New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd

Article abstract of DOI:10.1016/j.fitote.2015.04.007

Ten flavonoid glycosides, apetalumosides A (1), B₁-B₇ (2-8), and C (9), quercetin 3-O-(2,6-di-O-β-d-glucopyranosyl)-β-d-glucopyranoside (10), were obtained from the seeds of Lepidium apetalum Willd. Their structures were elucidated by chemical and spectroscopic methods (UV, IR, NMR, and HRESI-TOF-MS). 1-9 were new isolates. The NMR data for 10 was reported here for the first time. On the other hand, activity screening results showed that 1-3 and 10 had triglyceride accumulation inhibitory effects in HepG2 cells. Preliminary structure-activity relationship study revealed that 7-hydroxyl group is an essential moiety.

Full text of DOI:10.1016/j.fitote.2015.04.007

FITOTE-03164; No of Pages 9
Fitoterapia xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
1
Q1 New bioactive flavonoid glycosides isolated from the seeds of Lepidium
apetalum Willd
2
a
a
b
a
a
3
4
Q2 Pingping Shi , Liping Chao , Tingting Wang , Erwei Liua,b, Lifeng Han a,b, Qi Zong , Xiaoxia Li ,
a,
a,b,
Yi Zhang ⁎, Tao Wang
⁎
OOF
a
5
6
7
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshan Road, Nankai
District, Tianjin 300193, China
b
8
1
0
a r t i c l e i n f o
a b s t r a c t
1
1
1
1
1
Article history:
1 7
Ten flavonoid glycosides, apetalumosides A (1), B –B (2–8), and C (9), quercetin 3-O-(2,6-di-O-β-D- 15
2
3
4
Received 11 March 2015
Accepted in revised form 4 April 2015
Available online xxxx
glucopyranosyl)-β-D-glucopyranoside (10), were obtained from the seeds of Lepidium apetalum 16
Willd. Their structures were elucidated by chemical and spectroscopic methods (UV, IR, NMR, and 17
HRESI-TOF-MS). 1–9 were new isolates. The NMR data for 10 was reported here for the first time. On 18
the other hand, activity screening results showed that 1–3 and 10 had triglyceride accumulation 19
inhibitory effects in HepG2 cells. Preliminary structure–activity relationship study revealed that 20
2
2
2
2
2
3
4
5
6
7
Keywords:
Lepidium apetalum
Flavonoid glycosides
HepG2 cells
Triglyceride accumulation inhibitory effects
7-hydroxyl group is an essential moiety.
21
©
2015 Published by Elsevier B.V. 22
2 Q8 3 1. Introduction
1 7
new flavonoid glycosides, apetalumosides A (1), B –B (2–8), 44
and C (9), together with a known one, quercetin 3-O-(2,6-di-O- 45
β-D-glucopyranosyl)-β-D-glucopyranoside (10) were obtained 46
from it. The NMR data for 10 was reported for the first time. On 47
the other hand, activity screening results showed that 1–3 48
and 10 had triglyceride accumulation inhibitory effects in 49
HepG2 cells. This paper deals with their isolation and structure 50
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
Lepidium apetalum Willd belongs to the Brassicaceae family.
As a Chinese medical plant, it is widely distributed in Hebei,
Liaoning and Jilin provinces, and Inner Mongolia Autonomous
Region, etc. in China. The dry mature seeds of this plant have
been used in Traditional Chinese Medicine (TCM) to relieve
cough, prevent asthma, reduce edema, and promote urination.
Several studies have reported that it contains various types of
secondary metabolites, such as oil, flavonoids, sterols and
cardiac glycosides [1–3], and showed anti-oxidant [1], antibac-
terial [2], and cardiac [4] activities. Clinical research suggests
that it is useful for heart failure [5,6], pigmentation [7], and oral
elucidation and their TG accumulation inhibitory effect.
51
52
53
2
. Experimental
2.1. General
cancer [8]. But syste
U
matic
N
resea
C
rch for
O
chem
RREition CTED PR
UV and IR spectra were determined on a Varian Cary 54
50 UV–Vis and Varian 640-IR FT-IR spectrophotometer, respec- 55
ical co
mpos
and structure–activity relationship has not yet been reported.
