Two new compounds from the roots of Ilex pubescens and their cytotoxic activity

Article abstract of DOI:10.1007/s11418-016-0996-y

A new phenylethanoid, 1-O-β-d-(4-hydroxyphenyl)-ethyl-6-O-vanilloyl-glucopyranoside (1), and a new furofuran lignan, (7R,7′R,7′′S,8S,8′S,8′′R)-4′, 4′′-dihydroxy-3,3′,3′′,5-tetramethoxy-7,9′:7′9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (2), along with five known compounds (3–7) were isolated from the roots of Ilex pubescens. Their chemical structures were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, MS, and NMR experiments. In addition, compounds 2–7 were evaluated in vitro for their cytotoxic effects on human HeLa cells; among them, compounds 2, 3, 6, and 7 showed cytotoxic activity against HeLa cells in the test.

Full text of DOI:10.1007/s11418-016-0996-y

J Nat Med
DOI 10.1007/s11418-016-0996-y
NOTE
Two new compounds from the roots of Ilex pubescens and their
cytotoxic activity
1
1
1
1
1
•
Huan Xiong
1
Xue Chen
•
Fang Zhao
1
Yuncong Li
•
Jiayi Bi
Rui Yan
•
Yang Zhang
1
Qiang Zhao
•
Guiyun Zhao
1
1
1
•
•
•
•
Hua Qiao
•
Guogang Zhang
Received: 26 January 2016 / Accepted: 6 April 2016
Ó The Japanese Society of Pharmacognosy and Springer Japan 2016
Abstract A new phenylethanoid, 1-O-b-D-(4-hydrox-
yphenyl)-ethyl-6-O-vanilloyl-glucopyranoside (1), and a new
demonstrated broad biological properties, such as enlarging
blood vessels, decreasing plasma viscosity, inhibiting pla-
telet aggregation, preventing thrombosis, decreasing the
excitation of the cardiac conduction system and enhancing
anoxia resistance [2–4]. Previous studies on chemical
constituents of I. pubescens roots and leaves led to the
isolation of a series of diverse compounds, including
triterpene saponins [5, 6], lignans [7], lignin glycosides [8],
phenylethyl alcohol derivatives [9], secoiridoid glucosides
[10] and flavonoids [11]. To seek the bioactive con-
stituents, we carried out a phytochemical investigation on
the roots of I. pubescens, which resulted in two new
compounds (1 and 2), along with five known compounds
(3–7), which were isolated from this plant for the first time.
In this paper, we describe the isolation and structural elu-
cidation of these compounds and their in vitro cytotoxic
activity against HeLa cells.
0
00
0
00
0
00
furofuran lignan, (7R,7 R,7 S,8S,8 S,8 R)-4 , 4 -dihydroxy-
0 00 0 0 00 0
3
,3 ,3 ,5-tetramethoxy-7,9 :7 9-diepoxy-4,8 -oxy-8,8 -
0
0
00
sesquineolignan-7 ,9 -diol (2), along with five known
compounds (3–7) were isolated from the roots of Ilex pub-
escens. Their chemical structures were elucidated on the
basis of extensive spectroscopic analysis, including UV, IR,
MS, and NMR experiments. In addition, compounds 2–7
were evaluated in vitro for their cytotoxic effects on human
HeLa cells; among them, compounds 2, 3, 6, and 7 showed
cytotoxic activity against HeLa cells in the test.
Keywords Ilex pubescens Á Lignans Á Phenylethanoid Á
Cytotoxic activity
Introduction
Ilex pubescens is an evergreen shrub, mainly distributed in
the southern regions of China. In traditional Chinese
medicine, Mao-Dong-Qing, the dried roots of I. pubescens,
is used primarily for the treatment of cardiovascular dis-
ease and hypercholesterolemia [1]. Pharmacological stud-
ies on extracts prepared from the roots of I. pubescens
Results and discussion
The EtOH extract of the roots of I. pubescens was subjected
to silica gel CC, Sephadex LH-20, and reversed-phase HPLC
to afford a new phenylethanoid (1), and a new furofuran
lignan (2), together with five known compounds. Com-
0
00
0
00
pounds 3–7 were identified as (7R,7 R,7 R,8S,8 S,8 S)-
0
00 0 00 0 0
4
,4 -dihydroxy-3,3 ,3 ,5-tetramethoxy-7,9 :7 ,9-di-epoxy-
0
0
0
00 00
Electronic supplementary material The online version of this
article (doi:10.1007/s11418-016-0996-y) contains supplementary
material, which is available to authorized users.
