Two new secoiridoid glucosides and a new lignan from the roots of Ilex pubescens

Article abstract of DOI:10.1007/s11418-018-1227-5

Two new secoiridoid glucosides, ilexpublignoside (1), pubzenoside (2), and a new lignan, ilexlignan B (3), along with seven known compounds (4–10) were isolated from the roots of Ilex pubescens for the first time. Their chemical structures were elucidated on the basis of extensive spectroscopic methods, including IR, UV, HR-ESI–MS, CD, NMR experiments, as well as comparison with the reported data.

Full text of DOI:10.1007/s11418-018-1227-5

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1227-5
NOTE
Two new secoiridoid glucosides and a new lignan from the roots of Ilex
pubescens
1
1
1
1
1
1
Yang Zhang · Bowen Deng · Yida Cui · Xue Chen · Jiayi Bi · Guogang Zhang
Received: 27 August 2017 / Accepted: 15 July 2018
©
The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Two new secoiridoid glucosides, ilexpublignoside (1), pubzenoside (2), and a new lignan, ilexlignan B (3), along with seven
known compounds (4–10) were isolated from the roots of Ilex pubescens for the ꢀrst time. Their chemical structures were
elucidated on the basis of extensive spectroscopic methods, including IR, UV, HR-ESI–MS, CD, NMR experiments, as well
as comparison with the reported data.
Keywords Ilex pubescens · Secoiridoid glucosides · Lignan · Structural elucidation
Introduction
analyses. Here, we describe the isolation and structural elu-
cidation of these isolated compounds (Fig. 1).
Mao-dong-qing, the dried root of Ilex pubescens, is a Chi-
nese herbal medicine commonly used for the treatment of
cardiovascular diseases and hypercholesterolemia [1]. I.
pubescens Hook et Arn. is an evergreen shrub that belongs
to the Aquifoliaceae family and is mainly distributed in south
China. Previous phytochemical investigation of its roots and
leaves revealed the isolation of triterpene saponins [2–4],
lignan glycosides [5], phenylethyl alcohol derivatives [6] and
secoiridoid glucosides [7]. Pharmacological investigation
revealed several bioactivities, such as alleviating inꢁamma-
tion, inhibiting platelet aggregation, enlarging blood vessels,
improving microcirculation, preventing thrombosis, reduc-
ing cardiac ischemia, decreasing excitation of the cardiac
system, and enhancing anoxia resistance [8–11].
Results and discussion
Two new secoiridoid glucosides, ilexpublignoside (1), pub-
zenoside (2), and a new lignan, ilexlignan B (3), and seven
known compounds (4-10) were isolated from the n-BuOH
extract of the roots of I. pubescens by means of silica gel CC,
ODS CC, Phenomenex C-18 column, and reversed-phase
HPLC. Compounds 4-10 were identiꢀed as jasurolignoside
(4) [12], isolariciresinol 4′-O-β-d-glucopyranoside (5) [13],
(7S,8R)-balanophonin-4-O-β-d-glucopyranoside (6) [14],
phillyrin (7) [15], fraxiresinol-4′-O-β-d-glucopyranoside
(8) [16], (1S,2R)-1-(4′-O-β-d-glucopyranosyl-3′-
methoxyphenyl)-2-(4″-hydroxy-3″-methoxyphenyl)-1,3-
propanediol (9) [17], 4-(1,2,3-trihydroxypropyl)-2,6-
dimethoxyphenyl-1-O-β-d-glucopyranoside (10) [18], by
comparison of their spectral data with the literatures (Fig. 1).
These compounds were isolated for the ꢀrst time from I.
pubescens.
In this study, two new secoiridoid glucosides (1 and 2)
and a new lignan (3), together with seven known compounds
were isolated from the root of I. pubescens. Their structures
were identiꢀed by extensive spectroscopic and spectrometric
Compound 1 was obtained as a white amorphous solid.
Its molecular formula was established as C H O by pos-
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-018-1227-5) contains
supplementary material, which is available to authorized users.
