Five new benzylphenanthrenes from Cremastra appendiculata

Article abstract of DOI:10.1016/j.fitote.2015.03.003

Five new benzylphenanthrenes, cremaphenanthrenes L-P (1-5) were isolated from the EtOAc-soluble extract of the tubers of Cremastra appendiculata. Their structures were elucidated based on the spectroscopic data and chemical methods. Compounds 1-5 were tested in vitro for their cytotoxic effects against colon (HCT-116), cervix (Hela), and breast (MCF-7 and MDA-MB-231) cancer cell lines. Compound 1 showed moderate cytotoxic activities against HCT-116, MCF-7, and MDA-MB-231 cancer cell lines with IC₅₀ values ranging from 15.84 to 24.18 μM and weak cytotoxicity to Hela cell line with IC₅₀ value of 68.81 μM.

Full text of DOI:10.1016/j.fitote.2015.03.003

Fitoterapia 103 (2015) 27–32
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Five new benzylphenanthrenes from Cremastra appendiculata
Liang Liu a,b,c,d, Jun Li e, Ke-Wu Zenga, Yong Jianga, Peng-Fei Tua,c,
⁎
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, PR China
b
Medical College, Yangzhou University, Yangzhou 225009, PR China
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
c
d
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, PR China
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, PR China
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Five new benzylphenanthrenes, cremaphenanthrenes L–P (1–5) were isolated from the EtOAc-
soluble extract of the tubers of Cremastra appendiculata. Their structures were elucidated based on
the spectroscopic data and chemical methods. Compounds 1–5 were tested in vitro for their
cytotoxic effects against colon (HCT-116), cervix (Hela), and breast (MCF-7 and MDA-MB-231)
cancer cell lines. Compound 1 showed moderate cytotoxic activities against HCT-116, MCF-7,
and MDA-MB-231 cancer cell lines with IC50 values ranging from 15.84 to 24.18 μM and weak
cytotoxicity to Hela cell line with IC50 value of 68.81 μM.
Received 19 December 2014
Accepted in revised form 1 March 2015
Available online 7 March 2015
Keywords:
Benzylphenanthrene
Cremastra appendiculata
Cytotoxic activity
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
The tuber of Cremastra appendiculata (D. Don) Makino
(Orchidaceae), Shancigu in Chinese, is a famous traditional
Chinese medicine with a long history for treating cancers [1].
Previous phytochemical investigations suggested that phen-
anthrenes were the main constituents in this plant, which
have been reported to possess cytotoxic activities in vitro
[2–5]. As a continuing program to search for cytotoxic
phenanthrenes from C. appendiculata, UV-directed fraction-
ation was carried out to isolate phenanthrenes from the EtOAc-
soluble extract of this plant, which led to the isolation of five
new benzylphenanthrenes, namely cremaphenanthrenes L–P
(1–5) (Fig. 1). In this paper, we report the isolation and
structural elucidation as well as the cytotoxic activities of five
new benzylphenanthrenes.
2.1. General experimental procedures
UV spectra were run on a Shimadzu UV-2450 spectrometer.
IR spectra were recorded on a Nicolet NEXUS-470 FTIR
spectrometer. Optical rotations were measured on a Rudolph
AUTOPOL IV polarimeter. HR-ESI-MS was determined by a
Bruker APEX IVFT-MS (7.0 T) and a Waters Xevo G2 Q-TOF/
YCA mass spectrometers. NMR spectra were recorded on a
Varian Inova-500 and a Bruker Avance-600 FT NMR spec-
trometers. Semi-preparative HPLC was run on a Dionex
Ultimate 3000 instrument equipped with 170U UV Detector
and an ODS column (Thermo Scientific BDS-C18 column,
250 mm × 10 mm, 5 μm). GC analyses were performed on a
VARIAN CP-3800 instrument (DB-5, 0.25 mm × 30 m).
Column chromatography (CC) was performed using silica
gel (200–300 mesh, Qingdao Marine Chemistry Ltd., China),
Sephadex LH-20 (Amersham Biosciences, Sweden) and ODS
C18 (40–63 μm, Merck, Germany). TLC was carried out on
glass precoated silica gel (GF254) plates. Spots were visualized
under UV light and detected by spraying with 10% H2SO4 in
EtOH followed by heating.