4,8 -ox-y-8,8 -sesquineolignan-7 ,9 -diol (3) [12], erythro-
0
(
7S,8R)-guaiacylglycerol-b-O-4 -coniferyl alocohol (4) [13,
0
14], erythro-(7R,8S)-guaiacylglycerol-b-4 -coniferyl aloco-
&
Guogang Zhang
zggth@163.com
hol (5) [13, 14], erythro-(7R,8S)-guaiacylglycerol-b-con-
iferyl aldehyde ether (6) [14, 15], and erythro-(7S,8R)-
guaiacylglycerol-b-coniferyl aldehyde ether (7) [14, 15], by
comparison of their spectral data with those described in the
literature (Fig. 1). Reported herein are the extraction,
1
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wenhua Rd,
Shenyang 110016, People’s Republic of China
123

J Nat Med
isolation and structure elucidation of the compounds isolated
from the roots of I. pubescens.
the reduction of 3-hydroxyphenyl. The location of the other
functional groups and NMR data assignments of 1 were
determined by HMBC and HSQC spectroscopic analysis.
The sugar was identified as D-glucose by optical rotation
using chiral detection in HPLC analysis after acid hydrolysis
of 1. The b-anomeric configuration of the glucose was
Compound 1 was obtained as a white amorphous powder.
Its molecular formula was determined to be C H O
2 26 10
2
according to the positive-ion HRESIMS data at m/z 473.1432
?
M ? Na] . The IR spectrum of 1 showed characteristic
[
-
absorptions for hydroxy (3430 cm ) and aromatic moieties
1
1
determined by the J value in the H-NMR spectrum
-
1
1
1639, 1462 cm ). The H- and C-NMR spectrum
13
(
(J1 = 7.8 Hz). The location of the sugar moiety was
0 0
,2
00
assigned at C-8 and C-7 by the observed HMBC correla-
(
Table 1) of compound 1 strongly suggested that the com-
pound exhibited typical signals of three fragments, including
0
0
00
tions from H-1 to C-8, and H-6 to C-7 . On the basis of all
the evidence obtained, the structure of 1 was defined as 1-O-
b-D-(4-hydroxyphenyl)-ethyl-6-O-vanilloyl-
one p-hydroxyphenethyl group [A] [d 2.70 (2H, m), 3.79
H
(
1H, m), 3.60 (1H, m), 6.95 (2H, d, J = 8.4 Hz), 6.62 (2H, d,
J = 8.4 Hz)], one vanilloyl group [B] [dH 7.41 (1H, d,
J = 1.4 Hz), 7.46 (1H, dd, J = 8.3, 1.9 Hz), 6.83 (1H, d,
glucopyranoside.
Compound 2 was obtained as a yellow oil, and exhibited
a quasi-molecular ion peak at m/z 583.2189 in negative
HREIMS analysis, indicating a molecular formular of
C H O . The IR spectrum showed hydroxyl
J = 8.3 Hz), 3.70 (3H, s), d 165.5 (s)], as well as a sugar
C
moiety. In the HMBC spectrum, a long-range correlation
from H-7 to C-1/C-2/C-6, from H-8 to C-7/C-1 confirmed the
presence of a p-hydroxyphenethyl group. Moreover, the
3
1 36 11
-
1
-1
(3393 cm
) and benzene ring (1594, 1517 cm )
0
0
00
1
absorptions bands. The H-NMR spectrum (Table 1) of 2
HMBC correlation (Fig. 2) from OCH -3 to C-3 , from
3
0
0
00
00
00
00
00
00
00
H-2 to C-3 /C-4 /C-7 , from H-5 to C-1 /C-3 /C-4 ,
showed signals of four aromatic methoxy groups at d 3.76
(3H, s), 3.75 (6H, s), 3.73 (3H, s), along with eight olefinic
H
0
0
00
00
from H-6 to C-2 /C-4 revealed the presence of vanillyol.