Then studies of phytochemical and activity of constituents from
L. apetalum seeds were developed in our lab. As a result, nine
tively. Optical rotations were recorded on a Rudolph Autopol® IV 56
automatic polarimeter. NMR spectra were measured on a Bruker 57
1
5
00 MHz NMR spectrometer at 500 MHz for H and 125 MHz for 58
1
3
C NMR (internal standard: TMS). Negative-ion HRESI-TOF-MS 59
⁎
Corresponding authors at: Tel./fax: +86 22 5959 6163.
were obtained on an Agilent Technologies 6520 Accurate-Mass 60
Q-Tof LC/MS spectrometer. 61
E-mail addresses: zhwwxzh@263.net (Y. Zhang), wangtao@tjutcm.edu.cn
(T. Wang).
http://dx.doi.org/10.1016/j.fitote.2015.04.007
367-326X/© 2015 Published by Elsevier B.V.
0
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

2
P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
6
2
Column chromatographies (CC) were performed on
2.3.1 . Apetalumoside A (1)
117
25
63
macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin,
China), Silica gel (74–149 μm, Qingdao Haiyang Chemical Co.,
Ltd., Qingdao, China), ODS (50 μm, YMC Co., Ltd., Tokyo, Japan),
and Sephadex LH-20 (Ge Healthcare Bio-Sciences, Uppsala,
Sweden). Preparative high performance liquid chromatography
(PHPLC) column, Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm,
Nakalai Tesque, Inc., Tokyo, Japan) was used to separate the
constituents.
Yellow powder. [α]
D
−26.5° (c = 0.33, MeOH); IR νmax 118
−
1
64
(KBr) cm : 3343, 2923, 1655, 1604, 1507, 1457, 1355, 1290, 119
1200, 1073, 801; UV λmax (MeOH) nm (log ε): 354 (4.22), 268 120
65
66
(4.23, sh), 255 (4.29). 1H NMR (DMSO-d
6
, 500 MHz): δ 6.19 121
67
(1H, br. s, H-6), 6.43 (1H, br. s, H-8), 7.79 (1H, d, J = 1.5 Hz, H-2′), 122
6.92 (1H, d, J = 8.5 Hz, H-5′), 7.61 (1H, dd, J = 1.5, 8.5 Hz, H-6′), 123
5.69 (1H, d, J = 7.0 Hz, H-1″), 3.54 (1H, dd, J = 7.0, 9.5 Hz, H-2″), 124
3.49 (1H, dd, J = 9.5, 9.5 Hz, H-3″), 3.24 (1H, dd, J = 9.5, 9.5 Hz, 125
H-4″), 3.31 (1H, m, H-5″), [3.43 (1H, dd, J = 4.5, 11.5 Hz), 3.81 126
68
69
70
2
(1H, br. d, ca. J = 12 Hz), H -6″], 4.63 (1H, d, J = 7.5 Hz, H-1‴), 127
7
1
2.2. Plant material
3.05 (1H, dd, J = 7.5, 9.0 Hz, H-2‴), 3.10 (2H, dd, J = 9.0, 9.0 Hz, 128
H-3‴ and 4‴), 3.16 (1H, m, H-5‴), [3.49 (1H, dd, J = 4.5, 11.0 Hz), 129
7
7
7
7
7
2
3
4
5
6
The seeds of L. apetalum were collected from Anguo city,
China, and identified by Dr. Li Tianxiang (The Hall of TCM
Specimens, Tianjin University of TCM, China). The voucher
specimen was deposited at the Academy of Traditional Chinese
Medicine of Tianjin University of TCM (No. 20120501).
2
3.56 (1H, br. d, ca. J = 11 Hz), H -6‴], 4.02 (1H, d, J = 7.5 Hz, 130
H-1″″), 2.80 (1H, dd, J = 7.5, 8.0 Hz, H-2″″), 2.94 (1H, dd, J = 8.0, 131
8.0 Hz, H-3″″), 2.98 (1H, dd, J = 8.0, 8.0 Hz, H-4″″), 2.82 (1H, 132
m, H-5″″), [3.37 (1H, dd, J = 4.5, 12.0 Hz), 3.55 (1H, br. d, ca. 133
J = 12 Hz), H
2
-6″″], 12.60 (1H, br. s, 5-OH), 3.86 (3H, s, 3′-OCH
C NMR (125 MHz, DMSO-d ) spectroscopic data, see Table 1; 135
HRESI-TOF-MS: Negative-ion mode m/z 801.2114 [M–H] 136
3
); 134
13
6
−
7
7
2.3. Extraction and isolation
41
(calcd for C34H O22 801.2095).