4
3
56 21
+
itive-ion HR-ESI–MS at m/z 931.3232 ([M+Na] calcd for
C H O Na, 931.3245) and NMR data. The IR spectrum
4
3
56 21
*
Guogang Zhang
zggth@163.com
−1
of 1 showed the existence of hydroxyl (3422 cm ), carbonyl
−
1
−1
(
1706 cm ) and aromatic moieties (1628, 1512 cm ). The
1
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, People’s Republic of China
presence of d-glucose was conꢀrmed by acid hydrolysis of 1
with 2 M HCl, followed by HPLC analysis comparing with
Vol.:(0
1
123456789)
3

Journal of Natural Medicines
Fig. 1 Chemical structure for compound 1-10
1
authentic samples. The H NMR spectrum of 1 (Table 1)
at δ 3.74 and C-3′, and the methoxy signal at δ 3.77 and
H
H
showed five aromatic proton signals at δ 7.04 (1H, d,
C-3″, revealing that the methoxy groups were located at C-3′
and C-3″, respectively. The coupling constants of two ano-
meric protons demonstrated the presence of β-conꢀguration
of the d-glucose units. The location of the other functional
groups and NMR data assignments of 1 were determined by
HMBC and HSQC spectroscopic analysis (Table 1).
H
J=8.4 Hz), 6.97 (1H, br s), 6.89 (1H, dd, J=1.9, 8.4 Hz),
6
.68 (2H, s), two aromatic methoxyl signals at δ 3.77 (3H,
H
s), 3.74 (3H, s), four oxygenated methine proton signals at
δ 5.44 (1H, d, J=7.0 Hz), 3.61 (1H, m), 3.71 (1H, m), 3.41
H
(
2H, m), as well as a methine proton and two methylene pro-
ton signals at δ 3.40 (1H, m), 2.54 (2H, t, J=7.3 Hz), 1.71
The relative conꢀguration at C-7′ and C-8′ was deduced
H
1
(
2H, m). Furthermore, the H NMR spectrum of 1 exhib-
as threo from the coupling constant of J
= 7.0 Hz
H-7′, H-8′)
(
ited signals for an oleoside 11-methyl ester moiety [δ 7.49
[14, 15]. In the CD spectrum (Fig. 3), there was a positive
Cotton eꢂect at 279 nm, indicating the absolute conꢀgura-
tion at C-7′ and C-8′ of compound 1 was 7′S, 8′R forms [19,
20]. Thus, the structure of compound 1 was characterized as
shown in Fig. 1 and named as ilexpublignoside.
H
(
1H, s), 5.83 (1H, s), 5.96 (1H, q, J=6.6 Hz), 4.64 (1H, d,
J=7.8 Hz), 3.56 (3H, s), 1.62 (3H, d, J=6.8 Hz)] [15]. A
1
13
comprehensive analysis and comparison of its H and
C
NMR data with those of jasurolignoside [12] revealed that
they were similar in structure, except for the location of an
oleoside-type secoiridoid moiety. In the HMBC spectrum
Compound 2 was obtained as pale yellow solid, showing
UV absorption at λ
223 nm. It possessed the molecu-
max
(
Fig. 2), a long-range correlation from H-7′ to C-1′/C-8′/C-
lar formula C H O , as deduced from positive-ion HR-
51 68 29
+
9
′/C-4″, and from H-8′ to C-1′/C-7′/C-9′/C-5″ indicated the
ESI–MS (m/z=1167.3750 [M+Na] , calcd 1167.3744) and
NMR data. The IR spectrum of 2 demonstrated the pres-
existence of benzofuran lignans moiety. Moreover, the cor-
relation between H-1‴ and C-4′, H-6‴ and C-7, H-1″″ and
C-1 showed that one sugar moiety was located between C-4′
and C-7, and the other was assigned at C-1. The HMBC
spectrum showed a correlation between the methoxy signal
−
1
−1
ence of hydroxyl (3425 cm ), carbonyl (1707 cm ) and
−
1
oleꢀnic bonds (1631 cm ). The existence of d-glucose was
conꢀrmed by acid hydrolysis of 2 with 2 M HCl, followed
by the HPLC analysis comparing with authentic material.
1
3

Journal of Natural Medicines
Table 1 H and 13C NMR data
1
No.
δH (J in Hz)
δC
92.8
153.4
107.7
30.1
No.