⁎
Corresponding author at: State Key Laboratory of Natural and Biomimetic
Drugs, School of Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, PR China. Tel./fax: +86 10 82802750.
E-mail address: pengfeitu@vip.163.com (P.-F. Tu).
http://dx.doi.org/10.1016/j.fitote.2015.03.003
0367-326X/© 2015 Elsevier B.V. All rights reserved.

28
L. Liu et al. / Fitoterapia 103 (2015) 27–32
Fig. 1. Chemical structures of compounds 1–5.
2.2. Plant material
2.3.1. Cremaphenanthrene L (1)
Brown amorphous powder; IR (KBr) νmax (cm−1) 3356,
2934, 1611, 1514, 1454, 1368, 1277, and 1230; UV λmax (MeOH)
203 (0.75) and 264 (0.68) nm; negative ion HR-ESI-MS m/z
389.1398 [M−H]− (calcd for C24H21O5, 389.1395); 1H NMR
and 13C NMR data: Tables 1 and 2.
The tubers of C. appendiculata were collected in Yunnan
Province in June 2011. The plant materials were identified
by one of the authors (Prof. P.F. Tu), and a voucher specimen
(No. DJL20110628) was deposited at the Herbarium of Peking
University Modern Research Center for Traditional Chinese
Medicine.
2.3.2. Cremaphenanthrene M (2)
Brown amorphous powder; [α]20 D +35.5 (c 0.1, MeOH);
IR (KBr) νmax (cm−1) 3440, 2961, 1611, and 1509; UV λmax
(MeOH) 204 (0.78) and 263 (0.83) nm; negative ion HR-ESI-
MS, m/z 573.1554 [M+Cl]− (calcd for C29H30ClO10, 573.1533);
1H NMR and 13C NMR data: Tables 1 and 2.
2.3. Extraction and isolation
The powdered, dry tubers of C. appendiculata (30 kg) were
extracted with 95% EtOH (100 L) three times. After filtration
and evaporation, a dark brown residue was obtained. The
residue was suspended in distilled water and partitioned
successively with petroleum ether (PE, 3×10 L), EtOAc (3×10 L),
and n-BuOH (3×15 L). The EtOAc fraction was separated by a
silica gel CC (200–300 mesh) eluting with a gradient of
CHCl3:MeOH (50:1–0:100, v/v) to give 9 fractions (Fr. A–I)
based on TLC analysis. Fr. A (21.6 g) was chromatographed on a
silica gel column using a PE:acetone (5:2–0:100) gradient to get 13
fractions (Fr. A1–A13). Fr. A9 (1.8 g) was subjected to CC on a
Sephadex LH-20 eluting with CHCl3:MeOH (2:1) to yield 10
fractions (A9-1 to A9-10). Fr. A9-9 (58.5 mg) was purified by semi-
preparative HPLC, washing with MeOH:H2O (7:3, 2.0 mL/min) as
mobile phase to yield compound 1 (3.5 mg). Fr. G (5.2 g) was
applied to a RP-C18 CC eluting with a MeOH–H2O (1:1–100:0)
gradient to afford 10 fractions (Fr. G1–G10). Fr. G5 (1.34 g) was
further purified on a Sephadex LH-20 using MeOH to give 11
fractions (Fr. G5-1 to G5-11). Fr. G5-8 (75.0 mg) was purified
by semi-preparative HPLC, using CH3CN–H2O (1:4, 2.0 mL/min)
as mobile phase, to yield compound 2 (5.4 mg) and 3 (8.8 mg).
Fr. G5-9 (35.3 mg) and Fr. G5-11 (24.1 mg) was respectively
purified by semi-preparative HPLC, both eluting with CH3CN–
H2O (6:19, 2.0 mL/min) as mobile phase, to obtain compound
4 (2.3 mg) and compound 5 (2.0 mg), respectively.