137
7
7
8
8
8
8
8
8
8
9
0
1
2
3
4
5
The seeds of L. apetalum Willd (10 kg) were crashed to
coarse powder, and refluxed with 50% EtOH. The solvent was
evaporated under reduced pressure to yield the 50% EtOH
2.3.2 . Apetalumoside B
1
(2)
138
25
Yellow powder. [α]
D
−47.7° (c = 0.70, MeOH); IR νmax 139
−
1
(KBr) cm : 3341, 2922, 1698, 1654, 1602, 1513, 1450, 1361, 140
extract. Then, the extract was partitioned in a CHCl
3
–H
2
O
O
1271, 1169, 1074, 817; UV λmax (MeOH) nm (log ε): 331 (4.35), 141
269 (4.25, sh), 252 (4.32). H NMR (500 MHz, DMSO-d
1
mixture (1:1, v/v) to give CHCl and H O partitions. The H
3
2
2
6
): δ 6.14 142
layer was subjected to D101 macroporous resin CC and eluted
with H O and 95% EtOH, successively. As a result, H O and 95%
EtOH eluted fractions were obtained.
The EtOH fraction (80 g) was isolated by silica gel CC [CHCl
MeOH (100:0 → 100:3 → 100:5, v/v) → CHCl –MeOH–H
(1H, d, J = 1.5 Hz, H-6), 6.26 (1H, d, J = 1.5 Hz, H-8), 7.55 (1H, d, 143
J = 2.0 Hz, H-2′), 6.86 (1H, d, J = 8.5 Hz, H-5′), 7.58 (1H, dd, 144
J = 2.0, 8.5 Hz, H-6′), 5.64 (1H, d, J = 7.0 Hz, H-1″), 3.53 (1H, 145
dd, J = 7.0, 8.5 Hz, H-2″), 3.51 (1H, dd, J = 8.5, 9.0 Hz, H-3″), 146
3.27 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.30 (1H, m, H-5″), [3.44 147
2
2
86
3
–
87
3
2
O
88
(10:3:1 → 6:4:1, v/v/v) → MeOH], and 16 fractions (Fr. 1–16)
2
(1H, dd, J = 5.0, 11.5 Hz), 3.77 (1H, br. d, ca. J = 12 Hz), H -6″], 148
8
9
were given. Fraction 14 was separated by Sephadex LH-20 CC
4.66 (1H, d, J = 7.5 Hz, H-1‴), 3.14 (1H, dd, J = 7.5, 8.0 Hz, H-2‴), 149
3.24 (1H, dd, J = 8.0, 9.0 Hz, H-3‴), 3.22 (1H, dd, J = 9.0, 9.0 Hz, 150
H-4‴), 3.49 (1H, m, H-5‴), 4.22 (2H, m, H -6‴), 4.01 (1H, d, 151
2
J = 7.5 Hz, H-1″″), 2.81 (1H, dd, J = 7.5, 8.0 Hz, H-2″″), 2.95 152
(1H, dd, J = 8.0, 8.5 Hz, H-3″″), 2.99 (1H, dd, J = 8.5, 9.0 Hz, 153
H-4″″), 2.85 (1H, m, H-5″″), [3.38 (1H, dd, J = 5.5, 11.5 Hz), 154
90
[MeOH–H
Fraction 14-5 was purified by PHPLC [CH
(15:85, v/v)]. As a result, 18 fractions (Fr. 14-5-1–14-5-18) were
obtained. Fraction 14-5-6 was subjected to PHPLC [CH CN–1%
CH COOH (10:90, v/v)] to give apetalumoside A (1, 4.5 mg).
