Six new dihydrobenzofuran lignans from the branches and leaves of Illicium wardii and their cytotoxic activities

Article abstract of DOI:10.1016/j.phytol.2016.07.010

Six new dihydrobenzofuran lignans, named illiciumlignans A–F (compounds 1–6), along with 15 known compounds (7–21) were isolated from the branches and leaves of Illicium wardii. The structures of 1–6 were determined using a combination of 1D and 2D NMR, HR-ESI–-MS, and CD spectroscopic data. Illiciumlignan D (4) is the first reported dihydrobenzofuran lignan arabinofuranoside that is derivatized with the arabinofuranose moiety on C-9′. Compounds 1–21 were evaluated for cytotoxic activity against four human cancer cell lines. Compounds 8, 12 and 20 exhibited significant activity against human cancer cell lines (A549, SKOV3, HepG2 and HCT116), with IC₅₀ values ranging from 2.7 to 14.9μM.

Full text of DOI:10.1016/j.phytol.2016.07.010

Phytochemistry Letters 17 (2016) 263–269
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Short communication
Six new dihydrobenzofuran lignans from the branches and leaves of
Illicium wardii and their cytotoxic activities
Feng-mei Yea, Yang-guo Xiea, Jie Rena, Ji Yeb, Yi-gong Guoa, Shi-Kai Yana, Hui-Zi Jina,
Wei-Dong Zhang
,
*
a,b,**
a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China
Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Six new dihydrobenzofuran lignans, named illiciumlignans A–F (compounds 1–6), along with 15 known
compounds (7–21) were isolated from the branches and leaves of Illicium wardii. The structures of 1–6
were determined using a combination of 1D and 2D NMR, HR-ESI–-MS, and CD spectroscopic data.
Illiciumlignan D (4) is the first reported dihydrobenzofuran lignan arabinofuranoside that is derivatized
with the arabinofuranose moiety on C-90. Compounds 1–21 were evaluated for cytotoxic activity against
four human cancer cell lines. Compounds 8,12 and 20 exhibited significant activity against human cancer
Received 26 March 2016
Received in revised form 12 June 2016
Accepted 6 July 2016
Available online xxx
Keywords:
Illicium wardii
Illiciaceae
Dihydrobenzofuran lignans
Cytotoxic activity
cell lines (A549, SKOV3, HepG2 and HCT116), with IC50 values ranging from 2.7 to 14.9 mM.
ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction
compounds were tested for cytotoxicity against four tumor cell
lines using the MTT assay.
Illiciaceae is a single-genus family made up of the genus Illicium.
Globally, there are about fifty species of Illicium known with the
majority being found in the southeastern portions of Asia and
America. In China, there are about twenty-eight species and two
variants, and these are distributed in the southwestern part of the
country. Among these species, twenty-one are found exclusively in
China, and some of them are used to treat rheumatism, traumatic
injury, and stomach cold vomiting in Traditional Chinese Medicine
(Ye et al., 2015). Illicium wardii A.C. Smith, which grows as
evergreen trees or shrubs, was found as bushes in uncultivated
land near brooks at elevations of up to 1800–2700 m above sea
level (Gao et al., 2015). Our group has studied other plants of the
Illicium genus, but the phytochemical investigation of I. wardii has
seldom been reported, except for the isolation of fourteen
compounds from its fruits in 2007 and 2014 (Min et al., 2007;
Gao et al., 2014). In this work, phytochemical investigation of the
branches and leaves of I. wardii led to the isolation and
identification of six new dihydrobenzofuran lignans (1–6) and
15 (7–21) previously known lignins. Their structures were
elucidated by different spectroscopic methods including NMR,
CD, IR as well as HRESIMS analysis. In addition, these twenty-one
2. Results and discussion
The EtOAc-soluble portion of the 95% EtOH extract of the
branches and leaves of I. wardii was fractioned by silica gel, MCI,
Sephadex LH-20, and preparative HPLC to afford illicium lignans A–
F (1–6), as well as acernikol (7) (Toshio et al., 2003), isodunnianol
(8) (Konno et al., 1991), dihydrodehydrodiconiferylalcohol-9-O-
b-D
-xylopyranoside (9) (Konno et al., 1993), (70S, 80R)-dihydrode-
hydrodiconiferyl alcohol (10) (Seidel et al., 2000), hinokinin (11)
(Wenkert et al., 1976), honokiol (12) (Sun et al., 2005), (7S, 8R)-
dihydrodehydrodiconiferyl alcohol 40-O-
b
-
D
-glucopyranoside (13)
(Matsuda et al., 1996), (70R, 80R)-dihydrodehydrodiconiferyl alco-
hol-90-O-
-glucopyranoside (14) (Lee et al., 2014), (7S, 8R)-
dihydrodehydrodiconiferyl alcohol 9-O-
-(300-O-acetyl)xylopyr-
b-D
b-D
anoside (15) (Liu et al., 2011), (ꢀ)-massoniresinol (16) and (Shen
and Theander, 1985), (70S, 80S)-dihydrodehydrodiconiferyl alcohol
9-O-
yisolariciresinol-90-O-
b-D
-glucopyranoside (17) (Liu et al., 2011), (+)-50-methox-
a-L-rhamnopyranoside (18) (Fuchino et al.,
1995), 1-(4-hydroxy-3-methoxyphenol-2-[4-(3-rhamnopyrnoxy-
propyl)-2-methoxyphenoxyl]-1,3- propanediol(erythro)) (19)
* Corresponding author.