2.3.3. Cremaphenanthrene N (3)
Brown amorphous powder; [α]20 D −27.9 (c 0.15, MeOH);
IR (KBr) νmax (cm−1) 3399, 2924, and 1634; UV λmax (MeOH)
204 (0.90) and 263 (0.97) nm; negative ion HR-ESI-MS m/z
573.1520 [M+Cl]− (calcd for C29H30CIO10, 573.1533); 1H NMR
and 13C NMR data: Tables 1 and 2.
2.3.4. Cremaphenanthrene O (4)
Brown amorphous powder; [α]20 D −11.5 (c 0.12, MeOH);
IR (KBr) νmax (cm−1) 3392, 2920, 1613, 1460, and 1033; UV
λmax (MeOH) 204(0.97) and 264 (0.90) nm; positive ion
HR-ESI-MS m/z 577.1703 [M+Na]+ (calcd for C29H30O11Na,
577.1686); 1H NMR and 13C NMR data: Tables 1 and 2.
2.3.5. Cremaphenanthrene P (5)
Brown amorphous powder; [α]20 D −14.3 (c 0.07, MeOH);
IR (KBr) νmax (cm−1) 3452, 2965, 1509, and 1236; UV λmax
(MeOH) 204 (1.17), 262 (0.93), and 285 (0.74) nm; positive ion
HR-ESI-MS m/z 555.1870 [M+H]+ (calcd for C29H31O11,
555.1866); 1H NMR and 13C NMR data: Tables 1 and 2.

L. Liu et al. / Fitoterapia 103 (2015) 27–32
29
Table 1
1H NMR data of compounds 1–5.
Position
1a
2a
3a
4b
5b
3
6.96 s
6.97 s
7.33 s
6.93 s
6.93 s
5
6
8
9
10
2′
5′
6′
7′
9.38 d (9.5)
7.18 dd (9.5, 3.0)
7.33 d (3.0)
7.65 d (9.5)
7.81 d (9.5)
6.86 d (2.5)
6.57 d (8.5)
6.46 dd (8.5,2.5)
4.24 s
9.41 d (9.5)
7.29 dd (9.5, 3.0)
7.44 d (3.0)
7.62 d (9.5)
7.82 d (9.5)
6.86 d (2.5)
6.57 d (8.5)
6.46 dd (8.5, 2.5)
4.24 s
9.45 d (9.5)
7.09 dd (9.5, 3.0)
7.13 d (3.0)
7.51 d (9.5)
7.82 d (9.5)
6.80 d (2.5)
6.62 d (8.5)
6.61 dd (8.5, 2.5)
4.39 d (16.0)
4.54 d (16.0)
4.14 s
9.19 d (9.6)
7.14 d (9.6)
8.90 d (9.6)
7.43 d (9.6)
8.32 d (9.6)
7.78 d (9.6)
6.85 d (1.8)
6.55 d (7.8)
6.43 dd (7.8, 1.8)
4.18 d (15.6)
4.25 d (15.6)
4.02 s
8.00 d (9.6)
7.77 d (9.6)
6.85 d (1.8)
6.55 d (7.8)
6.44 dd (7.8, 1.8)
4.22 s
4-OCH3
7-OCH3
3′-OCH3
2-OH
4′-OH
1″
2″
3″
4″
5″
4.04 s
3.89 s
3.67 s
9.68 s
8.56 s
4.05 s
3.68 s
4.01 s
3.67 s
3.71 s
3.67 s
5.02 d (7.0)
3.31–3.38 m c
3.31–3.40 m c
3.20–3.22 m
3.39–3.41 m
3.73 br d
5.04 d (7.0)
3.54–3.58 m
3.50–3.54 md
3.36–3.41 m
3.50–3.54 md
3.94 br d
4.60 d (7.8)
3.43–3.46 m
3.24–3.29 me
3.23–3.27 me
3.16–3.20 m
3.62 br d
4.70 d (7.8)
3.33–3.38 f
3.26–3.33
3.18–3.20 m
3.33–3.37 f
3.76 br d
6″
3.48–3.51 m
3.68–3.72 m
3.49–3.52 m
3.50–3.52 m
c,d,e,fOverlapped signal.
a
1H NMR data were measured at 500 MHz in DMSO-d6 for 1–2, in CD3OD for 3.
