonances in the 13C NMR (Table
1), HSQC and HMBC spectra also supported the structure proposed.
The configuration at C-2 in compound 3 was determined following
the same procedure as that for 2. Thus, compound 3 was deter-
Compound 6 was obtained as a colorless amorphous powder. Its
molecular formula C37H44O17 was established by HRESIMS. Signals
in the 1H and 13C NMR spectra (Table 1) of 6 resembled those of 5,
except for resonances for an additional 4-b-D-glucopyranosyloxyb-
mined as 1-(4-b-
isobutylmalate.
D
-glucopyranosyloxybenzyl) 4-ethyl (2R)-2-
enzyl moiety. Correlations from HMBC and HSQC were consistent
with the structure proposed and the configuration of 6 was deter-
mined using the same method as that for 5. Hence compound 6
The molecular formula of 5 was determined by HRESIMS as
25H30O11. Compared with 2, signals for the extra benzyl at dc
C
was elucidated as 1,4-bis(4-b-D-glucopyranosyloxybenzyl) (2R)-2-
136.6 (C-100), 131.5 (or 131.4) (C-200, 600), 129.1 (C-300, 500), 127.9
(C-400) and 46.4 (C-700) in the 13C NMR spectrum and corresponding
proton resonances indicated the presence of a benzyl group in 5,
replacing the isobutyl. In the HMBC spectrum, the correlations
from H-700 (dH 3.01 and dH 2.93) to C-1 (dc 175.2), C-2 (dc 77.1),
C-3 (dc 44.1), C-200 [dc 131.5 (or 131.4)], and C-600 [dc 131.5 (or
131.4)] further confirmed that the benzyl moiety was positioned
at C-2 in 5. All chemical shifts were assigned by the combination
of analyses of the HSQC and HMBC spectra (Table 1). The absolute
configuration of 5 was also determined using the same method as
that for 2, with the hydrolytic product identified as (2R)-2-benzyl-
2-hydroxysuccinic acid (El Bialy et al., 2005). Thus, compound 5
benzylmalate.
Compound 9 was obtained as a colorless amorphous powder.
The HRESIMS gave a m/z of 427.135, corresponding to C21H24O8.
The UV spectrum showed maximum absorptions at 276 and
290 nm, this being characteristic of a 9,10-dihydrophenanthrene.
In the 1H NMR spectrum (Table 2), the deshielded aromatic proton
signal at dH 8.03 (1H, d, J = 8.0 Hz, H-5) also indicated that 9 pos-
sessed a 9,10-dihydrophenanthrene structure (Majumder and Lah-
iri, 1990). Seven proton resonances from dH 3.37 to dH 4.91, together
with the coupling constant of H-10 (7.5 Hz), suggested that 9 was a
9,10-dihydrophenanthrene derivative with one b-D-glucose substi-
tuent. The correlation from H-10 (dH 4.91) to C-2 (dc 157.9) in the
HMBC spectrum also established unequivocally that the glucose
moiety was connected to the 9,10-dihydrophenanthrene moiety
was determined as 1-(4-b-D-glucopyranosyloxybenzyl) 4-methyl
(2R)-2-benzylmalate.

272
Y. Wang et al. / Phytochemistry 94 (2013) 268–276
Table 2
1H and 13C NMR spectroscopic data of compounds 9–11.a
Pos.
9
10
11
dc
dH (J in Hz)
dc
dH (J in Hz)
dc
dH (J in Hz)
1
2
3
4
4a
4b
5
6
7
8
8a
9
109.5
157.9
101.0
158.8
119.4
125.7
130.4
113.6
156.5
115.1
140.8
31.1
6.62 (d, 2.5)
108.3
158.0
99.2
6.32 (d, 2.0)
108.5
157.2
100.0
157.7
116.8
126.6
129.0
114.1
156.0
115.8
139.4
29.8
6.59 (d, 2.0)
6.74 (d, 2.5)
6.41 (d, 2.0)
6.68 (d, 2.0)
159.4
116.3
128.9
129.8
115.0
156.7
116.6
140.4
31.2
8.03 (d, 8.0)
6.61 (dd, 3.0, 8.0)
8.09 (d, 6.8)
6.92 (dd, 2.0, 6.8)
8.05 (d, 8.4)
6.88 (dd, 2.8, 8.4)
6.63 (d, 3.0)
6.94 (d, 2.0)
6.90 (d, 2.8)
2.65 (2H, m)
2.67 (2H, m)
2.67 (2H, m)
2.66 (2H, m)
2.65 (2H, s)
2.65 (2H, s)
10
31.8
31.7
30.5
10a
141.6
102.6
75.0
78.1
71.6
142.2
102.4
75.0
78.0
71.5
140.5
101.0
73.7
77.1 or 77.2
70.4 or 70.2
77.5 or 77.7
61.2 or 61.3
10
4.91 (d, 7.5)
3.46 (mb)
4.91 (d, 7.5)
3.46 (mb)
4.88 (d, 7.6)
3.24 (mb)
3.28 (mb)
3.15 (mb)
3.33 (mb)
3.71 (m)
3.47 (m)
4.85 (d, 7.2)
3.82 (3H, s)
20(200)
30(300)
40(400)
50(500)
60(600)
3.47 (mb)
3.46 (mb)
3.37 (mb)
3.40 (mb)
78.3
62.7
3.47 (mb)
78.2
62.6
3.46 (mb)
3.70 (dd, 6.0, 12.0)
3.92 (dd, 2.5, 12.0)
3.71 (dd, 5.6, 12.4)
3.91 (dd, 2.0, 12.4)
100
4-OMe
101.0
55.9
56.0
3.85 (3H, s)
55.9
3.83 (3H, s)
a
1H NMR data were measured at 500 MHz in CD3OD for 9 and 10, at 400 MHz in DMSO-d6 for 11. 13C NMR data were measured at 125 MHz in CD3OD.
Overlapping signals.
b
at C-2. Therefore, structure 9 was determined as 7-hydroxy-4-
methoxy-9,10-dihydrophenanthrene-2-O-b- -glucopyranoside.
Compound 10 was obtained as a colorless amorphous powder.
This compound was identified as an isomer of 9 by its HRESIMS.
The 1H NMR and 13C NMR spectra (Table 2) of 10 highly resembled
those of 9. From the correlation from H-10 (dH 4.91) to C-7 (dc
156.7) in the HMBC spectrum, the b-D-glucose unit was shown to
be located at C-7 of the 9,10-dihydrophenanthrene moiety. There-
fore, compound 10 was determined as 7-hydroxy-5-methoxy-
9,10-dihydrophenanthrene-2-O-b-D-glucopyranoside.