δH (J in Hz)
δC
of ilexpublignoside (1)
1
3
4
5
6
5.83 (br s)
7.49 (s)
–
3.87 (dd, 4.1, 8.7)
2.66 (dd, 4.1, 14.5)
3″
4″
5″
6″
7″
8″
9″
1‴
2‴
3‴
4‴
5‴
6‴
–
–
–
143.4
145.5
128.9
116.4
31.6
34.7
60.2
100.1
73.1
77.3
69.9
73.6
6.68 (1H, s)
2.54 (t, 7.3)
1.71 (m)
3.41 (m)
4.91 (d, 7.1)
3.27 (m)
3.28 (m)
3.17 (m)
3.55 (m)
4.29 (d, 10.8)
4.03 (dd, 6.7, 11.8)
4.64 (d, 7.8)
3.08 (m)
3.15 (m)
3.07 (m)
3.19 (m)
3.65 (m)
3.44 (m)
3.74 (3H, s)
3.77(3H, s)
40.1
2
–
.38 (dd, 9.2, 14.5)
7
8
9
170.5
123.1
129.1
13.0
166.2
51.1
135.6
110.6
148.9
146.1
115.4
118.1
86.6
5.96 (q, 6.6)
–
1.62 (d, 6.8)
–
3.56 (3H, s)
–
6.97 (br s)
–
1
1
1
1
2
3
4
5
6
7
8
9
0
1
1-OCH3
′
′
′
′
′
′
′
′
′
63.6
1″″
2″″
3″″
4″″
5″″
6″″
98.9
73.3
77.3
69.8
76.5
61.0
–
7.04 (d, 8.4)
6.89 (dd, 1.9, 8.3)
5.44 (d, 7.0)
3.40 (m)
3.61 (m)
53.5
62.9
3
.71 (dd, 5.4, 10.7)
3′-OCH3
3″-OCH3
55.7
55.7
1
2
″
″
–
135.2
112.5
6.68 (s)
1H NMR at 600 MHz, 13C NMR at 150 MHz, recorded in DMSO-d6
1
13
The H and C NMR were similar to those of reticuloside
(Δδ + 2.4 ppm) and the upfield acylation shift of C-5‴
(Δδ−2.6 ppm) of 2 revealed the connections, which were
conꢀrmed by the HMBC correlation from H-6‴ to C-7′ and
H-9″ to C-7. In addition, the HMBC spectrum showed a cor-
[
21], except for the presence of one additional oleoside-type
1
secoiridoid moiety. The H NMR spectrum of 2 (Table 2)
revealed two characteristic oleoside-type secoiridoid moie-
ties [δ 5.88 (1H, s), 5.81 (1H, s), 7.53 (1H, s), 7.49 (1H,
relation between the proton signal at δ 3.76 and C-3″, 3.76
H
H
s), 5.98 (1H, q, J=6.8 Hz), 5.92 (1H, q, J=6.8 Hz), 1.68
and C-5″, 3.64 and C-1 as well as 3.63 and C-11′, indicating
the methoxy groups were located at C-3″, C-5″, C-11 and
C-11′, respectively. The coupling constants of the anomeric
protons manifested the presence of β-conꢀguration of the
d-glucose units. The location of the other functional groups
and NMR data assignments of 2 were determined by HMBC
and HSQC spectroscopic analysis (Table 2). Consequently,
the structure of compound 2 was characterized as shown in
Fig. 1 and named as pubzenoside.
(
3H, d, J=6.8 Hz), 1.56 (3H, d, J=6.8 Hz), 3.64 (3H, s),
3
.63 (3H, s), 4.65 (2H, d, J=8.1 Hz)] [15]. Moreover, the
1
H NMR data also showed the existence of a syringenin
moiety as deduced by aromatic proton signals at δ 6.75
H
(
2H, s), methoxy proton signals at δ 3.75 (6H, s), two
H
trans-conꢀguration oleꢀnic proton signals at δ 6.57 (1H,
H
d, J=15.8 Hz) and 6.29 (1H, dt, J=6.2, 15.8 Hz), and two
oxygenated proton signals at δ 4.68 (1H, m) and 4.63 (1H,
H
m). The HMBC correlation (Fig. 2) from H-1‴ to C-4″,
H-1″″ to C-1′, and H-1″‴ to C-1 manifested the connec-
tion of three glucopyranosyl moieties to the C-4″ of the
syringenin moiety, and C-1′ and C-1 of the two secoiridoid
moieties, respectively. The ester linkages between carbonyl
Compound 3 was obtained as pale yellow solid, showing
UV absorption at λ
230 and 277 nm. Its molecular for-
max
mula was determined to be C H O based on positive-ion
3
8
48 17
+
HR-ESI–MS (m/z=779.2783 [M+Na] , calcd 779.2789)
and NMR data. The IR spectrum displayed the existence of
−
1
−1
(
C-7′) of one secoiridoid moiety and C-6‴ of the glucopyra-
hydroxyl (3423 cm ) and oleꢀnic bonds (1631 cm ). The
presence of d-glucose was conꢀrmed by acid hydrolysis of
3 with 2 M HCl, followed by the HPLC analysis comparing
nosyl moiety, carbonyl (C-7) of the other one and C-9″ of
the syringenin moiety were determined by the comparison
1
3
1
13
of the C NMR data of 2 with those of syringin [22]. The
downꢀeld acylation shift of C-6‴ (Δδ+3.1 ppm) and C-9″
with authentic material. The H and C NMR was simi-
lar with those of erythro-guaiacylglycerol-β-syringaresinol
1
3

Journal of Natural Medicines
Fig. 2 HMBC spectrum for
compound 1-3
Fig. 3 CD spectrum for com-
pound 1
1
3

Journal of Natural Medicines
Table 2 H and 13C NMR data
1
No.