1H NMR data were measured at 600 MHz in DMSO-d6 for 4–5.
b
2.4. Complete acid hydrolysis and GC analysis of compounds 2–5
showed an ion at m/z 389.1398 [M−H]− and established
the molecular formula as C24H22O5. The IR spectrum showed
absorption bands for hydroxy (3356 cm−1), methylene
Compound 2–5 (1.0 mg, respectively) was stirred at 100 °C
for 8 h with 2 M CF3COOH (EtOH:H2O, 1:1, 2 mL). The solution
was cooled to room temperature and concentrated under
reduced pressure. The residue was then redissolved in 2 mL
pyridine with 0.1 M L-cysteine methyl ester hydrochloride and
stirred at 60 °C for 1.5 h. After being cooled and concentrated,
the mixture was treated with trimethylchlorosilane and
hexamethyl-disilazane (1:2 v/v, 0.5 mL) in pyridine (2 mL),
followed by stirring at 60 °C for 0.5 h. Then, the solution was
concentrated to dryness and added with H2O (2 × 3 mL),
followed by extraction with n-hexane (2 × 3 mL) [6]. The
n-hexane layer was analyzed by GC using a DB-5 column
(0.25 mm × 30 m). The temperature of both the injector
and detector was 220 °C. A temperature gradient system
was applied to the oven, starting at 140 °C for 1 min
and increasing up to 250 °C at a rate of 4 °C/min. Peaks of
the sugar derivatives were identified by comparison with
retention times to authentic sample of D-glucose (19.28 min)
treated as above.
Table 2
13C NMR data of compounds 1–5.
Carbon
1a
2a
3a
4b
5b
1
2
3
4
5
6
7
8
9
10
4a
4b
8a
10a
1′
2′
113.2
153.0
99.5
113.3
153.2
99.5
118.9
154.7
100.9
159.3
131.1
117.6
156.0
112.2
128.7
124.9
118.9
125.6
135.0
133.8
135.0
113.4
149.0
145.4
116.0
121.9
31.4
113.1
153.0
99.5
113.2
153.4
99.4
156.9
128.8
116.4
156.0
108.4
127.4
123.6
114.2
124.6
132.0
132.5
132.5
112.7
147.3
144.3
115.2
120.1
29.5
156.9
128.7
116.9
154.1
112.3
127.4
123.5
114.0
125.4
131.8
132.5
132.7
112.7
147.3
144.3
115.2
120.1
29.5
157.0
125.1
117.2
145.4
138.8
121.5
123.3
114.0
124.3
126.4
132.2
132.5
112.6
147.3
144.3
115.2
120.1
29.5
157.3
118.6
116.9
140.3
140.9
120.9
122.2
114.0
126.8
121.2
133.0
132.5
112.6
147.3
144.3
115.2
120.1
29.6
3′
4′
5′
6′
2.5. Cytotoxic bioassay
7′
Cytotoxic activity was evaluated by MTT assay as previously
reported [2]. Paclitaxel was served as positive control. Data are
presented as mean for three independent experiments. Each
concentration of compounds was tested in three parallel wells.
IC50 values were calculated using Microsoft Excel software.
4-OCH3
7-OCH3
3′-OCH3
1″
2″
3″
4″
5″
6″
55.8
55.0
55.5
55.6
56.5
55.5
55.5
55.5
100.5
73.3
76.7
69.7
77.1
60.7
56.4
104.1
75.3
78.5
71.9
78.7
63.0
55.6
106.3
74.1
76.1
69.5
77.2
60.6
55.6
103.6
73.5
75.7
69.9
77.4
60.4
3. Results and discussion
a
13C NMR data were measured at 125 MHz in DMSO-d6 for 1–2, in CD3OD for 3.
13C NMR data were measured at 150 MHz in DMSO-d6 for 4–5.
Compound 1 (Tables 1 and 2, Fig. 2) was obtained as
a brown amorphous powder. The negative ion HR-ESI-MS
b

30
L. Liu et al. / Fitoterapia 103 (2015) 27–32
structure of
1 was established as 1-(3′-methoxy-4′-
hydroxybenzyl)-4,7-dimethoxyphenanthrene-2-ol, and named
as cremaphenanthrene L.