1D NOE experiment was carried out. Irradiation of the protons at
dH 3.68 (4-OMe) enhanced the signals of H-3 (2.56%) and H-5
(0.34%), while irradiation of the protons at dH 4.13 (40-OMe) caused
enhancement of H-30 (2.86%) and H-50 (0.39%). This result was con-
sistent with those of other compounds, in which the methoxyl
group was positioned at C-4 (Bai et al., 1998; Yamaki et al.,
1990). Therefore, structure 27 was determined as 4,40-dime-
thoxy-9,10-dyhydro-[6,10-biphenanthrene]-2,20,7,70-tetraol.
Compound 29 was obtained as a brown amorphous powder.
The HREIMS gave an intense parent ion at m/z 420.1572, corre-
sponding to the molecular formula C25H24O6. The 1H and 13C
NMR spectroscopic data of 29 highly resembled those of pleionesin
C (30) (Dong et al., 2010). It differed from 30 though by the pres-
ence of the methylol moiety at C-3, instead of an acetoxymethyl
in 30. Accordingly, signals at dc 172.7 and dc 20.7 from 30 were ab-
sent in the 13C NMR spectrum of 29. The relative configuration of
H-2 and H-3 at the dihydrofuran ring was determined to be trans
by correlation between H-100 and H-2 in the NOESY experiment
(Ali et al., 2003; Dong et al., 2010; Yao et al., 2006). Thus, structure
29 was determined as (2,3-trans)-2-(4-hydroxy-3-methoxy-
phenyl)-3-hydroxymethyl-10-methoxy-2,3,4,5-tetrahydro-phenan
thro[2,1-b]furan-7-ol.
D
Compound 11 was obtained as a colorless amorphous powder
and the HRESIMS data established the molecular formula as
C
27H34O13. The 1H and 13C NMR spectra of 11 (Table 2) were more
complex than those of 9 and 10, including signals of two glucose
units and one 9,10-dihydrophenanthrene moiety. Correlations
from H-10 (dH 4.88) to C-2 (dc 157.2) and from H-100 (dH 4.85) to
C-7 (dc 156.0) observed in the HMBC spectrum suggested two b-
D-glucose units located at C-2 and C-7 of the 9,10-dihydrophe-
nanthrene moiety, respectively. The structure of 11 was therefore
determined as 4-methoxy-9,10-dihydrophenanthrene-2,7-di-O-b-
D-glucopyranoside.
Compound 27 was obtained as a brown amorphous powder.
The molecular formula C30H24O6 was deduced by HREIMS with
Compound 32 was assigned the molecular formula C40H34O8,
deduced by negative ion HRESIMS. Signals in the 1H and 13C
NMR spectra of 32 could be divided into two groups. One group
was very similar to those of 29, suggesting presence of the 2-(4-hy-
droxy-3-methoxyphenyl)-10-methoxy-2,3,4,5-tetrahydro-phenan-
thro[2,1-b]furan-7-ol unit. Besides that, characteristically
deshielded aromatic resonances at dH 9.42 (1H, d, J = 9.3 Hz, H-
500) in the 1H NMR spectrum suggested the presence of an addi-
tional phenanthrene unit (Majumder and Basak, 1990; Majumder
et al., 1997). In addition, signals in the 13C NMR spectrum including
three oxygenated quaternary aromatic carbons, five quaternary
aromatic carbons and six methenyl aromatic carbons, together
with an HMBC correlation from 4.09 (400-OMe) to 159.1 (C-400) con-
firmed the 2,7-dihydroxy-4-methoxy-phenanthrene as the other
unit. The two groups were connected through C-3 and C-100 with
[M]+ at m/z 480.1566 (calcd for 480.1573).
In the 1H NMR spectrum, the deshielded aromatic signals at dH
9.44 (1H, d, J = 9.2 Hz, H-50) and dH 8.00 (1H, s, H-5) suggested that
27 was a dimer formed from the combination of a phenanthrene
unit (Majumder and Basak, 1990; Majumder et al., 1997) and a
9,10-dyhydrophenanthrene unit (Majumder and Lahiri, 1990).
The HMBC spectrum showed a correlation from H-5 (dH 8.00) to
C-10 (dc 114.9), indicating that the phenanthrene group (C-10) con-
nected with the 9,10-dyhydrophenanthrene group at C-6. One
methoxyl at dH 3.68 (3H, s) located at C-4 was determined by cor-
relation from dH 3.68 (4-OMe) to dc 159.1 (C-4) in the HMBC spec-
trum. Although another correlation from dH 4.13 to dc 159.9 could
also be observed, no further prediction could be made due to the
uncertain assignment of the dc 159.9 signal. Therefore, a selective

Y. Wang et al. / Phytochemistry 94 (2013) 268–276
273
Table 3
a methylene which was deduced by correlations from dH 3.42
Cytotoxic activities of compounds 23, 24, 30–33 against
(H-1100) to dc 88.6 (C-2), dc 53.1 (C-3), dc 113.4 (C-100), dc 134.0
(C-1000a) and dc 154.5 (C-200) in the HMBC spectrum.
A549 cell line.a
Compound
IC50 value (lM)
The trans orientation of H-2 and H-3 at the dihydrofuran ring in
32 was determined by interactions between H-2/H-1100 and H-20/
H-3 in NOESY experiment (Ali et al., 2003; Dong et al., 2010; Iliya
et al., 2003; Nozaki et al., 2007; Yao et al., 2006). Therefore 32 was
determined as (2,3-trans)-3-[(2,7-dihydroxy-4-methoxy-phenan-
thren-1-yl)methyl]-2-(4-hydroxy-3-methoxyphenyl)-10-methoxy-
2,3,4,5-tetrahydro-phenanthro[2,1-b]furan-7-ol.