δH (J in Hz)
δC
93.0
153.5
107.7
30.2
No.
δH (J in Hz)
δC
of pubzenoside (2)
1
3
4
5
6
5.88 (br s)
7.53 (s)
–
3.90 (dd, 4.0, 9.2)
2.71 (dd, 4.0, 14.6)
6″
7″
8″
9″
6.75 (s)
104.6
122.9
133.4
64.7
6.57 (d, 15.8)
6.29 (dt, 6.2, 15.8)
4.68 (m)
40.6
4.63 (m)
2
–
.48 (dd, 9.4, 14.6)
1‴
2‴
3‴
4‴
5‴
6‴
4.87 (d, 7.0)
3.15 (m)
3.19 (m)
3.28 (m)
3.15 (m)
4.19 (d, 10.7)
4.00 (dd, 6.5, 11.8)
4.65 (d, 8.1)
3.05–3.29 (m)
3.17 (m)
102.5
74.0
76.3
70.0
73.8
63.8
7
8
9
170.5
123.1
129.1
13.1
166.2
51.3
5.98 (q, 6.8)
–
1.68 (d, 6.8)
–
3.63 (3H, s)
5.81 (1H, br s)
7.49 (s)
1
1
1
1
3
4
5
6
0
1
1-OCH3
′
′
′
′
′
92.8
1″″
2″″
3″″
4″″
5″″
6″″
98.9
73.3
77.4
70.0
76.5
61.1
153.4
107.7
30.0
–
3.78 (m)
2.55 (dd, 5.0, 14.7)
3.16 (m)
40.1
3.05–3.29 (m)
3.68 (d, 11.4)
3.45 (dd, 6.2, 11.4)
4.65 (d, 8.1)
3.05–3.29 (m)
3.06 (m)
2
–
.30 (dd, 9.2, 14.7)
7
8
9
1
1
1
1
2
3
4
5
′
170.4
123.1
129.1
12.9
166.2
51.3
131.8
104.6
152.8
134.2
152.8
′
5.92 (q, 6.8)
–
1.56 (d, 6.8)
–
3.63 (s)
–
6.75 (s)
–
–
–
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
99.1
73.3
77.4
70.0
76.5
61.1
′
0′
1′
3.06 (m)
1′-OCH3
3.05–3.29 (m)
3.68 (d, 11.4)
3.45 (dd, 6.2, 11.4)
3.76 (3H, s)
3.76 (3H, s)
″
″
″
″
″
3″-OCH3
5″-OCH3
56.3
56.3
1H NMR at 600 MHz, 13C NMR at 150 MHz, recorded in DMSO-d6
ether 4″-O-β-d-glucopyranoside [23], except for the diꢂerent
60.2. Furthermore, the HMBC spectrum represented long-
range correlations from H-7″ to C-1″/C-2″/C-6″/C-8″, H-9″
to C-7″/C-8″, H-8″ to C-7″/C-4′, suggesting that the aryl
glycerol portion with C-8″ was connected to C-4′ by an ether
linkage. The correlation between H-1‴ and C-4 in HMBC
manifested that the glucose was linked to C-4.