Fraction 14-5-16 was purified by PHPLC [MeOH–1% CH COOH
(34:66, v/v)] to yield apetalumoside B (2, 10.1 mg). Fraction
14-7 was purified by PHPLC [CH CN–1% CH COOH (14:86, v/v)],
and apetalumoside B (3, 5.9 mg) was given. Fraction 16 was
separated by PHPLC through gradient elution [MeOH–1%
COOH (20:80 → 25:75 → 30:70 → 35:65 → 40:60 →
01 50:50 → 60:40 → 100:0, v/v)] to obtain 26 fractions (Fr. 16-1–
02 16-26). Fraction 16-2 was further isolated by PHPLC [CH CN–1%
(4, 35.2 mg).
CN–1% CH COOH
(5, 10.8 mg) was given.
Fraction 16-5 was isolated by PHPLC [CH CN–1% CH COOH
(10:90, v/v)] to obtain 8 fractions (Fr. 16-5-1–16-5-8). Fractions
2
O (1:1, v/v)] to yield 7 fractions (Fr. 14-1–14-7).
91
3
CN–1% CH COOH
3
92
93
3
94
3
95
3
2
3.53 (1H, br. d, ca. J = 12 Hz), H -6″″], 7.11 (1H, d, J = 1.5 Hz, 155
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
7
8
9
1
H-2″‴), 6.73 (1H, d, J = 8.0 Hz, H-5″‴), 6.88 (1H, dd, J = 1.5, 156
8.0 Hz, H-6″‴), 7.36 (1H, d, J = 16.0 Hz, H-7″‴), 6.19 (1H, d, 157
J = 16.0 Hz, H-8″‴), 12.70 (1H, br. s, 5-OH), 3.76 (3H, s, 3″‴- 158
3
3
2
1
3
3 6
OCH ); C NMR (125 MHz, DMSO-d ) spectroscopic data, 159
00 CH
3
see Table 1; HRESI-TOF-MS: Negative-ion mode m/z 963.2395 160
−
[M–H] (calcd for C43
47
H O25 963.2412).
161
3
03
04
05
06
07
CH
Fraction 16-4 was purified by PHPLC [CH
(9:91, v/v)], and apetalumoside B
3
COOH (8:92, v/v)] to yield apetalumoside B
3
2.3.3 . Apetalumoside B
2
(3)
162
2
5
3
3
Yellow powder. [α]
D
−39.1° (c = 0.69, H
2
O); IR νmax (KBr) 163
cm− : 3315, 2885, 1702, 1654, 1603, 1513, 1452, 1361, 1271, 164
1
4
3
3
1170, 1073, 814; UV λmax (MeOH) nm (log ε): 331 (4.45), 270 165
(4.36, sh), 254 (4.44). 1H NMR (500 MHz, DMSO-d
): δ 6.17 166
6
08 16-5-5 and 16-5-6 were purified by PHPLC [MeOH–1%
09 CH COOH (19:81, v/v)] to give apetalumosides B (6,
10 19.5 mg) and B (8, 37.8 mg), respectively. Fraction 16-5-7
11 was separated by PHPLC [MeOH–1% CH COOH (20:80, v/v)]
12 to give quercetin 3-O-(2,6-di-O-β-D-glucopyranosyl)-β-D-
13 glucopyranoside (10, 13.6 mg) and apetalumoside C (9,
14 11.0 mg). Fraction law-16-8 was purified by PHPLC [CH
15 1% CH COOH (12:88, v/v)], and apetalumoside B (7, 14.9 mg)
16 was given.
(1H, d, J = 1.5 Hz, H-6), 6.38 (1H, d, J = 1.5 Hz, H-8), 7.59 (1H, d, 167
J = 2.0 Hz, H-2′), 6.85 (1H, d, J = 7.5 Hz, H-5′), 7.54 (1H, dd, 168
J = 2.0, 7.5 Hz, H-6′), 5.65 (1H, d, J = 7.5 Hz, H-1″), 3.52 (1H, 169
dd, J = 7.5, 8.5 Hz, H-2″), 3.39 (1H, dd, J = 8.5, 9.0 Hz, H-3″), 170
3.03 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.16 (1H, m, H-5″), [3.48 171
3
5
7
3
2
(1H, m, overlapped), 3.70 (1H, br. d, ca. J = 12 Hz), H -6″], 172
3
CN–
4.56 (1H, d, J = 7.5 Hz, H-1‴), 3.05 (1H, dd, J = 7.5, 8.5 Hz, H-2‴), 173
3.17 (1H, dd, J = 8.5, 9.0 Hz, H-3‴), 3.13 (1H, dd, J = 9.0, 9.0 Hz, 174
H-4‴), 3.08 (1H, m, H-5‴), [3.48 (1H, m, overlapped), 3.57 (1H, 175
3
6
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
3
t1:1
t1:2
Table 1
1
3C NMR data for 1–10 in DMSO-d
6
.