23
24
30
31
47.5
48.2
33.6
42.8
38.0
16.0
0.05
32
33
Bufalinb
Compound 33 was isolated as a brown amorphous powder. Po-
sitive mode HRESIMS at m/z 669.2470 [M+Na]+ (calcd for
669.2464) indicated its molecular formula as C40H38O8. Signals
from the 1H NMR spectrum of 33 could also be divided into two
groups, one of which was readily assigned to a 2-(4-hydroxy-3-
methoxyphenyl)-10-methoxy-2,3,4,5-tetrahydro-phenanthro[2,1-
b]furan-7-ol unit. Resonances in the 1H NMR spectrum of the
other group could be attributed to two methylenes [dH 2.80 (2H,
a
Compounds 1–22, 25–29 were inactive against
A549 cell line (IC50 > 50
lm).
b
Positive control.
showed moderate cytotoxic activity (IC50 16.0
cell line. All of these compounds were inactive towards Bel7402
cells (IC50 > 50 M).
lM) against A549
l
m, H-a
) and dH 2.70 (2H, m, H-a0)], one 1,2,3,5-tetrasubstituted
benzene moiety [dH 6.36 (1H, d, J = 2.4 Hz, H-300) and dH 6.34
(1H, d, J = 2.4 Hz, H-500)], one 1,3-meta-substituted benzene moiety
3. Conclusions
[dH 6.57 (1H, overlapped, H-2000), dH 6.58 (2H, overlapped, H-4000
,
This study afforded 11 previously unreported compounds from
the tubers of C. appendiculata. Among them, the structures of
compounds 32 and 33 were unusual as dimers, possessing a phen-
anthrene or bibenzyl unit connecting to C-3 of the 2,3,4,5-tetrahy-
dro-phenanthro[2,1-b]furan moiety; which is a novel discovery of
natural products from Orchidaceae plants. In addition to the com-
pounds described above, 23 known compounds were obtained at
the same time of which compounds 4, 13, 14, 20, 23, 24, 26, 28,
30 and 31 were isolated from tubers of C. appendiculata for the first
time.
H-6000), dH 7.04 (1H, t, J = 7.6 Hz, 8.4 Hz, H-5000)] and one methoxyl,
which indicated a bibenzyl moiety as the other group. In addition,
signals in the 13C NMR spectrum, including three quaternary
oxygenated aromatic carbons, three quaternary aromatic carbons,
six methenyl aromatic carbons and two methenyl carbons, further
confirmed the prediction. Moreover, correlations from dH 2.95
(H-700) to dc 144.0 (C-600), dc 158.1 (C-200), dc 89.5 (C-2), and dc
52.2 (C-3) in the HMBC spectrum suggested that the bibenzyl unit
and the dihydrophenanthrofuran moiety were connected through
C-3 and C-100 via a methylene.
According to the cytotoxicity assay, six phenanthrenes or 9,10-
dihydrophenanthrenes isolated from the herb showed weak or
moderate cytotoxic activity against A549 cells. These two types
of compounds were also reported to possess cytotoxic activity in
previous studies (Guo et al., 2009; Xia et al., 2005; Xue et al.,
2006). Moreover, traditional Chinese medicines are always charac-
terized by the synergistic effect of multiple components, thus two
classes of bioactive components including phenanthrenes and
9,10-dihydrophenanthrenes should be considered as the chemical
markers. Accordingly for quality control it is sensible to determine
the total amount of the phenanthrenes and 9,10-
dihydrophenanthrenes.
The relative orientation of H-2 and H-3 was determined as trans
by the interactions between H-2/H-700 observed in a NOESY
experiment (Ali et al., 2003; Dong et al., 2010; Yao et al., 2006).
Consequently structure 33 was determined as (2,3-trans)-3-[2-hy-
droxy-6-(3-hydroxyphenethyl)-4-methoxybenzyl]-2-(4-hydroxy-
3-methoxyphenyl)-10-methoxy-2,3,4,5-tetrahydro-phenanthro[2,
1-b]furan-7-ol.
Besides the previously unreported compounds, 23 known
compounds (Figs. 1 and 2) were identified as gastrodin (1) (Baek
et al., 1999), (ꢀ)-(2R,3S)-1-(4-b-D-glucopyranosyloxybenzyl)-4-
methyl-2-isobutyltartrate (4) (Zi et al., 2008), militarine (7) (Kizu
et al., 1999), loroglossin (8) (Kizu et al., 1999), coelonin (12)
(Majumder and Banerjee, 1988), 7-hydroxy-2,4-dimethoxy-9,10-
dihydrophenanthren (13) (Krautwurst and Tochtermann, 1981),
4,7-dihydroxy-1-p-hydroxybenzyl-2-methoxy-9,10-dihydrophe-
nanthrene (14) (Takagi et al., 1983), flavanthrinin (15) (Leong et al.,
1997), 2-hydroxy-5,7-dimethoxyphenanthrene (16) (Broering and
Morrow, 1999), 1-p-hydroxybenzyl-4-methoxyphenanthrene-2,7-
diol (17) (Yamaki et al., 1990), batatasin III (18) (Leong et al.,
1997), 3,30,5-trihydroxybibenzyl (19) (Honda and Yamaki, 2001),
3,30-dihydroxy-4-(p-hydroxybenzyl)-5-methoxybibenzyl (20) (Bai
et al., 1993), 3,30-dihydroxy-2-(p-hydroxybenzyl)-5-methoxybib-
enzyl (21) (Bai et al., 1993), 30,5-dihydroxy-2-(p-hydroxybenzyl)-
3-methoxybibenzyl (22) (Bai et al., 1993), blestriarene A (23)
(Yamaki et al., 1989), blestriarene B (24) (Yamaki et al., 1989), ble-
striarene C (25) (Yamaki et al., 1989), gymconopin C (26) (Matsuda
et al., 2004), blestrianol A (28) (Bai et al., 1991), pleionesin C (30)
(Dong et al., 2010), shanciol H (31) (Dong et al., 2010) and adeno-
sine (34) (Otsuka et al., 1989).
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polar-
imeter, UV spectra were recorded on a Shimadzu UV-2450 spec-
trometer. IR (4000–400 cmꢀ1) spectra were recorded as KBr disks
on a Perkin-Elmer 577 spectrometer. NMR spectra were measured
in CDCl3, CD3OD or DMSO-d6 on a Bruker AM-400 spectrometer for
1H, 1D NOE NMR spectra (400 MHz), on a Bruker AVANCE III 500
spectrometer for 1H, 13C, DEPT, HSQC and HMBC NMR spectra
(500 MHz), and on a Varian Inove-600 spectrometer for NOESY
NMR (600 MHz). High-resolution electrospray ionization mass
spectra (HRESIMS) were acquired using a Finnigan LCQDECA mass
spectrometer. HREIMS (70 ev) analyses were carried out on a Finn-
igan-MAT 95 mass spectrometer.