The above NMR spectroscopic data were very similar to
those of 1-O-b-D-(3, 4-dihydroxyphenyl)-ethyl-6-O-vanil-
loyl-glucopyranoside found in the literature [16], except for
protons signals at d 6.89 (1H, d, J = 1.4 Hz), 6.76 (1H,
dd, J = 8.2, 1.6 Hz), 6.72 (1H, d, J = 8.2 Hz), 6.91 (1H,
H
d, J = 1.4 Hz), 6.71 (1H, d, J = 8.2 Hz), 6.69 (1H, d,
Fig. 1 Structures of
compounds 1–7
OH
O
OCH3
H CO 3 OH
3
''
H
H
H CO
3
5''
O
1''
O
H O
O
HO
6
'
OCH3
H
5
'
O
HO
HO3
7
OH
1
' O
3
'
H
OH
8
1
HO
H
H
OH
5
OCH3
1
2: 7R,7'R,7''S,8S,8'S,8''R
3
: 7R,7'R,7''R,8S,8'S,8''S
H CO
3
H CO
3
CHO
OH
O
O
HO
HO
OH
OH
HO
HO
OCH3
OCH3
4
: erythro-7'S,8'R
6: erythro-7'S,8'R
7: erythro-7'R,8'S
5: erythro-7'R,8'S
1
23

J Nat Med
1
13
Table 1 H-NMR and C-
NMR spectral data of 1 and 2 (d
in ppm)
a
b
Position
1
Position
2
d (H)
d (C)
d (H)
d (C)
1
2
3
4
5
6
7
8
128.9
129.6
115.0
155.6
115.0
129.6
34.9
1
2
3
4
5
6
7
8
136.9
103.3
152.6
134.8
152.6
103.3
85.1
6.95 (d, 8.4)
6.62 (d, 8.4)
6.64 (s)
6.62 (d, 8.4)
6.95 (d, 8.4)
2.70 (m)
6.64 (s)
4.65 (d, 4.2)
3.05 (m)
3.60 (m)
70.2
53.7
3
.79 (m)
9
4.16 (m)
71.2
3
.79 (m)
0
0
1
4.26 (d, 7.8)
3.00 (m)
103.0
73.4
76.5
70.4
73.7
64.0
1
132.2
110.4
147.5
145.9
115.1
118.6
0
0
2
2
6.89 (d, 1.4)
0
0
3
3.15–3.21 (m)
3.15–3.21 (m)
3.48 (m)
3
0
0
4
4
0
0
5
5
6.72 (d, 8.2)
0
0
6
4.20 (dd, 11.6, 1.8)
6
6.76 (dd, 8.2, 1.6)
4
.56 (dd, 11.7, 7.1)
0
7
4.61 (d, 4.2)
3.05 (m)
85.1
53.5
71.0
0
8
0
9
4.16 (m)
3
.79 (m)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
1
2
3
4
5
6
7
120.3
112.5
147.4
152.0
115.2
123.5
165.5
1
133.3
110.9
147.0
145.3
114.6
119.4
72.1
00
7.41 (d, 1.4)
2
6.91 (d, 1.4)
00
3
00
4
00
6.83 (d, 8.3)
5
6.69 (d, 8.0)
6.71 (d, 8.2)
4.79 (t, 4.4)
4.10 (m)
00
7.46 (dd, 8.3, 1.9)
6
00
7
0
0
0
0
8
9
86.2
3.40 (m)
59.8
3
.70 (m)
0
OCH
3
-3
3.70 (3H, s)
55.4
OCH
OCH
OCH
OCH
3
-3
3
-5
3
-3
3
-3
3.75 (3H, s)
3.75 (3H, s)
3.76 (3H, s)
3.74 (3H, s)
56.0
56.0
55.5
55.6
0
0
0
a
1 13
H NMR at 600 MHz, C NMR at 100 MHz, obtained in DMSO-d