** Corresponding author at: School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China.
E-mail addresses: kimhz@sjtu.edu.cn (H.-Z. Jin), wdzhangy@hotmail.com (W.-D. Zhang).
http://dx.doi.org/10.1016/j.phytol.2016.07.010
1874-3900/ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

264
F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
(Liang et al., 2013), dunnianol (20) (Konno et al., 1993), and (70S,
80S)-dihydrodehydrodiconiferyl alcohol (21) (Liu et al., 2014). The
known compounds were identified by comparison of their NMR,
MS and CD spectroscopic data with the literature reports.
Illiciumlignan A (1) was obtained as a yellowish oil with
ESI–MS analysis (m/z 621.2313 [M+Na]+, 621.2306 calcd.). Analysis
of the NMR spectra revealed that 2 was almost identical to 1, except
for the presence of an ethanoyl moiety and lack of a methoxy
group. The ethanoyl moiety placement at C-9 was based upon the
observation of downfield shifts assigned to H2-9 (dH 4.06, t,
J = 6.6 Hz; 3.57, t, J = 6.5 Hz) and C-9 (dC 63.4, 62.3) while comparing
the 1H NMR and 13C NMR spectra of 2 with those of 1 (Fig. 2). The
absolute configurations at C-80, 800 and C-70, 700 were identical to 1
based on the CD spectrum (positive Cotton effects at 293 and
241 nm). On the basis of the above evidence, the absolute
configuration of 2 was determined and the compound was named
illiciumlignan B.
positive optical rotation ([a
]20D +3.0ꢁ). The molecular formula
was found to be (C30H34O9), which was determined by high
resolution ESI–MS analysis (m/z 561.2109 [M+Na]+, 561.2095
calcd.). The 1H NMR spectrum displayed signals at dH 6.93 (brs),
6.81 (d, J = 8.0, 1.7 Hz), and 6.76 (d, J = 8.1 Hz) suggestive of an ABX
spin system and four singlets (4H) at dH 6.93, 6.92, 6.73 ꢂ 2
corresponding to two tetrasubstituted aromatic rings with four
benzylic methines. The remaining 1H NMR signals showed two
&9472;OꢀꢀCHꢀꢀCHꢀꢀCH2ꢀꢀO-spin systems, three methoxy
groups, and an n-propyl group. The 13C and DEPT-NMR spectra
revealed the presence of three methoxy groups, five methylenes,
eleven methines, and eleven quaternary carbons (Table 1). By
comparing the IR, UV and NMR spectroscopic data for 1 with the
literature (Gu et al., 2008), 1 was found to be almost identical to
vitrifol A, except for the configuration at C-70, C-80, C-700 and C-800. In
the NOESY spectrum, the correlations between H-70 and H-80 as
well as H-700 and H-800 indicated erythro arrangements for phenyl
and hydroxymethylene groups at C-70 and C-80 and the two
hydroxyl groups at C-700 and C-800. Additionally, the absolute
configurations at C-80, 800 and C-70, 700 were elucidated as 80 (R),800(R)
and 70 (R),700(R) on the basis of CD data (a positive Cotton effect at
293 nm) (Zhang et al., 2014; Xiong et al., 2011; Huang et al., 2013).
On the basis of this evidence, the absolute configuration of 1 was
determined and the compound was named illiciumlignan A.