The cytotoxicities of compounds 1–33 were evaluated against
A549 and Bel7402 cell lines using the MTT method (Table 3), with
bufalin as a positive control. Among the compounds examined,
compounds 30, 32, 31, 23, 24 showed weak cytotoxic activity
Analytical and semi-preparative HPLC were performed on an
Agilent 1100 with an Agilent DAD spectrophotometer and a XDB-
C18 column (10 ꢁ 250 mm, 5
lm). Column chromatography (CC)
separations were carried out on silica gel (200–300 mesh, Qingdao
(IC50 33.6, 38.0, 42.8, 47.5, 48.2 lM respectively); compound 33
Haiyang Chemical Co., Ltd.) and Sephadex LH-20 gel (Amersham

274
Y. Wang et al. / Phytochemistry 94 (2013) 268–276
Biosciences), respectively. TLC was performed on precoated silica
gel sheets (GF 254, 100 ꢁ 100 mm, 0.15–0.20 mm, Sinopharm
Group Co., Ltd.). All solvents used were of analytical grade (Shang-
hai Sinopharm Chemical Reagent Company, Ltd.). Fractions were
monitored by TLC and spots were visualized under UV light or by
spraying with 10% H2SO4 in EtOH followed by heating.
for 1H NMR and 13C NMR spectroscopic data, see Table 1; HRESIMS
at m/z 495.1833 [M+Na]+ (calcd for C22H32O11Na 495.1842).
4.3.2. 1-(4-b-D-Glucopyranosyloxybenzyl) 4-ethyl (2R)-2-
isobutylmalate (3)
20
Colorless gum; [
a]
ꢀ23 (c 0.11, MeOH); UV (MeOH) kmax
D
(loge) 223 (4.03), 270 (3.25), 276 (3.22) nm; IR (KBr) mmax 3427,
2938, 2843, 1682, 1614, 1520, 1462, 1425, 1325, 1213, 1115,
1059, 991, 829, 702 cmꢀ1; for 1H NMR and 13C NMR spectroscopic
data, see Table 1; HRESIMS at m/z 509.2017 [M+Na]+ (calcd for C23
H34O11Na 509.1999).
4.2. Plant material
Tubers of C. appendiculata were collected in Guizhou Province,
People0s Republic of China, in July 2009. The plant identity was ver-
ified by Professor De-an Guo (one of the authors). A voucher spec-
imen (No. 2009016) was deposited at Shanghai Research Center for
TCM Modernization, National Engineering Laboratory for TCM
Standardization Technology, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences.
4.3.3. 1-(4-b-D-Glucopyranosyloxybenzyl) 4-methyl (2R)-2-
benzylmalate (5)
23
Colorless amorphous powder; [
a
]
ꢀ34 (c 0.05, MeOH); UV
D
(MeOH) kmax (loge) 215 (3.92), 264 (2.66) nm; IR (KBr) mmax
3427, 2924, 1741, 1614, 1514, 1439, 1367, 1236, 1072, 833, 702,
602 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table
1; HRESIMS at m/z 529.1673 [M+Na]+ (calcd for C25H30O11Na
529.1686).
4.3. Extraction and isolation
Powdered and air-dried tubers of C. appendiculata (30 kg) were
successively extracted with EtOH–H2O (20 L ꢁ 3, 95:5 and 70:30,
v/v). The dark brown residue resulting (310 g) was partitioned into
EtOAc-soluble (120 g) and H2O-soluble (190 g) fractions. The EtOAc
fraction was applied to a silica gel column and eluted with a
CHCl3–MeOH gradient (from 100:1–2:1) to give 12 fractions (A1–
4.3.4. 1,4-Bis(4-b-
(6)
Colorless amorphous powder; [
(MeOH) kmax (log
3427, 2920, 2515, 1797, 1732, 1612, 1514, 1421, 1236, 1169,
1074, 876, 702, 542, 474 cmꢀ1; for 1H NMR and 13C NMR spectro-
scopic data, see Table 1; HRESIMS at m/z 783.2495 [M+Na]+ (calcd
for C37H44O17Na 783.2476).
D
-glucopyranosyloxybenzyl) (2R)-2-benzylmalate
23
a]
ꢀ40 (c 0.05, MeOH); UV
D
e
) 219 (4.18), 269 (2.76) nm; IR (KBr) mmax
A12). Fraction A6 (50 g) was subjected to a silica gel column washed
with a gradient of increasing EtOAc (1–50%) in petroleum ether
(PE) (60–90 °C) to give five fractions (A6B1 to A6B5). Fraction A6B1
(5 g) was separated by silica gel CC [CH2Cl2–EtOAc (8:1 to 2:1)]
producing four fractions (A6B1C1 to A6B1C4). Fraction A6B1C2 was
applied to a Sephadex LH-20 column eluted with PE–CH2Cl2–
MeOH (2:1:1) and further purified by prep. HPLC [MeOH–H2O
(45:55), k 210 nm] to yield compound 29 (4 mg). Fraction A7
(10 g) was subjected to silica gel CC eluted with CH2Cl2–EtOAc–
MeOH (100: 6: 0.8) to give ten fractions (A7B1 to A7B10). Fraction
A7B2 was applied to a Sephadex LH-20 column [PE–CH2Cl2–MeOH
(2:1:1)] and further purified by prep. HPLC [MeOH–H2O (60:40), k
210 nm] to obtain compound 27 (4 mg). Fraction A7B3 was sub-
jected to Sephadex LH-20 CC [PE–CH2Cl2–MeOH (2:1:1)] and fur-
ther separated by prep. HPLC [MeOH–H2O (30:70), k 210 nm] to
yield compounds 32 (8 mg) and 33 (2 mg). Fraction A10 (6 g) was
applied to a silica column washed with CH2Cl2–EtOAc–MeOH
(100:6:0.8) to give four fractions (A10B1 to A10B4). Fraction A10B4
was eluted with MeOH through a Sephadex LH-20 column to afford
three subfractions (A10B4C1–A10B4C3). Subfraction A7B4C3 was fur-
ther separated by prep. HPLC [MeOH–H2O (30:70), k 210 nm] to
obtain compounds 9 (8 mg) and 10 (3 mg). The aqueous fraction
(190 g) was applied to a MCI resin column with successive elution
with EtOH–H2O (20:80; 50:50; 80:20, v/v). The EtOH–H2O (50:50,
v/v) fraction was separated by reversed-phase silica gel CC eluting
with a step gradient of 10%, 20%, 30% and 40% MeOH in H2O to af-
ford four fractions. Part of the MeOH–H2O (40:60, v/v) fraction
(320 mg) was subjected to Sephadex LH-20 CC [MeOH–H2O
(1:1)] to give three fractions, with the first and second fractions
4.3.5. 7-Hydroxy-4-methoxy-9,10-dihydrophenanthrene-2-O-b-D-
glucopyranoside (9)
Colorless amorphous powder; [
(MeOH) kmax (log
(KBr) mmax 3413, 2920, 1614, 1462, 1271, 1171, 1082, 856,
476 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table
2; HRESIMS at m/z 427.1355 [M+Na]+ (calcd for C21H24O8Na
427.1369).