1
location of the glucopyranosyl moiety. The H NMR of com-
pound 3 showed signals of ꢀve aromatic methoxy groups at
δ 3.76 (6H, s), 3.75 (6H, s) and 3.71 (3H, s), along with
H
ꢀ
ve aromatic proton signals at δ 6.96 (1H, s), 6.78 (1H,
H
d, J = 8.1 Hz), 6.68 (1H, d, J = 8.1 Hz), 6.66 (2H, s) and
.64 (2H, s), indicating the existence of two 1,3,4,5-tetrasu-
6
The small coupling constants of J
= J
H-7, H-8)
(
bstituted benzene rings and a 1,3,4-trisubstituted benzene
=5.2 Hz and the chemical shifts for bridge carbons
H-7′, H-8′)
(
1
13
ring. In the H and C NMR (Table 3) spectra, the signals
(C-8/C-8′) revealed that two aryl substituents were equatorial
and cis-oriented with H-8, H-8′ [24, 25]. Except that the cou-
pling constant (J=4.0 Hz) observed between H-7″ and H-8″
suggested the relative conꢀguration was deduced as erythro
[25, 27].The positive Cotton eꢂect at 277 nm indicated that
the absolute conꢀguration of the furofuran unit was 7S, 7′S,
8R, 8′R [26]. The CD spectrum of 3 (Fig. 4) showed a nega-
tive Cotton eꢂect at 230 nm, demonstrating that the absolute
conꢀguration at C-7″ and C-8″ was to be 7″S and 8″R form
[25, 27]. Thus, the structure of 3 was identiꢀed as (7S, 8R,
7′S, 8′R, 7″S, 8″R)-guaiacylycerol-β-syringaresinol ether
at δ 4.67 (2H, t, J=5.2 Hz), 4.21 (2H, m), 3.82 (2H, dd,
H
J=3.3, 8.9 Hz), 3.06 (2H,m) and δ 85.1, 71.2, 71.3, 53.7,
C
5
3.6 suggested the presence of a furofuran lignan skeleton
with 7, 7′-diequatorial diaryl substitution with diꢂerent aryl
groups, which was further conꢀrmed by the HMBC cor-
relation (Fig. 2) from H-7 to C-2/C-8/C-9, H-7′ to C-2′/C-
1
8
′/C-9′, H-9′ to C-7 and H-9 to C-7′. The H NMR spec-
trum of 3 revealed typical signals of the glyceryl moiety
of 8-O-4′ system neolignan at δ 4.84 (1H,m), 3.99 (1H, d,
H
J=4.0 Hz), 3.19 (1H,m), 3.62 (1H,m) and δ 71.4, 87.0 and
C
1
3

Journal of Natural Medicines
Table 3 1H and 13C NMR data of ilexlignan B (3)
No. δH (J in Hz) δC No. δH (J in Hz) δC
4-O-β-d-glucopyranoside, named as ilexlignan B. The cou-
pling constant of the anomeric protons manifested the exist-
ence of β-conꢀguration of the d-glucose unit. The location
of the other functional groups and NMR data assignments
of 3 were determined by HMBC and HSQC spectroscopic
analysis (Table 3).