Illiciumlignan B (2) was obtained as yellowish solid with a
Illiciumlignan C (3) was obtained as yellowish solid with a
negative optical rotation ([
a
]20D ꢀ3.33ꢁ). The molecular formula
was (C25H32O10), which was ascertained via high resolution ESI–
MS analysis (m/z 537.1985 [M+COOH]+, 537.1978, calcd.). The 1H
NMR spectrum displayed signals at dH 6.98 (1H, brs), 6.84 (1H, d,
J = 8.2 Hz), 6.76 (1H, d, J = 8.2 Hz) corresponding to an ABX spin
system and two singlets (2H) at dH 6.77 (1H, brs, H-6) and 6.72 (1H,
brs, H-2), suggestive of a tetra substituted aromatic ring with two
benzylic methines. The remaining 1H NMR signals showed one
&9472;OꢀꢀCHꢀꢀCHꢀꢀCH2ꢀꢀO-spin system, two methoxy groups,
and n-propyl and xylose moieties. The 13C and DEPT-NMR
spectrum revealed the presence of two methoxy groups, four
methylenes, seven methines, seven quaternary carbons, and a
xylose moiety (Table 3). Acidic hydrolysis of 3 in HCl-methanol
(2 M) yielded (70S, 80S)-dihydrodehydrodiconiferyl alcohol and a
xylose, identified as
b-D-xylopyranose by GC comparison with an
authentic sample and both showed the same 1H NMR coupling
00 00
constant [J(1 ,2 ) = 7.5 Hz] (Li et al., 1984; Jiang et al., 2004; Wang
]20D ꢀ11.2ꢁ).
2 possessed the
et al., 2014). The cross-peak between H-100 and C-90 in the HMBC
spectrum indicated that the xylopyranose group is linked to C-90.
negative optical rotation ([
molecular formula (C32H38O11), as revealed by high resolution
a
Table 1
1H-, 13C- and 2D-NMR data for compound 1 recorded at 500 (1H) and 125 (13C, 2D) MHz in CD3OD.
No.
1
dc
DEPT
CH
dH (J in Hz)
HMBC
COSY
NOESY
1
2
3
4
5
6
7
8
135.6
112.7
144.0
146.4
128.4
116.5
31.5
6.73 brs
C-7,6,3,4
CH
6.73 brs
2.62 t (7.7)
1.82 m
C-7,2,3,4
C-1,2,6,8,9
C-1,7,9
CH2
CH2
CH2
H-8
H-7,9
H-8
34.4
60.8
H-2,6,7,9
9
3.56 t (6.5)
C-7,8
10
135.4
110.4
143.8
147.8
128.9
114.8
87.8
20
CH
6.91 brs
C-60,40,30
30
40
50
60
CH
CH
CH
CH2
6.93 brs ovl
5.52 d (6.2)
3.50 m ovl
3.86,3.80 m
C-70,20
70
C-90,80,60,20,10,5,4
C-90,70,60,10,6,5,4
C-80,70,5
H-80
H-70,90
H-80,90
H-70,90
80
54.0
63.6
90
1”
2”
3”
4”
5”
6”
7”
132.9
109.2
147.8
146.0
114.4
118.3
87.9
CH
6.93 brs
C-7”,6”,4”,3”,1”
CH
CH
CH
CH
CH2
OMe
OMe
OMe
6.76 d (8.2)
6.81 dd (1.7,8.2)
5.52 d (6.2)
3.50 m ovl
3.76 m
3.83 s
3.85 s
3.80 s
C-6”,4”,3”
C-7”,2”
C-9”,8”,6”,2”,1”,50,40
C-9”,7”,6”,1”,60,50,40
H-8”
H-7”,9”
H-6”,8”,9”
H-60,7”,9”
8”
53.8
9”
63.4
3-OMe
30-OMe
3”-OMe
55.0
55.4
55.3
C-3
C-30
C-3”
Ovl, overlap.

F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
265
Fig. 1. Structures of compounds 1–21.
By comparing IR, UV and NMR spectroscopic data for 3 with
literature (Konno et al., 1993), 3 was nearly identical to 2,3-
the absolute configuration of the 80-position of 3 was in the S
orientation (Zhang et al., 2014; Xiong et al., 2011; Huang et al.,
2013). In the NOESY spectrum, the correlations between H-70 and
H-80 indicated erythro arrangements for phenyl and hydroxy-
methylene groups at C-70 and C-80, respectively. Hence, the
dihydro-7-methoxy-2-(40-hydroxy-30-methoxyphenyl)-3a-O-
b-D-
xylopyranosyloxymethyl-5- benzofuranprop-anol, except for the
stereochemistry at C-70 and C-80. Moreover, the CD spectrum of 3
showed a negative Cotton effect at 293 nm, which indicated that
absolute configuration at the 70-position of
3 was in the

266
F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
Fig. 2. Key 1H–1H COSY and HMBC correlations for 1–6.