6
b
1 13
H NMR at 600 MHz, C NMR at 150 MHz, obtained in DMSO-d
6
J = 8.0 Hz), and 6.64 (2H, s), revealing the presence of
supported by the HMBC correlations from H-7 to C-1/C-2/
0 0 0 0 0 0
two 1,2,4-trisubstituted benzene rings and a 1,2,3,5-tetra-
C-8 /C-9 /, from H-7 to C-8/C-9/C-2 /C-1 /C-6 , from H-9
0
1
substituted benzene ring. The H- and C-NMR (Table 1)
1
0
to C-8, H-9 to C-7/C-8 . It also showed signals at d 4.79
H
spectrum showed signals at d 4.65 (1H, d, J = 4.2 Hz),
(1H, t, J = 4.4 Hz), 4.10 (1H, m), 3.70 (1H, m), 3.40 (1H,
0
H
0
00
00
4
.61 (1H, d, J = 4.2 Hz), 3.05 (2H, m), 4.16 (2H, m), 3.79
0
m) and dc 86.2 (C-8 ), 72.1 (C-7 ), and 59.8 (C-9 ), which
0
0
(
(
2H, m) and d 85.1 (C-7,7 ), 71.2 (C-9), 71.0 (C-9 ), 53.7
C-8), 53.5 (C-8 ), suggesting the presence of a furofuran
0
are typical signals of the glyceryl moiety of 8-O-4 system
C
0
neolignan. Furthermore, the HMBC experiment (Fig. 2)
00 00 00
lignan skeleton with 7,7 -diequatorial diayl substitution
with different aryl groups. This deduction was further
showed long-range correlations from H-7 to C-9 /C-8 /C-
00
0
0
00
00
00
2 /C-6 /C-1 ; correlations from H-8 to C-4/C-1
123

J Nat Med
Fig. 2 Selected HMBC
correlations of compounds 1
and 2
OCH3
3
'
OH
1
'
O
5
'
9
7'
H CO
3
OH
H
3''
H
7
9'
H CO
3
5
1
5''
O
1''
9
''
O
H O
O
3
HO
8''
''
OCH3
6
'
7
OH
H
5
'
O
5
''
HO
HO3
7
1
' O
1''
3
'
H
OH
8
1
HO
H
H
3''
OCH3
OH
5
1
2
1
13C
HMBC H
confirmed the deduction, revealing that the aryl glycerol
0
Table 2 Inhibitory effects of compounds 2–7 isolated from Ilex
pubescens on HeLa cell (n = 3)
0
portion with C-8 was linked to C-4 by an ether linkage.
Comparison of NMR data of 2 with the known hedyotol C
17] demonstrated that 2 possessed the same planar struc-
Compound
Cytotoxic activity [IC50 (lM)]
[
0
0
2
50.60 ± 6.47
51.17 ± 8.12
[100
ture but different absolute configuration between H-7 and
0
0
3
H-8 . The configuration of 2 was supported by CD spec-
, J 00 00
.