t1:3
No.
1
2
3
4
5
6
7
8
9
10
t1:4
2
3
4
5
155.9
132.7
177.2
161.1
98.7
155.4
132.8
177.1
161.1
98.6
156.0
132.6
177.3
161.1
98.9
156.3
133.1
177.4
160.8
99.2
156.6
133.0
177.5
160.9
99.4
156.0
133.1
177.3
160.7
99.2
156.1
133.1
177.3
160.7
99.2
156.8
132.9
177.6
160.8
99.4
156.3
133.0
177.4
160.7
99.2
155.6
132.8
177.2
161.2
98.6
t1:5
t1:6
t1:7
t1:8
6
t1:9
7
8
164.2
93.7
164.1
93.4
164.6
93.8
162.7
94.4
162.9
94.8
162.6
94.3
162.6
94.3
162.9
94.9
162.6
94.4
164.2
93.5
t1:10
t1:11
t1:12
t1:13
t1:14
t1:15
t1:16
t1:17
t1:18
t1:19
t1:20
t1:21
t1:22
t1:23
t1:24
t1:25
t1:26
t1:27
t1:28
t1:29
t1:30
t1:31
t1:32
t1:33
t1:34
t1:35
t1:36
t1:37
t1:38
t1:39
t1:40
t1:41
t1:42
t1:43
t1:44
t1:45
t1:46
t1:47
t1:48
t1:49
t1:50
t1:51
t1:52
t1:53
t1:54
t1:55
t1:56
t1:57
9
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
156.3
103.9
121.0
112.9
146.9
149.4
115.2
122.7
98.3
81.5
76.3
69.3
76.2
67.7
103.3
74.1
76.5
69.6
76.7
60.7
103.1
73.2
76.5
156.1
103.7
121.0
116.1
144.8
148.5
115.2
121.8
98.0
83.1
76.1
69.2
76.5
67.8
104.4
74.4
76.4
69.5
73.9
63.1
103.2
73.2
76.4
156.2
103.7
120.8
116.1
144.8
148.7
115.5
121.6
98.0
81.8
76.2
69.3
78.0
66.6
103.8
74.2
76.4
69.6
76.6
60.7
100.0
73.6
74.1
155.8
105.5
120.8
116.3
144.7
148.6
115.3
121.8
98.0
82.2
76.3
69.1
76.2
67.8
104.0
74.2
76.4
69.5
76.6
60.6
103.1
73.1
76.3
155.7
105.5
120.6
116.4
144.7
148.8
115.3
121.7
98.0
82.1
76.2
69.3
77.8
66.6
104.0
74.2
76.4
69.5
76.6
60.6
100.0
73.4
74.0
156.0
105.5
120.8
116.4
144.7
148.7
115.2
121.8
97.9
83.4
76.1
69.1
76.3
67.8
104.5
74.4
76.2
69.5
73.9
63.3
103.2
73.1
76.2
155.7
105.5
120.9
116.4
144.8
148.7
115.2
121.9
97.9
83.3
76.1
69.2
76.3
67.8
104.5
74.4
76.2
69.5
73.9
63.2
103.2
73.1
76.2
155.7
105.5
120.6
116.3
144.8
148.8
115.3
121.7
97.9
155.7
105.5
120.5
131.0
115.2
160.1
115.2
131.0
98.0
156.2
103.8
121.0
116.1
144.7
148.5
115.3
121.8
98.1
82.2
76.3
69.1
76.1
67.8
103.9
74.2
76.4
69.5
76.7
60.6
103.1
73.2
76.4
O
81.9
O
8
F
2.8
76.2
69.2
78.0
66.7
103.9
74.2
76.4
69.5
76.5
60.7
100.1
73.4
74.0
69.6
76.2
60.1
99.9
73.0
76.3
69.5
77.0
60.6
76.1
69.2
76.3
67.7
104.2
74.3
76.2
69.6
74.0
63.3
103.2
73.1
76.3
69.6
76.4
60.7
99.7
73.0
76.2
69.5