S
orientation. On the basis of this evidence, the absolute
correlations between H-70 and H-80, indicated erythro arragements
for phenyl and hydroxymethylene groups at C-70 and C-80,
respectively (Fig. 2). Hence, the absolute configuration at the 70-
position of 4 was in the R orientation. Thus, the absolute
configuration of 4 was elucidated and the compound was named
illiciumlignan D, which is the first reported dihydrobenzofuran
lignin containing an arabinofuranoside (positioned at C-90).
Illiciumlignan E (5) was obtained as yellowish solid with a
stereochemistry of 3 was elucidated and the compound was
named illiciumlignan C.
Illiciumlignan D (4) was obtained as yellowish solid with a
negative optical rotation ([
a
]20D ꢀ26.4ꢁ). The molecular formula
was found to be (C25H32O10), which was determined by high
resolution ESI–MS analysis(m/z 515.1888 ([M+Na]+, 515.1888).
Analysis of the 1H and 13C NMR spectra indicated the presence
of the same skeleton as 3. The major difference in the NMR spectra
negative optical rotation ([
a
]20D ꢀ0.55ꢁ). The molecular formula
b-D
-arabinofuranosyl at C-90 and the absolute configuration
was found to be (C30H36O10), which was based on high resolution
ESI–MS analysis (m/z 579.2214 [M+Na]+, 579.2201 calcd.). Analysis
of the NMR spectra revealed that 5 was almost identical to 7, except
for the absence of a methoxy signal. In the NOESY spectrum, the
correlations observed between H-70 and H-80 as well as H-700 and
H-800 indicated erythro arrangements for both the phenyl and
hydroxymethylene groups at C-70 and C-80, and the two hydroxyl
groups at C-700 and C-800 (Fig. 2). Moreover, the CD spectrum of 5
showed a positive Cotton effect at 290 nm and a negative Cotton
effect at 240 nm, which indicated the absolute configurations of
the 80, 800 and 70, 700 positions of 5 were 80 (R),800(R) and 70 (R),700(S),
respectively (Zhang et al., 2014; Xiong et al., 2011; Huang et al.,
were
at C-70 and C-80. Acidic hydrolysis of 4 in HCl-methanol (2 M)
yielded (70R, 80R)-dihydrodehydrodiconiferyl alcohol and arabino-
furanose, which was identified as
b-D-arabinofuranose by GC
comparison with an authentic sample and both showed the same
1H NMR coupling costant (J(1 ,2 ) = 1.4 Hz) (Kaeothip et al., 2013).
00 00
The cross-peak between H-100 and C-90 observed in the HMBC
spectrum indicated that the arabinofuranose moiety was linked to
C-90. Moreover, the CD spectrum of 4 showed a positive Cotton
effect at 290 nm, which indicated that the absolute configuration of
the 80-position was in the R orientation (Zhang et al., 2014; Xiong
et al., 2011; Huang et al., 2013). In the NOESY spectrum, the

F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
267
Table 3
2013). Therefore, the absolute configuration of 6 was elucidated
and the compound was named illiciumlignan E.
1H and 13Cꢀꢀ NMR data for compounds 3, 4 and 6, recorded at 500 (1H) and 125 (13C)
MHz in CD3OD.
Illiciumlignan F (6) was obtained as yellowish crystal with a
negative optical rotation ([
a
]20D ꢀ2.0ꢁ) and the molecular
No.
3
4
6
formula (C27H34O11), which was deduced from high resolution
ESI–MS analysis (m/z 579.2101 [M+COOH]+, 579.2083 calcd.).
Analysis of the NMR spectra revealed that 6 was almost identical
with 4, except for small differences in the chemical shifts which
were attributed to the ethanoyl moiety. The ethanoyl moiety was
positioned at C-9 based upon the observation of downfield shifts in
the signals assigned to H2-9 (dH 4.05, t, J = 6.6 Hz; 3.56, t, J = 6.4 Hz)
and C-9 (dC 63.6, 60.8) while comparing the 1H NMR and 13C NMR
spectra of 6 with those of 3. In the NOESY spectrum, the
correlations observed between H-70 and H-80 indicated erythro
arrangements for phenyl and hydroxymethylene groups at C-70
and C-80 (Fig. 2). Moreover, the CD spectrum of 6 showed a positive
Cotton effect at 293 nm, which indicated the absolute config-
urations of the 80 and 70-position of 6 were 80 (R) and 70 (R),
respectively. Therefore, 6 was elucidated as shown and the
compound was named illiciumlignan F.