1
2
3
4
5
6
7
8
9
–
133.7 2″
104.2 3″
152.6 4″
132.9 5″
152.6 6″
104.2 7″
85.1 8″
6.96 (s)
–
–
6.68 (d, 8.1) 114.6
6.78 (d, 8.1) 119.1
4.84 (m)
3.99 (d, 4.0) 87.0
3.19 (m)
3.62 (m)
110.9
146.8
145.3
6.66 (s)
–
–
–
6.66 (s)
4.67 (d, 5.2)
3.06 (m)
71.4
Experimental
53.7 9″
60.2
3.82 (dd, 3.3, 8.9) 71.2
General experimental procedures
HR-ESI–MS was determined with a Bruker micrOTOF-
4
.21 (m)
1‴
135.3 2‴
103.3 3‴
152.5 4‴
136.9 5‴
152.5 6‴
103.3
4.88 (d, 7.7) 102.6
1′
2′
3′
4′
5′
6′
7′
8′
9′
–
3.19 (m)
3.02 (m)
3.12 (m)
3.19 (m)
3.58 (m)
3.40 (m)
74.2
77.2
69.9
76.5
60.9
1
13
6.64 (s)
–
–
–
6.64 (s)
4.67 (d, 5.2)
3.06 (m)
Q mass spectrometer. H, C and 2D NMR spectra were
recorded on a Bruker AV-600 spectrometer. IR spectra were
recorded on a Bruker IFS 55 spectrometer. UV spectra were
obtained from a Shimadzu UV-1700. CD measurements
were performed on a Bio-Logic Science MOS-450 spec-
trometer. HPLC analyses were carried out using an Agi-
lent 1100 series system (Agilent Technologies, Palo Alto,
CA, USA) coupled with a diode array detector. RP-HPLC
separations were achieved using an LC-10AT VP liquid
chromatography with a Phenomenex Pack ODS-A column
(250 mm×10 mm, 5 µm) and SPD-10A VP UV/VIS detec-
tor. Silica gel (200–300 mesh) for column chromatography
85.1 3, 5-OCH3 3.76 (6H, s)
53.6 3′, 5′-OCH3 3.75 (6H, s)
56.4
55.9
55.5
3.82 (dd, 3.3, 8.9) 71.3 3″-OCH3
3.71 (3H, s)
8.74 (s)
4
–
.20 (m)
4″-OH
–
1
″
132.9
1H NMR at 600 MHz, 13C NMR at 150 MHz, recorded in DMSO-d6
50 MHz, obtained in DMSO-d6
1
(
CC) and silica gel GF254 used for TLC analysis were pur-
chased from Qingdao Marine Chemical Factory (Qingdao,
Fig. 4 CD spectrum for com-
pound 3
1
3

Journal of Natural Medicines
People’s Republic of China). HPLC-grade methanol, ace-
tonitrile and water were obtained Kangkede and Wahaha,
respectively. All analytical grade reagents were purchased
from Tianjin Damao Company.
Compound 2: pale yellow solid; UV (MeOH) max (log
−
1
ε): 223 (2.61); [α]-16.5°(CH OH, c=0.5);IR (KBr) cm :
3
3422, 2921, 2852, 1706, 1628, 1512, 1447, 1384, 1306,
−
1 1
1266, 1209, 1076, 856, 814, 772 cm ; H NMR (DMSO-
1
3
d , 600 MHz) and C NMR (DMSO-d , 150 MHz) see
6
6
+
,
Plant material
Table 2; HR-ESI–MS: m/z 1167.3750 [M+Na] calcd for
+
[
M+Na] , 1167.3744.
The roots of I. pubescens were purchased from Bozhou,
Anhui province and identified by Professor Jincai Lu,
Department of Pharmaceutical Botany, School of Traditional
Chinese Materia Medica, Shenyang Pharmaceutical Univer-
sity. A voucher specimen (AP-20120601) was deposited in
the herbarium of the Department of Natural Products Chem-
istry, Shenyang Pharmaceutical University.
Compound 3: pale yellow solid; UV (MeOH) max (log
ε): 230 (2.66) and 277 (2.89); [α]-8.0°(CH OH, c=0.5);IR
3
−
1
(KBr) cm : 3425, 2922, 1707, 1631, 1504, 1440, 1384,
−
1
1305, 1260, 1159, 1077, 854, 817, 770, 617, 419 cm ;
1
13
H NMR (DMSO-d , 600 MHz) and C NMR (DMSO-
6
d , 150 MHz) see Table 3; HR-ESI–MS: m/z 779.2783
6
+
,
+
[M+Na] calcd for [M+Na] , 779.2789.
Extraction and isolation
Acid hydrolysis of compounds
The dried roots of I. pubescens(15 kg) were powered and
reꢁux extracted with 70% EtOH (90 L × 2 h) three times.
The ꢀltrate was concentrated and yielded the crude sam-
ple extract after removing the solvent under vacuum. The
obtained crude extract was suspended in water (15L) and
then partitioned successively with petroleum ether, EtOAc,
and n-BuOH (15L × 3). The n-BuOH extract was evapo-
rated under reduced pressure to obtain a residue (300 g),
which was subjected to macroporous resin eluting with
Compounds 1, 2 and 3 (each 2 mg) were dissolved with
2
M HCl (5 ml) and heated under reꢁux for 5 h at 90 ℃ with
magnetic stirring, respectively. The mixture was partitioned
with CHCl three times after cooling to yield the aglycon
3
and the sugar, respectively. The residue of sugar was dis-
solved in water (1 ml) and analyzed by HPLC with chiral
detection (JAsco OR-4090 detector). Separations were car-
ried out on a Shodex Asahipak NH2P-50 4E at a ꢁow rate of
0
.8 ml/min using acetonitrile–water (3:1) as mobile phase.