Compound 2 (Tables 1 and 2, Fig. 3) was present to be a
brown amorphous powder, with the molecular formula of
C29H30O10 determined from the negative ion HR-ESI-MS at
m/z 573.1554 [M+Cl]−. In the 13C NMR spectrum, six carbon
signals (δC 100.5, 73.3, 76.7, 69.7, 77.1, and 60.7) assigned
to a glucopyranosyl moiety were observed. The coupling
constant of the anomeric proton (7.0) found in 1H NMR
spectrum indicated it to be a β-glc, acid hydrolysis of 2 gave
D-glucose, confirmed by comparison with an authentic
sample. Meanwhile, twenty aromatic, one methylene, and
two methoxy carbon signals in the 13C NMR spectrum
suggested the presence of a benzylphenanthrene moiety,
whose NMR spectroscopic data were quite similar to those of
1-(3′-methoxy-4′-hydroxybenzyl)-4-methoxyphenanthrene-
2,7-diol [4], the existence of the same benzylphenanthrene
moiety was further supported by the HSQC, HMBC, and NOESY
experiments. The glucopyranosyl moiety was determined to
be located at C-7 based on the HMBC correlation between
H-1″ (δH 5.02) and C-7 (δC 154.1) and the NOESY correla-
tions between H-1″ (δH 5.02) and H-6 (δH 7.29), H-8 (δH
7.44). Thus, the structure of 2 was elucidated as 1-(3′-methoxy-
4′-hydroxybenzyl)-4-methoxyphenanthrene-2-ol-7-O-β-D-
glucopyranoside, and named as cremaphenanthrene M.
Compound 3 (Tables 1 and 2, Fig. 3) was isolated as a brown
amorphous powder. The molecular formula of 3 was determined
as C29H30O10 on the basis of the negative ion HR-ESI-MS at m/z
573.1520 [M+Cl]−. The same molecular formula and the highly
similar IR, UV, 1D NMR spectra of 3 with those of 2 indicated that
3 was also a benzylphenanthrene glycoside. The main difference
was the linkage position of glucopyranosyl moiety. The HMBC
correlations between H-1″ (δH 5.02) and C-2 (δC 154.7) as well as
the NOESY correlations between H-1″ (δH 5.02) and H-3 (δH
7.33) confirmed that the glucopyranosyl moiety was connected
to C-2. The β-D-glc unit was further deduced by the coupling
constant of the anomeric proton (7.0) and acid hydrolysis.
Fig. 2. Key HMBC correlations of compound 1.
(2934 cm−1), and aromatic functional groups (1611, 1514,
1454, 1368, 1277, and 1230 cm−1), respectively [7]. The UV
spectrum showed absorption maxima at 203 and 264 nm.
The 1H NMR spectrum displayed two exchangeable phenolic
hydroxy singlet signals at δH 9.68 (1H, s, 2-OH) and 8.56
(1H, s, 4′-OH); nine aromatic proton signals consisting of
one singlet signal at δH 6.96 (1H, s, H-3), a pairs of doublet
signals at δH 7.65 (1H, d, J = 9.5 Hz, H-9) and 7.81 (1H, d, J =
9.5 Hz, H-10), two sets of ABX coupling systems at δH 9.38
(1H, d, J = 9.5 Hz, H-5), 7.18 (1H, dd, J = 9.5, 3.0 Hz, H-6),
7.33 (1H, d, J = 3.0 Hz, H-8), 6.86 (1H, d, J = 2.5 Hz, H-2′),
6.57 (1H, d, J = 8.5 Hz, H-5′), and 6.46 (1H, dd, J = 8.5,
2.5 Hz, H-6′); three methoxy singlet signals at δH 4.04 (3H, s,
H-4-OCH3), 3.89 (3H, s, H-7-OCH3) and 3.67 (3H, s, H-3′-
OCH3); and a benzylic methylene singlet signal at δH 4.24
(2H, s, H-7′). In the 13C NMR spectrum, twenty aromatic,
one methylene, and three methoxy carbon signals were
observed. This information, especially the presence of the
deshielded proton signals δH 9.38 (H-5), indicated the
characteristics of benzylphenanthrene for compound 1.