77.0
60.5
4‴
5‴
6‴
1″″
2″″
3″″
4″″
5″″
6″″
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
7″‴
8″‴
9″‴
1‴‴
2‴‴
3‴‴
4‴‴
5‴‴
6‴‴
7‴‴
8‴‴
9‴‴
3′-OCH
69.7
76.5
60.8
69.6
76.5
60.7
69.6
76.2
60.1
69.6
76.4
60.7
99.7
73.0
76.3
69.5
77.0
69.6
76.3
60.1
99.9
73.1
76.3
69.5
77.0
69.6
76.4
60.7
99.7
73.0
76.2
69.5
77.0
69.6
76.4
60.7
99.8
73.1
76.2
69.5
77.0
69.6
76.4
60.6
125.3
111.0
147.7
149.2
115.3
122.7
144.8
113.9
166.4
125.6
110.8
147.9
149.2
115.3
122.8
144.8
114.5
165.5
60.5
60.6
60.5
60.6
125.1
130.0
115.8
159.7
115.8
130.0
144.5
114.1
165.4
125.3
114.9
145.3
148.1
115.6
120.9
144.9
113.5
166.4
125.3
110.8
147.7
149.1
115.3
122.9
144.8
113.9
166.4
125.6
110.0
147.9
149.2
115.5
122.8
144.7
114.4
165.5
125.2
114.8
145.4
148.2
115.5
120.9
145.0
113.4
166.3
3
55.8
3″‴-OCH
3‴‴-OCH
3
55.5
55.7
3
55.5
55.7
UNCORRECTED PR
2.3.4. Apetalumoside B (4)
1
1
1
1
1
1
1
1
1
1
76
2
br. d, ca. J = 12 Hz), H -6‴], 4.28 (1H, d, J = 8.0 Hz, H-1″″), 4.42
186
187
3
25
77 (1H, dd, J = 8.0, 8.5 Hz, H-2″″), 2.84 (1H, dd, J = 8.5, 9.0 Hz,
78 H-3″″), 3.13 (1H, dd, J = 9.0, 9.0 Hz, H-4″″), 2.68 (1H, m, H-5″″),
79 3.44 (2H, m, H -6″″), 7.34 (1H, d, J = 1.5 Hz, H-2″‴), 6.86 (1H, d,
2
Yellow powder. [α] −52.6° (c = 0.96, MeOH); IR ν
D
max
−1
(KBr) cm : 3366, 2921, 1653, 1601, 1493, 1344, 1302, 1199, 188
1072, 815; UV λ (MeOH) nm (log ε): 356 (4.25), 270 (4.25, 189
max
1
80 J = 7.5 Hz, H-5″‴), 7.12 (1H, dd, J = 1.5, 7.5 Hz, H-6″‴), 7.48
81 (1H, d, J = 16.0 Hz, H-7″‴), 6.22 (1H, d, J = 16.0 Hz, H-8″‴),
sh), 256 (4.37). H NMR (500 MHz, DMSO-d ): δ 6.43 (1H, br. s, 190
6
H-6), 6.73 (1H, br. s, H-8), 7.60 (2H, m, overlapped, H-2′ and 191
13
82 12.78 (1H, br. s, 5-OH), 3.89 (3H, s, 3″‴-OCH
3
); C NMR
6′), 6.89 (1H, d, J = 8.5 Hz, H-5′), 5.64 (1H, d, J = 7.5 Hz, H-1″), 192
3.57 (1H, dd, J = 7.5, 8.5 Hz, H-2″), 3.49 (1H, dd, J = 8.5, 9.0 Hz, 193
H-3″), 3.26 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.31 (1H, m, H-5″), 194
[3.44 (1H, dd, J = 5.0, 11.5 Hz), 3.81 (1H, br. d, ca. J = 12 Hz), 195
83 (125 MHz, DMSO-d
84 TOF-MS: Negative-ion mode m/z 963.2424 [M–H] (calcd for
6
) spectroscopic data, see Table 1; HRESI-
−
85
43 47
C H O25 963.2412).