7 ,8
1
trum, and the H-NMR coupling constant J7,8, J
4
0
0
7 ,8
5
[100
The coupling constant of J7,8 and J
0
0
(4.2 Hz) and the
0
7 ,8
6
23.88 ± 4.52
29.14 ± 3.01
19.55 ± 3.47
chemical shift for the bridge carbons (C-8/C-8 , 53.7/53.5)
indicated that the aryl groups were pseudoequatorial and
7
a
0
Fluorouracil
cis-oriented with H-8 and H-8 [12, 18]. A negative cotton
a
effect at 278 nm in the CD spectrum revealed that the
absolute configuration of the furofuran unit was
Positive control
0
0
7
R,7 R,8S,8 S [12, 18]. The large and small J values for
0
Experimental
H-7 and H-8 of 8-O-4 neolignan diastereoisomers corre-
spond to the threo form and erythro form, respectively. The
small coupling constant (J = 4.4 Hz) observed between
General experimental procedures
0
0
00
00
H-7 and H-8 indicated that the aryl glycerol-8 -yloxy
moiety was determined to be in the erythro configuration
UV spectra were recorded on an Agilent UV 1100 spec-
trophotometer. IR spectra were recorded on a Bruker IFS
[14]. In addition, the CD spectrum of compound 2 showed
negative Cotton effects at 239.5 nm, indicating that the
55 spectrometer. CD spectra were determined with a Bio-
0
0
00
Logic Science MOS-450 spectrometer. NMR experiments
were performed on Bruker ARX-400 and AV-600 spec-
trometers. The chemical shifts are stated relative to TMS
and expressed in d values (ppm), with coupling constants
reported in Hertz. HRESIMS were obtained on an Agilent
absolute configurations at C-7 and C-8 of compound 2
0
0
00
were the 7 S and 8 R forms [14, 18]. Consequently, the
0
00
0
00
structure of 2 was defined as (7R,7 R,7 S,8S,8 S,8 R)-
0
00 0 00 0 0
4
4
,4 -dihydroxy-3,3 ,3 ,5-tetramethoxy-7,9 :7 9-diepoxy-
0
0
0
00 00
,8 -ox-y-8,8 -sesquineolignan-7 ,9 -diol.
6200 TOF mass spectrometer. Silica gel GF254 prepared
Compounds 2–7 were tested for their cytotoxic activities
for TLC and silica gel (200–300 mesh) for column chro-
matography (CC) were obtained from Qingdao Marine
Chemical Factory (Qingdao, People’s Republic of China).
Sephadex LH-20 was a product of Pharmacia. RP-HPLC
separations were conducted using an LC-10AT VP liquid
against HeLa cells by the MTT assay (Table 2). Com-
pounds 6 and 7 showed mild inhibitory effects, whilst the
activities of 2 and 3 were very weak. Compounds 4 and 5
exhibited no significant inhibitory activity, with IC50 val-
ues higher than 100 lM concentration. The IC50 values of
chromatograph with a Phenomenex Pack ODS-A column
˚
1 were not acquired due to the paucity of the available
sample.
(250 9 10 mm, 5 lm, 120 A) and SPD-10A VP UV/VIS
1
23

J Nat Med
1
3
detector. All reagents were HPLC or analytical grade, and
were purchased from Tianjin Damao Chemical Company.
Spots were detected on TLC plates under UV light, or by
heating after spraying with anisaldehyde-H SO reagent.
600 MHz) and C-NMR (DMSO-d , 100 MHz) spectrum
6
?
data see Table 1; HRESIMS: m/z 473.1432 [M ? Na] ; calcd
?
for [M ? Na] , 473.1424.
Compound 2: pale yellow oil; UV (MeOH) max (log e)
57 (2.74), 230 (2.66); IR (KBr) cm : 3393, 1594, 1517,
2
4
-
1
2
1
Plant material
1463, 1126, 1029; H-NMR (DMSO-d , 600 MHz) and
6
1
3
C-NMR (DMSO-d , 150 MHz) spectrum data see
6
-
Table 1; HRESIMS: m/z 583.2189 [M - H] ; calcd for
The roots of I. pubescens were collected from Bozhou,
Anhui province, China, and identified by Professor Jincai
Lu, Department of Pharmaceutical Botany, School of
Traditional Chinese Materia Medica, Shenyang Pharma-
ceutical University. A voucher specimen (AP-20120601)
has been deposited in the herbarium of the Department of
Natural Products Chemistry, Shenyang Pharmaceutical
University.
-
[M - H] , 583.2179.