d
c
dH (J in Hz)
d
c
dH (J in Hz)
dc
dH (J in Hz)
1
2
3
4
5
6
7
8
133.2
135.5
133.1
112.8 6.72 brs
143.8
112.7 6.71 brs
143.8
112.7 6.71 d (1.5)
143.8
146.1
146.1
146.2
128.2
128.2
128.2
116.7 6.77 brs
116.6 6.72 brs
2.61 t (6.4)
34.4 1.81 m
116.7 6.76 brs
31.4
2.62 t (7.6) 31.5
31.5
2.63 t (7.3)
34.3 1.82 m
60.8 3.56 t (6.4) 60.8 3.56 t (6.5)
135.5
30.3 1.91 m
63.6 4.05 t (6.6)
134.8
9
10
20
30
40
50
60
70
80
90
1”
2”
3”
4”
5”
133.2
109.3 6.98 brs
147.6
146.0
109.2 6.96 d (1.7)
147.7
146.1
109.2 6.97 d (1.9)
147.6
146.1
114.7 6.76 d (8.2) 114.7 6.76 d (8.2)
114.6 6.75 d (8.2)
118.3 6.84 d (8.2) 118.3 6.82 dd (8.2,1.9) 118.3 6.83 dd (8.1,1.9)
87.8
51.5
5.55 d (6.3) 87.9
3.62 m 51.5
5.49 d (6.4)
3.62 m
87.8
51.5
5.54 d (6.4)
3.61 m
70.7 4.00,3.83 m 69.0 3.90,3.71 m
103.3 4.27 d (6.9) 107.9 4.93 d (1.4)
70.9 4.00,3.83 m
103.5 4.29 d (7.5)
73.5 3.20 m ovl
76.5 3.33 m
69.8 3.50 m
65.6 3.88, 3.20 m
55.3 3.85 s
Compounds 1–21 were tested for their cytotoxicity against
human lung cancer (A549), colon cancer (HCT116), ovarian
neoplasm (SKOV3), and hepatocellular carcinoma (HepG2) cell
lines using the MTT assay using Doxorubicin as a positive control.
The results of this survey are as follows: Doxorubicin had IC50
72.9 3.20 m ovl 82.1
77.3 3.33 m 77.4
68.2 3.50 m
65.6 3.87,3.20 m 61.6
55.1
4.01 m
3.85 m
84.3 3.93 m
3.63, 3.75 m
3.83 s
3-OMe 55.3 3.85 s
30-
OMe
Me
C¼O
Ovl, overlap.
values of 0.72, 0.16, 3.09, and 0.10
m
M against the A549, HCT116,
55.0 3.82 s
55.4 3.80 s
55.0 3.81 s
SKOV3, and HepG2 cell lines, respectively. 8, 12, and 20 showed
moderate activity against the A549 cell line with IC50 values of
19.4 2.03 s
171.7
14.88, 26.04, and 28.22
mM, respectively. 8 and 20 had better
cytotoxicity against the HCT116 cell line with respective IC50 values
of 2.78 and 2.70 mM than 10 and 12 with respective IC50 values of
80.47 and 20.48
values ranging from 6.08
m
M. Compounds 4, 5, 8, 10, 18, and 20 showed IC50
M to 13.31 M against the SKOV3 cell
line. 8, 12, and 20 showed moderate activity against the HepG2 cell
line with IC50 values of 5.65, 14.61, and 11.31 M, respectively.
None of the remaining compounds showed significant cytotoxicity
against the above cell lines (IC50 > 100 M) (Figs. 1 and 2, Table 4).
m
m
Table 2
1H and 13Cꢀꢀ NMR data for compounds 2 and 5, recorded at 500 (1H) and 125 (13C)
MHz in CD3OD.
m
No.
2
5
m
dc
dH (J in Hz)
dc
dH (J in Hz)
This study describes the isolation of six new dihydrobenzofuran
lignans from the branches and leaves of I. wardii. The structures of
these compounds were determined by extensive IR, UV, CD and
NMR spectroscopic analysis. To the best of our knowledge,
illiciumlignan D (4) is the first reported dihydrobenzofuran lignan
arabinofuranoside in which the arabinofuranose moiety was
derivatized on C-90. Additionally, the cytotoxic properties of the
21 isolated compounds were assessed. Among them, compound 8
is the most promising with the most potent cytotoxicity against
SKOV3 and HepG2 cell lines, while 20 is the most promising against
HCT116.