23
a
]
ꢀ54 (c 0.05, MeOH); UV
D
e
) 217 (4.24), 276 (4.07), 290 (3.98) nm; IR
4.3.6. 7-Hydroxy-5-methoxy-9,10-dihydrophenanthrene-2-O-b-D-
glucopyranoside (10)
Colorless amorphous powder; [
(MeOH) kmax (log
(KBr) mmax 3423, 2929, 1614, 1462, 1348, 1259, 1223, 1076,
584 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table
2; HRESIMS at m/z 427.1356 [M+Na]+ (calcd for C21H24O8Na
427.1369).
23
a
]
ꢀ36 (c 0.05, MeOH); UV
D
e
) 208 (4.48), 276 (4.24), 290 (4.14) nm; IR
4.3.7. 4-Methoxy-9,10-dihydrophenanthrene-2,7-di-O-b-D-
glucopyranoside (11)
Colorless amorphous powder; [
(MeOH) kmax (log
3379, 2927, 2519, 2133, 1797, 1612, 1587, 1567, 1462, 1429,
1300, 1086, 1024, 611 cmꢀ1; for 1H NMR and 13C NMR spectro-
scopic data, see Table 2; HRESIMS at m/z 589.1895 [M+Na]+ (calcd
for C27H34O13Na 589.1897).
23
a]
ꢀ28 (c 0.05, DMSO); UV
D
e
) 278 (4.14), 292 (4.09) nm; IR (KBr) mmax
separated by prep. HPLC [MeOH–H2O (10:90) to (20:80),
k
210 nm] to yield compounds 2 (34 mg) and 3 (5 mg), 5 (28 mg)
and 6 (8 mg), respectively. Part of the MeOH–H2O (20:80, v/v) frac-
tion (64 mg) was subjected to Sephadex LH-20 CC [MeOH–H2O
(1:1)] to give compound 11 (15 mg).
4.3.8. 4,40-Dimethoxy-9,10-dyhydro-[6,10-biphenanthrene]-2,20,7,70-
tetraol (27)
20
Brown amorphous powder; [
(MeOH) kmax (log
nm; IR (KBr) mmax 3427, 2933, 2850, 1614, 1589, 1460, 1437,
a
]
+5 (c 0.17, MeOH); UV
D
e
) 210 (4.79), 260 (4.84), 310 (4.38), 375 (3.64)
4.3.1. 1-(4-b-D-Glucopyranosyloxybenzyl) 4-methyl (2R)-2-
isobutylmalate (2)
1348, 1280, 1224, 1157, 1086, 1020,980, 829, 538 cmꢀ1 1H NMR
;
20
Colorless gum; [
a]
ꢀ19 (c 0.12, MeOH); UV (MeOH) kmax
(CD3OD, 400 MHz) d 9.44 (1H, d, J = 9.2 Hz, H-50), 8.00 (1H, s, H-
5), 7.44 (1H, d, J = 9.2 Hz, H-90), 7.44 (1H, d, J = 9.2 Hz, H-100),
7.12 (1H, d, J = 2.8 Hz, H-80), 7.09 (1H, dd, J = 9.2, 2.8 Hz, H-60),
D
(log
e) 221 (4.01), 269 (3.00), 275 (2.91) nm; IR (KBr) mmax 3427,
2920, 1736, 1616, 1514, 1439, 1371, 1234, 1172, 1074, 577 cmꢀ1
;

Y. Wang et al. / Phytochemistry 94 (2013) 268–276
275
6.93 (1H, s, H-30), 6.86 (1H, s, H-8), 6.36 (1H, d, J = 2.4 Hz, H-3), 6.35
(1H, d, J = 2.4 Hz, H-1), 4.13 (3H, s, 40-OMe), 3.68 (3H, s, 4-OMe),
overlapped, H-6), 6.58 (3H, overlapped, H-11/H-6000/H-4000), 6.57
(1H, overlapped, H-2000), 6.47 (1H, dd, J = 2.0, 8.0 Hz, H-60), 6.36
(1H, d, J = 2.4 Hz, H-300), 6.34 (1H, d, J = 2.4 Hz, H-500), 6.16 (1H, d,
J = 2.0 Hz, H-20), 5.51 (1H, d, J = 2.4 Hz, H-2), 3.86 (3H, s, 10-
OMe), 3.73 (3H, s, 400-OMe), 3.64 (1H, m, H-3), 3.60 (3H, s, 30-
13
2.76 (4H, s, H-9/10); C NMR (CD3OD, 125 MHz) d 159.9 (C-40),
159.1 (C-4), 157.6 (C-2), 155.4 (C-70), 154.3 (C-7), 153.4 (C-20),
141.9 (C-10a), 140.2 (C-8a), 134.7 (C-10a0), 134.5 (C-8a0), 133.9
(C-5), 130.6 (C-50), 128.0 (C-90), 126.5 (C-4b), 126.4 (C-100), 125.6
(C-4b0), 121.6 (C-6), 117.2 (C-60), 116.7 (2C, C-4a/4a0), 115.7 (C-8),
114.9 (C-10), 112.0 (C-80), 108.4 (C-1), 100.3 (C-30), 99.3 (C-3),
56.0 (C-40-OCH3), 55.9 (C-4-OMe), 31.8 (C-10), 31.0 (C-9); HREIMS
at m/z 480.1566 [M]+ (calcd for C30H24O6 480.1573).
OMe), 2.95 (2H, m, H-700), 2.80 (2H, m, H- ), 2.70 (2H, m, H-a0),
a
2.68 (2H, m, H-4), 2.48 (2H, m, H-5); 13C NMR (CD3OD, 125 M) d
160.4 (C-11a), 160.2 (C-400), 159.0 (C-10), 158.4 (C-3000), 158.1
(C-200), 156.0 (C-7), 148.8 (C-30), 146.5 (C-40), 144.7 (C-1000), 144.0
(C-600), 140.4 (C-5a), 137.6 (C-3b), 136.3 (C-10), 130.3 (C-5000),
130.2 (C-9), 126.3 (C-9a), 120.7 (C-3a), 120.7 (C-6000), 117.9 or
118.0 (C-9b or C-100), 117.7 (C-60), 116.3 (C-2000), 115.9 (C-50),
114.9 (C-6), 113.8 or 113.6 (C-8 or C-4000), 109.1 (C-20), 107.1 (C-
500), 100.4 (C-300), 93.8 (C-11), 89.5 (C-2), 56.1 or 56.2 (C-30-OMe
4.3.9. (2,3-trans)-2-(4-Hydroxy-3-methoxyphenyl)-3-hydroxymethyl-
10-methoxy-2,3,4,5-tetrahydro-phenanthro[2,1-b]furan-7-ol (29)
20
Brown amorphous powder; [
(MeOH) kmax (log
(KBr) mmax 3425, 2920, 2850, 1610, 1516, 1454, 1350, 1286, 1238,
1199, 1116, 1028, 548 cmꢀ1 1H NMR (CD3OD, 400 MHz) d 8.00
a
]
+5 (c 0.133, MeOH); UV
D
e
) 205 (4.39), 283 (4.00), 306 (3.81) nm; IR
or C-10-OMe), 55.5 (400-OMe), 52.2 (C-3), 38.7 (C-a0), 36.7 (C-
a),
32.1 (C-700), 30.9 (C-5), 28.1 (C-4); HRESIMS at m/z 669.2470
;
[M+Na]+ (calcd for [C40H38O8Na]+ 669.2464).