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

4
P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
1
1
1
1
2
2
2
2
2
2
2
2
2
2
96
H
2
-6″], 4.62 (1H, d, J = 7.5 Hz, H-1‴), 3.08 (1H, dd, J = 7.5,
(1H, dd, J = 8.0, 9.0 Hz, H-3″‴), 3.20 (1H, dd, J = 9.0, 9.0 Hz, 255
H-4″‴), 3.48 (1H, m, H-5″‴), [3.49 (1H, dd, J = 4.5, 11.5 Hz), 3.72 256
(1H, br. d, ca. J = 12 Hz), H -6″‴], 6.89 (1H, br. s, H-2‴‴), 6.71 257
2
97 8.0 Hz, H-2‴), 3.21 (1H, dd, J = 8.0, 8.0 Hz, H-3‴), 3.16 (1H, dd,
98 J = 8.0, 9.0 Hz, H-4‴), 3.14 (1H, m, H-5‴), [3.48 (1H, m,
99 overlapped), 3.56 (1H, br. d, ca. J = 10 Hz), H
00 J = 7.5 Hz, H-1″″), 2.81 (1H, dd, J = 7.5, 8.5 Hz, H-2″″), 2.91
01 (1H, dd, J = 8.5, 9.0 Hz, H-3″″), 2.99 (1H, dd, J = 9.0, 9.0 Hz,
02 H-4″″), 2.84 (1H, m, H-5″″), [3.38 (1H, dd, J = 5.0, 11.5 Hz),
2
-6‴], 4.00 (1H, d,
(1H, d, J = 8.0 Hz, H-5‴‴), 6.78 (1H, br. d, ca. J = 8 Hz, H-6‴‴), 258
7.30 (1H, d, J = 16.0 Hz, H-7‴‴), 6.01 (1H, d, J = 16.0 Hz, H-8‴‴), 259
12.61 (1H, br. s, 5-OH); 13C NMR (125 MHz, DMSO-d
6
) spectro- 260
scopic data, see Table 1; HRESI-TOF-MS: Negative-ion mode m/z 261
−
03 3.52 (1H, br. d, ca. J = 12 Hz), H
2
-6″″], 5.09 (1H, d, J = 7.0 Hz,
1111.2780 [M–H] (calcd for C48
55
H O30 1111.2784).
262
04
05
06
07
08
09
H-1″‴), 3.27 (1H, dd, J = 7.0, 7.5 Hz, H-2″‴), 3.32 (1H, dd, J = 7.5,
9.0 Hz, H-3″‴), 3.19 (1H, dd, J = 9.0, 9.0 Hz, H-4″‴), 3.46 (1H, m,
H-5″‴), [3.48 (1H, m, overlapped), 3.72 (1H, br. d, ca. J = 11 Hz),
2.3.7. Apetalumoside B
6
(7)
263
25
Yellow powder. [α]
D
−41.6° (c = 0.99, H
2
O); IR νmax (KBr) 264
H
2
-6″‴], 12.64 (1H, br. s, 5-OH); 13C NMR (125 MHz, DMSO-d
6
)
cm−1: 3319, 2889, 1705, 1654, 1600, 1515, 1453, 1343, 1272, 265
1174, 1073, 816; UV λmax (MeOH) nm (log ε): 330 (4.45), 270 266
spectroscopic data, see Table 1; HRESI-TOF-MS: Negative-ion
mode m/z 949.2481 [M–H] (calcd for C39
−
(4.39, sh), 254 (4.46). 1H NMR (500 MHz, DMSO-d
49
H O27 949.2467).