1
2
3
4
5
6
7
8
134.8
112.6
143.9
147.2
128.4
116.5
31.5
135.6
112.7
143.8
146.1
128.4
116.6
31.5
6.71 brs ovl
6.71 brs
6.71 brs ovl
2.63 t (7.5)
1.92 m
6.70 brs
2.62 t (7.5)
1.80 m
30.3
63.4
34.4
60.9
9
4.06 t (6.6)
3.56 t (6.6)
10
132.6
109.6
150.5
145.5
114.2
117.9
87.2
132.6
109.9
147.3
147.5
114.3
118.1
87.2
20
6.93 d (1.9)
6.95 brs
30
40
50
6.90 d (8.4)
6.82 m ovl
5.50 d (5.5)
3.43 m
6.72 d (8.2)
6.82 m ovl
5.50 d (6.1)
4.37 m
60
3. Experimental
70
80
54.1
63.6
54.0
63.6
3.1. General experimental procedures
90
3.75 m
3.81,3.75 m
1”
2”
3”
4”
5”
6”
7”
8”
136.1
110.4
147.5
146.2
117.4
119.6
72.7
84.8
60.8
55.3
54.9
136.1
110.4
150.5
145.6
117.5
119.7
72.7
84.8
60.9
55.4
55
IR spectra were obtained on Bruker FTIR Vector 22 spectrome-
ter. UV spectra were acquired in MeOH on SHIMADZU UV-2550
spectrometer. Optical rotations were measured on Jasco P-2000
Polarimeter. Melting points were carried out on XT4A micro-
melting point apparatus. ESIMS spectra were performed using an
Agilent 1100 series mass and Autospec-Ultima ETOF apparatus,
while high resolution ESI–MS spectra were recorded on Q-TOF
micro-mass spectrometer (Waters, USA). 1H and 13C NMR spectra
were acquired in CD3OD on Bruker Avance-500 spectrometers (1H
at 500 Hz and 13C at 125 MHz). TLC analysis was performed on
6.99 d (1.9)
7.00 brs
6.82 m ovl
6.71 m ovl
4.80 d (5.9)
4.36 m
3.82 m
3.85 s
6.89 d (8.4)
6.83 m ovl
4.83 d (5.3)
4.37 m
3.78, 3.85 m
3.80 s
9”
3-OMe
30-OMe
50-OMe
3”-OMe
Me
C¼O
Ovl, overlap.
3.74 s
3.74 s
55.0
19.4
171.7
3.77 s
2.02 s
55.2
3.70 s
HSGF254 silica gel plates (10–40 mm, Yantai, China), and com-
pounds were visualized by spraying the dried plates with 10%
H2SO4, followed by heating, or using iodine vapor. Sephadex LH-20

268
F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
(GE Healthcare Bio-Sciences AB, Sweden) was used and column
chromatography (CC) was performed on silica gel (200–300 mesh,
Yantai, China). GC data were recorded on an Agilent N6890
instrument. A preparative column (Shimadzu PRC-ODS EV0233,
yield 2 (7.0 mg, MeOH/H2O 60/40), 4 (20.0 mg, MeOH/H2O 40/60),
5 (30.0 mg, MeOH/H2O 40/60), and 6 (6.0 mg, MeOH/H2O 60/40).
Compounds 3 (6.0 mg, MeOH/H2O 35/65), 16 (20.0 mg, MeOH/H2O
40/60), and 17 (8.0 mg, MeOH/H2O 50/50) were obtained from Fr.7
C
18, 5
m
m, 250 ꢂ 21.2 mm, 500
mL/inj.) was used for preparative
by preparative HPLC. Fr.8 was subjected to preparative HPLC (C18
,
HPLC (Shimadzu LC-20A).
5
m
m, 250 ꢂ 21.2 mm, 500
m
L/inj, flow rate 8.0 mL/min) to yield 18
(4.0 mg, MeOH/H2O 30/70), 19 (8.0 mg, MeOH/H2O 35/65) and 21
3.2. Plant material
(6.0 mg, MeOH/H2O 45/55).
The branches and leaves of I. wardii were collected in Nujiang
County of Yunnan Province, PR China in August 2011 and were
subsequently authenticated by Prof. Zhou Yuan-chuan, the director
of Nujiang Nationality Medicine Plants Institution. An authentic
specimen (No.201108GSBJ) was deposited at the School of
Pharmacy, Shanghai Jiao Tong University.
3.4. Acid hydrolysis of compounds 3, 4, 6
Each compound (5.0 mg) was refluxed in 2 M HCl/MeOH (v/v 1/
1, 2 mL) at 60 ꢁC for 3 h, and the solution was evaporated under N2.