EtOH-H O (0:100, 30:70, 50:50, 70:30, 90:10) to yield
2
The peaks of the hydrolysates of 1, 2 and 3 and the retention
times were consistent with d-glucose.
four fractions (Fr. A-D). Fraction B was subjected to silica
gel CC (8×120 cm) eluted with CH Cl -MeOH (100:0 to
2
2
0
:100, v/v) to give 7 fractions (Fr. B1-B7). Fraction B6
Acknowledgements The authors are grateful to the analytical detec-
tive center of Shenyang Pharmaceutical University and Northeastern
University for help with the NMR measurements.
was fractionated on silica gel CC (4 × 60 cm) eluted with
CH Cl -MeOH (100:0 to 0:100, v/v) to give 4 fractions
2
2
(
(
Fr. B61-B64). Fraction B63 was subjected to ODS CC
4 × 60 cm) using MeOH-H O (10–100%, v/v) to yield 5
2
References
fractions (Fr. B631-B635). Compound 1 (14 mg) and com-
pound 4 (32 mg) were obtained from Fr. B632 through
1
.
Jiangsu New Medical College (1977) Directory of Chinese Mate-
ria Medica. Shanghai Scientiꢀc and Technological Press, Shang-
hai, p 441
semi-preparative RP-HPLC with 50% MeOH/H O as elut-
2
ing system, respectively. These fractions were then treated
with similar methods, respectively; ꢀrst, CC over silica
gel eluted with CH Cl -MeOH (100:0 to 0:100, v/v) and
2. Wu P, Gao H, Li ZH, Liu ZQ (2015) Two new triterpene saponins
from the roots of Ilex pubescens. Phytochem Lett 12:17–21
2
2
3
. Zhou Y, Chai XY, Zeng KW, Zhang JY, Li N, Jiang Y, Tu PF
then CC over ODS using MeOH-H O (10% to 90%, v/v)
2
(
2013) Ilexpublesnins C-M, eleven new triterpene saponins from
as mobile phase and then semi-preparative HPLC on a
the roots of Ilex pubescens. Planta Med 79:70–77
Phenomenex C-18 column (eluted with MeOH-H O from
4. Zang CX, Lin CZ, Xiong TQ, Zhu CC, Yang JY, Zhao ZX (2010)
New triterpene saponins from the root of Ilex pubescens. Fitotera-
pia 81:788–792
2
2
0:80 to 50:50) to yield compounds 2 (17 mg), 3 (7.6 mg),
5
(12 mg), compound 6 (5 mg),7 (9 mg), 8 (18 mg), 9 (3 mg)
5
.
Zhou YB, Wang JH, Li XM, Li X (2008) A new lignan derivative
and 10 (18 mg).
from the root of Ilex pubescens. Chin Chem Lett 19:550–552
Compound 1: white amorphous solid; UV (MeOH)
6. Zhou YB, Wang JH, Li XM, Fu XC, Yan Z, Cong Y, Li X (2008)
Two new 2-phenylethyl alcohol derivatives and one new lignan
derivative from the root of Ilex pubescens. Helv Chim Acta
max (log ε): 229 (2.65) and 279 (3.12); [α]-20°(CH OH,
3
−
1
c = 0.5);IR (KBr) cm : 3423, 2922, 2851, 2169, 1697,
9
1:1244–1250
1
6
631, 1403, 1384, 1338, 1271, 1115, 865, 833, 703, 688,
7
.
Yang X, Ding Y, Zhang DM (2007) Isolation and identiꢀcation
−
1
1
13
17 cm ; H NMR (DMSO-d , 600 MHz) and C NMR
of iridoid glycosides from Ilex pubescens. Chin J Med Chem
6
1
7:173–177
(
DMSO-d , 150 MHz) see Table 1; HR-ESI–MS: m/z
6
+
+
9
31.3232 [M+Na] ; calcd for [M+Na] , 931.3245.
1
3

Journal of Natural Medicines
8
9
.
.