(1H, d, J = 9.2 Hz, H-9), 6.90 (1H, d, J = 2.0 Hz, H-20), 6.79 (1H, dd,
J = 8.2, 2.0 Hz, H-60), 6.74 (1H, d, J = 8.2 Hz, H-50), 6.63 (1H, d,
J = 2.8 Hz, H-6), 6.62 (1H, dd, J = 9.2, 2.8 Hz, H-8), 6.55 (1H, s, H-
11), 5.65 (1H, d, J = 3.0 Hz, H-2), 3.85 (1H, m, H-100a), 3.85 (3H, s,
10-OMe), 3.80 (3H, s, 30-OMe), 3.59 (1H, m, H-100b), 3.46 (1H, m,
H-3), 2.62 (4H, m, H-4/5); 13C NMR (CD3OD, 125 MHz) d 160.6
(C-11a), 159.4 (C-10), 156.1 (C-7), 149.0 (C-30), 147.1 (C-40), 140.2
(C-5a), 137.5 (C-3b), 135.7 (C-10), 130.2 (C-9), 126.1 (C-9a), 119.0
(C-60),117.9 (C-9b), 116.7 (C-3a), 116.1 (C-50), 114.9 (C-6), 113.7
(C-8), 110.0 (C-20), 93.8 (C-11), 88.7 (C-2), 64.9 (C-100), 56.3 (C-30-
OMe), 56.2 (C-10-OMe), 54.6 (C-3), 30.9 (C-5), 27.9 (C-4); HREIMS
at m/z 420.1572 [M]+ (calcd for C25H24O6 420.1573).
4.4. Alkaline hydrolysis of 2, 3, 5, 6
To each compound (15–20 mg) was individually added 3%
aqueous NaOH solution (5 mL) with the mixture stirred at room
temperature for 2 h. The reaction mixture was then acidified to
pH 4 by 2 N HCl and partitioned with EtOAc. The EtOAc phase
was concentrated under reduced pressure to give (2R)-2-hydro-
xy-2-(2-methylpropyl) butanedioic acid from 2 and 3 (5 mg,
6 mg), (2R)-2-benzyl-2-hydroxysuccinic Acid from 5 and 6 (4 mg,
6 mg). The 1H NMR, ESIMS and optical rotation data of (2R)-2-hy-
droxy-2-(2-methylpropyl) butanedioic acid (Yin et al., 2010) and
(2R)-2-benzyl-2-hydroxysuccinic acid (El Bialy et al., 2005) were
identical to those reported in the literature.
4.3.10. (2,3-trans)-3-[(2,7-Dihydroxy-4-methoxy-phenanthren-1-
yl)methyl]-2-(4-hydroxy-3-methoxyphenyl)-10-methoxy-2,3,4,5-
tetrahydro-phenanthro[2,1-b]furan-7-ol (32)
4.5. Cytotoxicity assay
20
Brown amorphous powder; [
(MeOH) kmax (log
315 (4.22) nm; IR (KBr) mmax 3431, 2931, 2840, 1612, 1583, 1516,
1462, 1381, 1352, 1284, 1232, 1200, 1130, 1028, 542, 476 cmꢀ1
a
]
+5 (c 0.175, MeOH); UV
D
e) 205 (4.84), 250 (4.63), 265 (4.75), 300 (4.39),
Cytotoxicities against the A549 and Bel7402 tumor cell lines
were evaluated by the MTT method according to the protocols de-
scribed (Alley et al., 1988), with bufalin and 0.5% DMSO (aq) as a
positive and negative control respectively.
;
1H NMR (CD3OD, 400 MHz) d 9.42 (1H, d, J = 9.3 Hz, H-500), 8.03
(1H, d, J = 9.2 Hz, H-9), 7.95 (1H, d, J = 9.3 Hz, H-1000), 7.64 (1H, d,
J = 9.3 Hz, H-900), 7.17 (1H, d, J = 2.8 Hz, H-800), 7.11 (1H, dd,
J = 9.3, 2.8 Hz, H-600), 6.93 (1H, s, H-300), 6.66 (1H, d, J = 2.4 Hz, H-
6), 6.64 (1H, dd, J = 2.4, 9.2 Hz, H-8), 6.60 (1H, s, H-11), 6.47 (1H,
d, J = 8.2 Hz, H-50), 6.38 (1H, dd, J = 8.2, 2.0 Hz, H-60), 5.55 (1H, d,
J = 2.4 Hz, H-2), 5.48 (1H, d, J = 2.0 Hz, H-20), 4.09 (3H, s, 400-OMe),
3.87 (3H, s, 10-OMe), 3.70 (1H, m, H-3), 3.42 (2H, m, H-1100), 2.95
(3H, s, 30-OMe), 2.80 (2H, m, H-4), 2.67 (2H, m, H-5); 13C NMR (CD3-
OD, 125 M) d 160.3 (C-11a), 159.1 (2C, C-10/400), 156.1 (C-7), 155.5
(C-700), 154.5 (C-200), 148.6 (C-30), 146.1 (C-40), 140.3 (C-5a), 137.4
(C-3b), 136.4 (C-10), 134.2 (C-800a), 134.0 (C-1000a), 130.6 (C-500),
130.2 (C-9), 129.0 (C-900), 126.4 (C-9a), 125.9 (C-400b), 123.9 (C-
1000), 120.6 (C-3a), 117.9 (C-9b), 117.6 (C-600), 117.0 (C-60), 116.8
(C-400a), 115.5 (C-50), 115.0 (C-6), 113.6 (C-8), 113.4 (C-100), 112.1
(C-800), 108.4 (C-20), 100.2 (C-300), 93.8 (C-11), 88.6 (C-2), 56.2 or
56.0 (C-10-OMe or C-400-OMe), 55.4 (C-30-OMe), 53.1(C-3), 31.0
(C-5), 30.9 (C-1100), 28.2 (C-4); HRESIMS at m/z 641.2137 [MꢀH]ꢀ
(calcd for [C40H33O8]ꢀ 641.2175).