The residue was dissolved in pyridine (100
mL), 1 M L-cysteine
methyl ester hydrochloride (100 L) was added, and the mixture
m
was heated to 60 ꢁC for 2 h. After the reaction mixture was dried in
vacuo, the trimethylsilylation reagent HMDS-TMCS (hexamethyl-
disilazane/Me3SiCl/pyridine 2/1/10) was added, and heating at
60 ꢁC was continued for another 2 h. Finally, the mixture was
partitioned between hexane and H2O (2 mL each), and the hexane
extract was analyzed by gas chromatography (GC) under the
following conditions: column temperature, 255 ꢁC; injection
temperature, 255 ꢁC; flow rate 1.0 mL/min; N2 carrier gas. In the
acid hydrolysate of 4, identification of D-arabinofuranose and D-
xylopyranose was carried out by comparison of the retention time
with those of authentic samples prepared in a similar way. tR:
6.8 min (D-arabinofuranose), 9.7 min (D-xylopyranose) (Li et al.,
1984; Wang et al., 2014).
3.3. Extraction and isolation
The dried branches and leaves of I. wardii (17.4 kg) were
pulverized and extracted with 95% EtOH under reflux for 3ꢂ2 h.
The combined EtOH extracts were concentrated in vacuo to yield
the crude material (1.6 kg), which was successfully portioned with
petroleum ether (5 L ꢂ 4), methylene chloride (5 L ꢂ 4), and ethyl
acetate (5 L ꢂ 4). The CH2Cl2 fraction (100.0 g) was subjected to CC
on silica gel (1200 g, 100–200 mesh, 100 mm ꢂ 60 cm) with a
CH2Cl2/MeOH (100/1–2/1, v/v) gradient eluent to obtain fourteen
subfractions (Fr.1–Fr.14). Compound 8 (10.0 mg) was obtained from
Fr.1 (10.0 g) after CC on silica gel (140 g, 200–300 mesh, 60 mm
ꢂ 60 cm, CH2Cl2/MeOH 20/1) and recrystallized in methanol. Fr.4
(6.0 g) was purified over Sephadex LH-20 (5.0 ꢂ150 cm, MeOH,
3.5. Structural elucidation of new products
1 mL/min) followed by preparative HPLC (C18
,
5
m
m,
250 ꢂ 21.2 mm, 500 L/inj, flow rate 8.0 mL/min) to yield
m
7
Illiciumlignan A (1): Yellowish oil, C30H34O9, ESI–MS m/z: 561
[M+Na]+, HR-ESI–MS: m/z 561.2109 ([M+Na]+, calcd. 561.2095); UV
(MeOH) lmax (log e):217 (2.05), 281 (0.54) nm, [a]20D +3.0 (c 0.1,
MeOH); CD [MeOH c 0.005, De (nm)], ꢀ1.85 (201), +1.19 (209),
+2.18 (220), +5.19 (245), +1.52 (292); IR (KBr) ymax 3434, 2924,1619,
1499,1463,1383,1275,1211,1141,1032, 813 cmꢀ1; 1H NMR (CD3OD,
500 MHz) and 13C NMR data (CD3OD, 125 MHz) (Table 1).
(25.0 mg, MeOH/H2O 45/55), 1 (16.0 mg, MeOH/H2O 45/55), 9
(20.0 mg, MeOH/H2O 45/55), 10 (50.0 mg, MeOH/H2O 45/55), 11
(12.0 mg, MeOH/H2O 75/25), and 12 (12.0 mg, MeOH/H2O 85/15).
Fr.5 (4.0 g) was purified using MCI CC (3.0 ꢂ 60 cm, MeOH/H2O 90/
10) followed by preparative HPLC to yield 13 (30.0 mg, MeOH/H2O
40/60),14 (15.0 mg, MeOH/H2O 40/60),15 (45.0 mg, MeOH/H2O 70/
30) and 20 (20.0 mg, MeOH/H2O 85/15). The EtOAc fraction (100 g)
was subjected to CC on silica gel (1200 g, 100–200 mesh, 100 mm
ꢂ 60 cm) with a CH2Cl2/MeOH (100/1–2/1, v/v) gradient eluent to
obtain ten subfractions (Fr.1ꢀ Fr.10). Fr.6 (5.0 g), Fr.7 (3.0 g) and Fr.8
(4.0 g) were purified over MCI CC (3.0 ꢂ 60 cm, MeOH/H2O 90/10)
followed by Sephadex LH-20 (5.0 ꢂ150 cm, MeOH, 1 mL/min).