The 1D NMR data were similar to those of 1-(3′-methoxy-
Accordingly, we declared the structure of
2 as 1-(3′-
4′-hydroxybenzyl)-4-methoxyphenanthrene-2,7-diol
[4]
methoxy-4′-hydroxybenzyl)-4-methoxyphenanthrene-7-ol-2-
O-β-D-glucopyranoside, and named as cremaphenanthrene N.
Compound 4 (Tables 1 and 2, Fig. 4) was also obtained as a
brown amorphous powder. A positive ion at m/z 577.1703
except that a methoxyl replaced the hydroxyl at C-7.
This was further confirmed by HMBC correlations be-
tween H-5 and C-7, H-7-OCH3 and C-7. Therefore, the
Fig. 3. Key HMBC and NOESY correlations of compounds 2–3.

L. Liu et al. / Fitoterapia 103 (2015) 27–32
31
[M+Na]+ was found in the HR-ESI-MS spectrum of 4,
Table 3
Cytotoxicities of compounds 1 against tumor cell lines, IC50 value (μM).
indicating the molecular formula of C29H30O11. The IR and
UV spectra of 4 were similar to those of 3. Six carbon signals
(δC 106.3, 74.1, 76.1, 69.5, 77.2, and 60.6) assignable to a
glucopyranosyl moiety were also observed in the 13C NMR
spectrum. Acid hydrolysis of 4 yielded D-glucose, identified
by comparison with an authentic sample. In the 13C NMR
spectrum, twenty aromatic, one methylene and two methoxy
carbon signals were observed. All the above data indicated 4 to
be a benzylphenanthrene glycoside. The 1H NMR spectrum
displayed eight aromatic proton signals including one set of
ABX coupling systems at δH 6.85 (1H, d, J = 1.8 Hz, H-2′), 6.55
(1H, d, J = 7.8 Hz, H-5′), and 6.43 (1H, dd, J = 7.8, 1.8 Hz, H-6′);
one singlet signal at δH 6.93 (1H, s, H-3); two pairs of doublet
signals at δH 8.32 (1H, d, J = 9.6 Hz, H-9), 7.78 (1H, d, J =
9.6 Hz, H-10), 9.19 (1H, d, J = 9.6 Hz, H-5) and 7.14 (1H, d, J =
9.6 Hz, H-6). Two methoxy singlet signals at δH 4.02 (3H, s,
H-4-OCH3) and 3.67 (3H, s, H-3′-OCH3) were also observed
in 1H NMR spectrum of 4. The substituent positions of
benzylphenanthrene moiety were deduced by 2D-NMR exper-
iments. The HMBC correlations from H-3 to C-1, C-2, C-4, and
C-4a, H-5 to C-4a, C-7, and C-8a, H-9 to C-4b, C-8, C-8a and
C-10a, H-10 to C-1, C-4a, C-8a and C-10a, H-4-OCH3 to C-4, H-2′
to C-1′, C-3′, C-4′, C-6′ and C-7′, H-5′ to C-1′, C-3′, C-4′ and C-6′,
H-6′ to C-2′, C-4′, C-5′ and C-7′, H-7′ to C-1′, C-2′, C-6′ and C-2,
H-3′-OCH3 to C-3′, The NOESY correlations between H-4-OCH3
(δH 4.02) and H-3 (δH 6.93) and H-5 (δH 9.19), H-3′-
OCH3 (δH 3.67) and H-2′ (δH 6.85), these data showed
the benzylphenanthrene moiety to be 1-(3′-methoxy-4′-
hydroxybenzyl)-4-methoxyphenanthrene-2,7,8-triol. The
glucopyranosyl moiety was determined to connect at C-8 on
the basis of the HMBC correlation between H-1″ (δH 4.60) and
C-8′ (δC 138.8) as well as the NOESY correlation between H-1″
(δH 4.60) and H-9 (δH 8.32). Subsequently, the structure
of 4 was established as 1-(3′-methoxy-4′-hydroxybenzyl)-4-
methoxyphenanthrene-2,7-diol-8-O-β-D-glucopyranoside, and
named as cremaphenanthrene O.