Acknowledgments
This study was financially supported by the Twelfth Five-Year
National Science
&
Technology Support Program
(No.:
2012BAI29B06) and Major Projects of Knowledge Innovation Pro-
gram of the Chinese Academy of Sciences (No.: KSCX2-YW-R-
166). This work was also partially supported by National Science
& Technology Major Project ‘‘Key New Drug Creation and Manufac-
turing Program’’, China (No.: 2011ZX09307-002-03).
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.phytochem.2013.
06.001.
References
4.3.11. (2,3-trans)-3-[2-Hydroxy-6-(3-hydroxyphenethyl)-4-
methoxybenzyl]-2-(4-hydroxy-3-methoxyphenyl)-10-methoxy-
2,3,4,5-tetrahydro-phenanthro[2,1-b]furan-7-ol (33)
Ali, Z., Tanaka, T., Iliya, I., Iinuma, M., Furusawa, M., Ito, T., Nakaya, K.-I., Murata, J.,
Darnaedi, D., 2003. Phenolic constituents of Gnetum klossii. J. Nat. Prod. 66, 558–
560.
Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L.,
Abbott, B.J., Mayo, J.G., Shoemaker, R.H., Boyd, M.R., 1988. Feasibility of drug
23
Brown amorphous powder; [
(MeOH) kmax (log
nm; IR (KBr) mmax 3423, 2921, 2852, 2517, 2158, 1612, 1454,
a
]
ꢀ2.04 (c 0.049, MeOH); UV
D
e
) 206 (4.66), 281 (4.06), 306 (3.81), 316 (3.66)
screening with panels of human tumor cell lines using
tetrazolium assay. Cancer Res. 48, 589–601.
a microculture
1286, 1200, 978, 598 cmꢀ1 1H NMR (CD3OD, 400 MHz) d 8.00
;
Baek, N.-I., Choi, S.Y., Park, J.K., Cho, S.-W., Ahn, E.-M., Jeon, S.G., Lee, B.R., Bahn, J.H.,
Kim, Y.K., Shon, I.H., 1999. Isolation and identification of succinic semialdehyde
dehydrogenase inhibitory compound from the rhizome of Gastrodia elata
Blume. Arch. Pharm. Res. 22, 219–224.
(1H, d, J = 8.4 Hz, H-9), 7.04 (1H, t, J = 7.6, 8.4 Hz, H-5000), 6.62
(1H, overlapped, H-8), 6.61 (1H, overlapped, H-50), 6.60 (1H,

276
Y. Wang et al. / Phytochemistry 94 (2013) 268–276
Bai, L., Kato, T., Inoue, K., Yamaki, M., Takagi, S., 1991. Blestrianol A, B and C,
biphenanthrenes from Bletilla striata. Phytochemistry 30, 2733–2735.
Bai, L., Kato, T., Inoue, K., Yamaki, M., Takagi, S., 1993. Stilbenoids from Bletilla
striata. Phytochemistry 33, 1481–1483.
Bai, L., Masukawa, N., Yamaki, M., Takagi, S., 1998. Four stilbenoids from Pleione
bulbocodioides. Phytochemistry 48, 327–331.
Broering, T.J., Morrow, G.W., 1999. Oxygenated dihydrophenanthrenes via quinol
acetals: a brief synthesis of orchinol. Synth. Commun. 29, 1135–1142.
Dong, H.-L., Guo, S.-X., Wang, C.-L., Yang, J.-S., Xiao, P.-G., 2007. Advances in studies
on chemical constituents in plants of Pseudobulbus Cremastrae seu Pleiones and
their pharmacological activities. Chin. Tradit. Herb. Drugs 38, 1734–1738.
Dong, H.-L., Wang, C.-L., Li, Y., Guo, S.-X., Yang, J.-S., 2010. Complete assignments of
1H and 13C NMR data of three new dihydrophenanthrofurans from Pleione
yunnanensis. Magn. Reson. Chem. 48, 256–260.
Mao, T.-F., Liu, T., Liu, Z.Y., Zhu, G.S., Huang, Y.H., 2007. Rapid propagation of
Cremastra appendiculata in vitro. J. Chin. Med. Mater. 30, 1057–1059.
Matsuda, H., Morikawa, T., Xie, H.-H., Yoshikawa, M., 2004. Antiallergic
phenanthrenes and stilbenes from the tubers of Gymnadenia conopsea. Planta
Med. 70, 847–855.
Nozaki, H., Hayashi, K.-I., Kido, M., Kakumoto, K., Ikeda, S., Matsuura, N., Tani, H.,
Takaoka, D., Iinuma, M., Akao, Y., 2007. Pauferrol A, a novel chalcone trimer with
a cyclobutane ring from Caesalpinia ferrea mart exhibiting DNA topoisomerase II
inhibition and apoptosis-inducing activity. Tetrahedron Lett. 48, 8290–8292.
Otsuka, H., Takeuchi, M., Inoshiri, S., Sato, T., Yamasaki, K., 1989. Phenolic
compounds from Coix lachryma-jobi var. Ma-yuen. Phytochemistry 28, 883–886.
Qi, Y.X., Cao, M.L., Wang, X.D., Gao, Y.S., Xia, Z.L., 2006. Determination and
comparison of 3 active components in the cultivated salvia miltiorrhiza bge and
wild salvia miltiorrhiza bge in taisha. Pharm. Biotechnol. 13, 279–282.
Shim, J.S., Kim, J.H., Lee, J., Kim, S.N., Kwon, H.J., 2004. Anti-angiogenic activity of a
homoisoflavanone from Cremastra appendiculata. Planta Med. 70, 171–173.
Takagi, S., Yamaki, M., Inoue, K., 1983. Antimicrobial agents from Bletilla striata.
Phytochemisty 22, 1011–1015.
El Bialy, S.A.A., Braun, H., Tietze, L.F., 2005. Enantioselective synthesis of
a-
alkylmalates as the pharmacophoric group of several natural alkaloids and
glycosides. Eur. J. Org. Chem. 2005, 2965–2972.
Guo, D.-G., Wang, H., Zhu, X.-H., Zhou, Y., 2009. Initial study on the in vitro
antibacterial
Lishizhen Med. Mater. Med. Res. 220, 594–595.
activity
of
Pseudobulbus
Cremastrae
seu
Pleiones.
Toh, C.-A., 1994. Pharmacognostical studies on Orchidaceae plants. Korean J.
Pharmacogn. 25, 293–304.
Hahn, K.N., Fadeyi, O.O., Cho, H.P., Lindsley, C.W., 2012. Synthesis and biological
evaluation of cremastrine and an unnatural analogue. Tetrahedron Lett. 53,
3577–3580.