Illiciumlignan B (2): Yellowish solid, C32H38O11, m.p. 105.4–
106.2 ꢁC, ESI–MS: m/z 621 [M+Na]+, HR-ESI–MS: m/z 621.2313 ([M
+Na]+, calcd. 621.2306); UV (MeOH) lmax (log
e): 214 (1.95), 230
(1.51), 282 (0.55) nm, [
a
]20 D ꢀ11.2 (c 0.13, MeOH); CD [MeOH c
0.005, De (nm)]: +3.76 (202), +2.55 (212), +1.34 (240), 0.63 (292);
IR (KBr) ymax 3432, 2932, 1733, 1607, 1514, 1456, 1429, 1367, 1266,
1213, 1141, 1032 cmꢀ1 1H NMR (CD3OD, 500 MHz) and 13C NMR
;
Then, the Fr.6 was subjected to preparative HPLC (C18, 5
m
m,
250 ꢂ 21.2 mm, 500 L/inj, flow rate 8.0 mL/min, MeOHꢀꢀH2O) to
m
data (CD3OD, 125 MHz) (Table 2).
Illiciumlignan C (3): Yellowish solid, C25H32O10, m.p. 95.7–
96.6 ꢁC, ESI–MS m/z: 515 [M+Na]+, HR-ESI–MS: m/z 537.1985 ([M
+COOH]+, calcd. 537.1978); UV (MeOH) lmax (log
e
): 213 (1.74), 230
Table 4
(1.72), 284 (0.43) nm, [
a
]20D ꢀ3.33 (c 0.21, MeOH); CD [MeOH c
IC50 values of compounds 1–21 against four human tumor cell lines.
0.005, De (nm)]: +5.52 (212), ꢀ4.55 (242), ꢀ2.55 (292); IR (KBr)
ymax 3415, 2924, 1607,1517, 1499, 1452, 1430, 1384, 1215, 1211,
1040 cmꢀ1; 1H NMR (CD3OD, 500 MHz) and 13C NMR data (CD3OD,
125 MHz) (Table 3).
Compounds
IC50
(mM)
A549
HCT116
SKOV3
HepG2
1
2
3
4
5
6
7
8
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
14.88
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
2.78
>100.00
>100.00
>100.00
8.67
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
5.65
Illiciumlignan D (4): Yellowish solid, C25H32O10, m.p. 84.4–
85.3 ꢁC, ESI–MS m/z: 515 [M+Na]+, HR-ESI–MS: m/z 515.1888 ([M
+Na]+, calcd. 515.1888); UV (MeOH) lmax (log
e): 214 (1.98), 232
10.66
>100.00
>100.00
6.08
(1.58), 283 (0.61) nm; [
a
]20D ꢀ26.4 (c 0.22, MeOH); CD [MeOH c
0.005, De (nm)]: ꢀ3.19 (202), ꢀ1.24 (223), +4.41 (240), +2.03 (290);
IR (KBr) ymax 3419, 2936, 1607, 1518, 1498, 1455, 1275, 1211, 1141,
1034 cmꢀ1; 1H NMR(CD3OD, 500 MHz) and 13C NMR data (CD3OD,
125 MHz) (Table 3).
10
12
18
20
DOX
>100.00
26.04
>100.00
28.22
80.47
20.48
>100.00
2.70
9.36
36.25
11.47
13.31
3.09
>100.00
14.61
>100.00
11.31
Illiciumlignan E (5): Yellowish solid, C30H36O10, m.p. 90.2–
0.72
0.16
0.10
91.0 ꢁC, ESI–MS: m/z 579 [M+Na]+, HR-ESI–MS: m/z 579.2214 ([M
+Na]+, calcd. 579.2201); UV (MeOH) lmax (log
e): 214 (2.01), 230
Doxorubicin (DOX) was used as a positive control.

F.- Ye et al. / Phytochemistry Letters 17 (2016) 263–269
269
(1.72), 283 (0.62) nm; [
a
]20D ꢀ0.55 (c 0.91, MeOH); CD [MeOH c
References
0.005, De (nm)]: +0.83 (223), ꢀ1.30 (240), +1.44 (290); IR (KBr)
ymax 3417, 2934, 1605, 1513, 1463, 1428, 1269, 1210, 1139,
1029 cmꢀ1; 1H NMR (CD3OD, 500 MHz) and 13C NMR data (CD3OD,
125 MHz) (Table 2).
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Author contributions
Feng-mei Ye and Yang-Guo Xie contributed equally to this work
and should be considered co-first authors.
Acknowledgments
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The work was supported by the NCET Foundation, NSFC
(81230090), Shanghai Leading Academic Discipline Project
(B906), Key laboratory of drug research for special environments,
PLA, Shanghai Engineering Research Center for the Preparation of
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Appendix A. Supplementary data
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Supplementary data associated with this article, including the
HRESIMS, 1D and 2D NMR data for compounds 2–7, can be found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.
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