Compounds
HCT-116
Hela
MCF-7
MDA-MB-231
1
19.01
2.33
68.81
0.08
24.18
0.52
15.84
0.002
Paclitaxel
4, which suggested that 5 was a benzylphenanthrene glycoside.
The presence of D-glucose was elucidated by GC analysis of its
acid hydrolysis derivation. The glucopyranosyl moiety linkage
was determined to be C-7 due to the HMBC correlation
between H-1″ (δH 4.70) and C-7 (δC 140.3) as well as the
NOESY correlation between H-1″ (δH 4.70) and H-6 (δH 7.43).
Eventually, we established the structure of 5 as 1-(3′-methoxy-
4′-hydroxybenzyl)-4-methoxyphenanthrene-2,8-diol-7-O-β-D-
glucopyranoside, and named as cremaphenanthrene P.
The cytotoxicities of compounds 1–5 were evaluated by
MTT Method, using paclitaxel as a positive control. Their
cytotoxicities against colon (HCT-116), cervix (Hela), and
breast (MCF-7, MDA-MB-231) cell lines were determined.
The results (Table 3) indicated that 1 showed moderate
cytotoxic activities against HCT-116, MCF-7 and MDA-MB-
231 cancer cell lines with IC50 values ranging from 15.84 to
24.18 μM, and weak cytotoxicity to Hela cell line with IC50
value of 68.81 μM, while compounds 2–5 were inactive
(IC50 N 100 μM).
Phenanthrenes have been reported mainly from the
Orchidaceae family [8]. Our findings afford further evi-
dences that phenanthrenes can serve as the taxonomic
marker of this family [8]. Benzylphenanthrenes have been
previously reported mainly from three genera of the
Orchidaceae family: Bletilla, Gymnadenia, and Spiranthes,
all of which belong to the Orchidoideae subfamily [8,9]. The
occurrence of benzylphenanthrenes in C. appendiculata sug-
gested that the Cremastra genus could have a close genetic
relationship with the genera Bletilla, Gymnadenia, and Spiranthes.
Compound 5 (Tables 1 and 2, Fig. 4) was isolated as a brown
amorphous powder. The molecular formula of 5 was deter-
mined as C29H30O11 from the positive ion HR-ESI-MS at m/z
555.1870 [M+H]+. The molecular formula is identical to that of
4, the IR, UV, and 1D-NMR spectra of 5 were similar to those of
Acknowledgments
This research work was supported by National Key
Technology R&D Program “New Drug Innovation” of China
(Nos. 2012ZX09301002-002-002 and 2012ZX09304-005).
Fig. 4. Key HMBC and NOESY correlations of compounds 4–5.

32
L. Liu et al. / Fitoterapia 103 (2015) 27–32
tartrate benzyl ester glucosides from tubers of Cremastra appendiculata.
Appendix A. Supplementary data
Phytochemistry 2013;94:268–76.
[4] Liu L, Li J, Zeng KW, Li P, Tu PF. Three new phenanthrenes from Cremastra
appendiculata (D. Don) Makino. Chin Chem Lett 2013;24:737–9.
[5] Liu L, Ye J, Li P, Tu PF. Chemical constituents of tubers of Cremastra
appendiculata. Chin J of Chin Mater Med 2014;39:250–3.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2015.03.003.
[6] Nan ZD, Zeng KW, Shi SP, Zhao MB, Jiang Y, Tu PF. Phenylethanoid
glycosides with antiinflammatory activities from the stems of Cistanche
deserticola cultured in Tarim desert. Fitoterapia 2013;89:167–74.
[7] Matsuda H, Morikawa T, Xie HH, Yoshikawa M. Antiallergic phenanthrenes
and stilbenes from the tubers of Gymnadenia conopsea. Planta Med 2004;
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activity. Phytochemistry 2008;69:1084–110.
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China, vol. 17. Beijing: Science Press; 1999 1–4.
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