Lin X, Chen J, Wu SJ, Wang BJ, Xiong TQ, Zhu CC, Zhao ZX
19. Liu QB, Huang XX, Bai M, Chang XB, Yan XJ, Zhu T, Zhao W,
Peng Y, Song SJ (2014) Antioxidant and anti-inꢁammatory active
dihydrobenzofuran neolignans from the seeds of Prunus tomentos.
J Agric Food Chem 62:7796–7803
(
2016) Vasorelaxant action of n-BuOH extract of the radix of
Ilex pubescens Hook et arn and mechanism. Pharmacol Clin Chin
Materia Medica 32:146–150
Wang LL (2007) Study on anti-inꢁammatory eꢂects of maodong-
qing oral liquid. J Med Forum 28:22–23
20. Xiong L, Zhu CG, Li YR, Tian Y, Lin S, Yuan SP, Hu JF, Hou Q,
Chen NH, Yang YC, Shi JG (2011) Lignans and neolignans from
Sinocalamus afnis and their absolute conꢀgurations. J Nat Prod
74:1188–1200
1
0. Chen YY, Xiong TQ, Zhao YM, Liao JY, Lin X, Tan QL, Zhao
ZX (2015) Antithrombotic activity of Ilex pubescens total extract
and the action of mechanism based on ADP. Chin Arch Trad Chin
Med 33:1092–1096
21. Bi XY, Li W, Sasaki T, Li Q, Mitsuhata N, Asada Y, Zhang QB,
Koike K (2011) Secoiridoid glucosides and related compounds
from Syringa reticulata and their antioxidant activities. Bioorg
Med Chem Lett 21:6426–6429
1
1. Zhang SW, Li RM, Xu JW, Wang F, Qiang J, Li AQ, Zhang J
(
2015) Eꢂects of ilexoside E on hypoxia/reoxygenation injury of
H9C2 myocardial cells and its mechanism. Trad Chin Drug Res
Clin Pharmacol 26:591–595
22. Liu XQ, Li WW, Sheng KX, Liu J, Chen FK (2006) Studies on
the chemical constituents of the n-BuOH extract of Polygonum
bistorta L. J Shenyang Pharmaceut Univ 23:15–17
1
1
2. Shen YC, Hsieh PW, Kuo YH (1998) Neolignan glucosides from
Jasminum urophyllum. Phytochemistry 48:719–723
23. Yukinori M, Tsuyoshi T (1998) Studies on constituents of Scutel-
i)
3. Guyot RB, Trendel JM, Albrecht P, Schaeꢂer A (2000) Glycosides
and phenylpropanoid glycerol in Vitis vinifera Cv.gewurztraminer
wine. J Agric Food Chem 48:6178–6182
laria species X I X lignan glycesides of roots of Scutellaria
baicalensis GEORGI. Nat Med 52(1):82–86
24. Sakushima A, Coskun M, Maoka T, Nishibe S (1996) Dihyd-
robenzofuran lignans from Boreava orientalis. Phytochemistry
43:1349–1354
1
1
4. Han JJ, Liu H, Guo P, Cong YQ, Fang Y (2016) Chemical con-
stituents from Serissa japonica. J Chin Med Mater 39:94–97
5. Kwak JH, Kang MW, Roh JH, Choi SU, Zee OP (2009) Cytotoxic
phenolic compounds from Chionanthus retusus. Arch Pharm Res
25. Tezuka Y, Kasimu R, Li JX, Basnet P, Tanaka K, Namba T, Kad-
ota S (1998) Constituents of roots of Salvia deserta Schang. Chem
Pharm Bull 46:107–112
3
2:1681–1687
1
1
6. Li S, An TY, Li J, Shen Q, Lou FC, Hu LH (2006) PTP1B inhibi-
tors from Saussurea lappa. J Asian Nat Prod Res 8:281–286
7. Jiang JS, Feng ZM, Wang YH, Zhang PC (2005) New phenolics
from the roots of Symplocos caudata W all. Chem Pharm Bull
26. Hofer O, Scholm R (1981) Stereochemistry of tetrahydrofurofunan
derivatives-circular dichroism and absolute conformation. Tetra-
hedron 37(6):1181–1186
27. Huo CH, Liang H, Zhao YY, Wang B, Zhang QY (2008) Neol-
ignan glycosides from Symplocos caudata. Phytochemistry
69:788–795
5
3:110–113
1
8. Feng J, Yang XW (2012) Constituents from the leaves of Aqui-
laria sinensis. Chin J Chin Mater Med 37:230–234
1
3