Acid hydrolysis of compound 1
Compound 1 (3 mg) was dissolved with 2 M HCl (1 mL)
and heated for 3 h at 95 °C. After cooling, the reaction
mixture was extracted with CHCl three times, and the
3
aqueous layer was evaporated repeatedly to dryness with
EtOH until neutral. Then, the residue of sugar was dis-
solved in water (1 mL) and analyzed by HPLC with chiral
detection under the following conditions. HPLC was con-
ducted using a Shodex Asahipak NH2P-50 4E column and
a JAsco OR-4090 detector, the mobile phase consisted of
acetonitrile and water (3:1), the flow rate was kept constant
at 0.8 mL/min. The peak shape (positive peak) and reten-
tion time (14.9 min) was consistent with D-glucose.
Extraction and isolation
The dried roots of I. pubescens (10 kg) were extracted with
7
0 % EtOH (90 L 9 2 h 9 3). The obtained extract was
then concentrated under reduced pressure in a rotary
evaporator to remove the ethanol, yielding the crude
sample extract. Then, the resulting extract was suspended
in H O (15 L), and partitioned successively with petroleum
2
ether, EtOAc, and n-BuOH (15 L 9 3). The EtOAc extract
(
102 g) was subjected to silica gel CC (10 9 80 cm) and
Inhibitory activities on HeLa bioassay
eluted with CH Cl -CH OH (100:0, 100:1, 50:1, 20:1,
2
2
3
1
0:1, 5:1, 1:1 and 0:1 v/v) to obtain six fractions (IE1–IE6).
Fraction IE3 (20 g) was subjected to a silica gel column
6 9 80 cm) and eluted with PE-EtOAc (from 30:1 to 0:1)
Cytotoxicity for compounds 2–7 was performed in HeLa cells
by the MTT colorimetric assay. Briefly, HeLa cells were
seeded into 96-well tissue culture plates at a density of
(
4
to produce six fractions (IE31–IE36). Fraction IE34 (4.8 g)
was further subjected to silica gel CC (3 9 25 cm) and
eluted successively with gradient of petroleum ether-actone
3 9 10 cells/well. The cells were allowed to grow for 24 h
and subsequently treated with the test compounds at different
concentrations for 48 h. Fluorouracil was used as the positive
control while vehicle control DMSO was used as the negative
control. After incubation, 10 lL/well MTT was added and
further incubated for 4 h. Thereafter, growth medium was
removed and the blue formazan product was solubilized in
DMSO. Absorbance was measured on a microplate reader at
570 nm. Data are presented as mean ± SD. Each experiment
was performed at least three times.
(
10:1–1:1) to afford fractions IE341–IE346. Fraction IE343
was chromatographed over Sephadex LH-20 (CH Cl /
2
2
MeOH, 1:1; 1.5 9 30 cm) and then separated by RP-
HPLC (50 % MeOH/H O, 2 mL/min) to afford compounds
2
2
(18 mg, t = 39 min) and 3 (27.6 mg, t = 36 min).
R R
Fraction IE345 was chromatographed over Sephadex LH-
0 (CH Cl /MeOH, 1:1; 1.5 9 30 cm) and then separated
2
2
2
by RP-HPLC (45 % MeOH/H O, 2 mL/min) to afford
2
Acknowledgment The authors are grateful to the analytical detec-
tive center of Shenyang Pharmaceutical University for help with the
NMR measurements.
compounds 4 (14 mg, t = 49 min) and 5 (20 mg,
R
tR = 53 min). Fraction IE346 was purified by RP-HPLC
(
(
(
48 % MeOH/H O, 2 mL/min) to afford compounds 6
2
47 mg, t = 42 min) and 7 (15 mg, t = 46 min). IE5
R
R
38 g) was subjected to RP-C18 silica gel CC (10 9 80 cm)
and eluted with MeOH/H O (1:9–8:2) to produce six
2
References
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LH-20 column and RP-HPLC (50 % MeOH/H O, 2 mL/
2
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. Fujimoto T, Fujimura K, Kuramoto A (1991) Electrophysiolog-
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-1
max (log e) 263.9 (2.94), 220.5 (3.35); IR (KBr) cm : 3430,
1
920, 2850, 1639, 1462, 1384, 1130; H-NMR (DMSO-d ,
2
6
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J Nat Med
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