Xia, W.-B., Xue, Z., Li, S., Wang, S.-J., Yang, Y.-C., He, D.-X., Ran, G.-L., Kong, L.-Z., Shi,
J.-G., 2005. Chemical constituents from tuber of Cremastra appendiculata. China
J. Chin. Mater. Med. 30, 1827–1830.
Honda, C., Yamaki, M., 2001. Stilbenoids from Dendrobium plicatile. Nat. Med. 55,
68–70.
Xie, Z.-W., Fan, C.-S., Zhu, Z.-Y., et al., 1996. Compilation of National Herbal
Medicine, second ed. The People’s Medical Publishing House, Beijing, pp. 117–
119.
Xue, Z., Li, S., Wang, S.-J., Wang, Y.-H., Yang, Y.-C., Shi, J.-G., He, L., 2006. Mono-, bi-,
and triphenanthrenes from the tubers of Cremastra appendiculata. J. Nat. Prod.
69, 907–913.
Xue, Z., Li, S., Wang, S.-J., Yang, Y.-C., He, D.-X., Ran, G.-L., Kong, L.-Z., Shi, J.-G., 2005.
Studies on chemical constituents from the corm of Cremastra appendiculata.
China J. Chin. Mate. Med. 30, 511–513.
Yamaki, M., Bai, L., Inoue, K., Takagi, S., 1989. Biphenanthrenes from Bletilla striata.
Phytochemistry 28, 3503–3505.
Ikeda, Y., Nonaka, H., Furumai, T., Igarashi, Y., 2005. Cremastrine, a pyrrolizidine
alkaloid from Cremastra appendiculata. J. Nat. Prod. 68, 572–573.
Iliya, I., Ali, Z., Tanaka, T., Iinuma, M., Furusawa, M., Nakaya, K., Murata, J., Darnaedi,
D., Matsuura, N., Ubukata, M., 2003. Stilbene derivatives from Gnetum gnemon
Linn.. Phytochemistry 62, 601–606.
Kim, J.H., Kim, K.H., Kim, J.H., Yu, Y.S., Kim, Y.-M., Kim, K.-W., Kwon, H.J., 2007.
Homoisoflavanone inhibits retinal neovascularization through cell cycle arrest
with decrease of cdc2 expression. Biochem. Biophys. Res. Commun. 362, 848–
852.
Kizu, H., Kaneko, E., Tomimori, T., 1999. Studies on nepalese crude drugs: XXVI.
Chemical constituents of Panch aunle, the roots of Dactylorhiza hatagirea D. Don.
Chem. Pharm. Bull. 47, 1618–1625.
Krautwurst, K.-D., Tochtermann, W., 1981. Polycyclische verbindungen: XX. Eine
einfache Orchinol-synthese. Eur. J. Inorg. Chem. 114, 214–219.
Leong, Y.-W., Kang, C.-C., Harrison, L.J., Powell, A.D., 1997. Phenanthrenes,
dihydrophenanthrenes and bibenzyls from the orchid Bulbophyllum
vaginatum. Phytochemistry 44, 157–165.
Li, S., Xue, Z., Wang, S.-J., Yang, Y.-C., Shi, J.-G., 2008. Terpenoids from the tuber of
Cremastra appendiculata. J. Asian Nat. Prod. Res. 10, 677–683.
Liu, J., Yu, Z.-B., Ye, Y.-H., Zhou, Y.-W., 2008. Chemical constituents from the tuber of
Cremastra appendiculata. Acta Pharm. Sin. 43, 181–184.
Majumder, P.L., Banerjee, S., 1988. Structure of flavanthrin, the first dimeric 9,10-
dihydrophenanthrene derivative from the orchid Eria flava. Tetrahedron 44,
7303–7308.
Yamaki, M., Bai, L., Inoue, K., Takagi, S., 1990. Benzylphenanthrenes from Bletilla
striata. Phytochemistry 29, 2285–2287.
Yang, H., Sung, S.-H., Kim, Y.-C., 2007. Antifibrotic phenanthrenes of Dendrobium
nobile stems. J. Nat. Prod. 70, 1925–1929.
Yao, C.-S., Lin, M., Wang, L., 2006. Isolation and biomimetic synthesis of anti-
inflammatory stilbenolignans from Gnetum cleistostachyum. Chem. Pharm. Bull.
54, 1053–1057.
Yin, R.-J., Huang, X.-F., Kong, L.-Y., 2010. Constituents from the heartwoods of
Osmanthus armatus. Nat. Prod. Res. 24, 948–952.
Yu, Y., Lang, Q.B., Chen, Z., Li, B., Yu, C.Q., Zhu, D.Z., Huang, X.Q., Zhai, X.F., Ling, C.Q.,
2009. Prognostic analysis of transarterial chemoembolization combined with a
traditional Chinese herbal medicine formula for treatment of unresectable
hepatocellular carcinoma. Chin. Med. J. 122, 1990–1995.
Zhang, M.-S., Peng, S.-W., Wang, W., 2010. Macro research on growth and
development of Cremastra appendiculata (D. Don) Makino (Orchidaceae). J.
Med. Plants Res. 4, 1837–1842.
Majumder,
P.L.,
Basak,
M.,
1990.
Cirrhopetalin,
a
phenanthrene
derivative from Cirrhopetalum andersonii. Phytochemistry 29, 1002–1004.
Majumder, P.L., Guha, S., Sen, S., 1999. Bibenzyl derivatives from the orchid
Dendrobium amoenum. Phytochemistry 52, 1365–1369.
Zhang, Y., Huang, B., Zhao, Z., Zhou, Y., 2011. Study on the chemical constituents
from the ethyl acetate extracts of Cremastra appendiculata. J. Chin. Med. Mater.
34, 1882–1883.
Majumder, P.L., Kar, A., Banerjee, S., Pal, A., Joardar, M., 1997. Application of 2D NMR
spectroscopy in structural elucidation of some phenanthrene derivatives from
Indian orchidaceae plants. J. Indian Chem. Soc. 74, 908–916.
Zi, J.-C., Li, S., Liu, M.-T., Gan, M.-L., Lin, S., Song, W.-X., Zhang, Y.-L., Fan, X.-N., Yang,
Y.-C., Zhang, J.-J., Shi, J.-G., Di, D.-L., 2008. Glycosidic constituents of the tubers
of Gymnadenia conopsea. J. Nat. Prod. 71, 799–805.
Majumder, P.L., Lahiri, S., 1990. Lusianthrin and lusianthridin, two stilbenoids from
the orchid Lusia indivisa. Phytochemistry 29, 621–624.