6
): δ 6.38 267
1H, d, J = 2.0 Hz, H-6), 6.55 (1H, d, J = 2.0 Hz, H-8), 7.59 (1H, d, 268
(
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10 2.3.5. Apetalumoside B
4
(5)
J = 2.0 Hz, H-2′), 6.88 (1H, d, J = 8.0 Hz, H-5′), 7.58 (1H, dd, 269
J = 2.0, 8.0 Hz, H-6′), 5.63 (1H, d, J = 7.0 Hz, H-1″), 3.52 (1H, dd, 270
J = 7.0, 8.0 Hz, H-2″), 3.50 (1H, dd, J = 8.0, 8.0 Hz, H-3″), 3.25 271
(1H, dd, J = 8.0, 9.0 Hz, H-4″), 3.32 (1H, m, H-5″), [3.44 (1H, dd, 272
25
11
Yellow powder. [α]
D
2
−19.8° (c = 0.52, H O); IR νmax (KBr)
12 cm− : 3370, 2923, 1710, 1652, 1601, 1514, 1454, 1342, 1272,
1
13 1172, 1073, 827; UV λmax (MeOH) nm (log ε): 317 (4.26), 270
14 (4.27, sh), 254 (4.30). H NMR (500 MHz, DMSO-d
1
6
): δ 6.46
2
J = 5.5, 11.0 Hz), 3.77 (1H, br. d, ca. J = 11 Hz), H -6″], 4.66 273
15
(1H, d, J = 1.5 Hz, H-6), 6.71 (1H, d, J = 1.5 Hz, H-8), 7.62 (1H, d,
(1H, d, J = 7.5 Hz, H-1‴), 3.16 (1H, dd, J = 7.5, 8.0 Hz, H-2‴), 3.25 274
(1H, dd, J = 8.0, 8.5 Hz, H-3‴), 3.23 (1H, dd, J = 8.5, 8.5 Hz, H-4‴), 275
3.50 (1H, m, H-5‴), [4.20 (1H, br. d, ca. J = 12 Hz), 4.25 (1H, dd, 276
16 J = 2.0 Hz, H-2′), 6.85 (1H, d, J = 8.0 Hz, H-5′), 7.55 (1H, dd,
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
J = 2.0, 8.0 Hz, H-6′), 5.60 (1H, d, J = 7.5 Hz, H-1″), 3.53 (1H, dd,
J = 7.5, 9.0 Hz, H-2″), 3.40 (1H, dd, J = 9.0, 9.0 Hz, H-3″), 3.04
(1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.16 (1H, m, overlapped, H-5″),
2
J = 4.5, 12.0 Hz), H -6‴], 3.99 (1H, d, J = 7.5 Hz, H-1″″), 2.80 277
(1H, dd, J = 7.5, 8.5 Hz, H-2″″), 2.90 (1H, dd, J = 8.5, 9.0 Hz, 278
H-3″″), 2.98 (1H, dd, J = 9.0, 9.0 Hz, H-4″″), 2.84 (1H, m, H-5″″), 279
[3.38 (1H, dd, J = 5.0, 11.5 Hz), 3.54 (1H, br. d, ca. J = 12 Hz), 280
H -6″″], 5.01 (1H, d, J = 7.0 Hz, H-1″‴), 3.28 (1H, dd, J = 7.0, 281
2
8.5 Hz, H-2″‴), 3.32 (1H, dd, J = 8.5, 8.5 Hz, H-3″‴), 3.18 (1H, dd, 282
J = 8.5, 8.5 Hz, H-4″‴), 3.42 (1H, m, H-5″‴), [3.49 (1H, dd, J = 6.0, 283
2
[3.48 (1H, m, overlapped), 3.71 (1H, br. d, ca. J = 12 Hz), H -6″],
4.56 (1H, d, J = 8.0 Hz, H-1‴), 3.07 (1H, dd, J = 8.0, 8.0 Hz, H-2‴),
3.16 (1H, m, overlapped, H-3‴), 3.16 (1H, m, overlapped, H-4‴),
3.08 (1H, m, H-5‴), [3.48 (1H, m, overlapped), 3.55 (1H, br. d, ca.
J = 11 Hz), H -6‴], 4.26 (1H, d, J = 8.0 Hz, H-1″″), 4.42 (1H, dd,
2
J = 8.0, 9.0 Hz, H-2″″), 2.80 (1H, dd, J = 9.0, 9.0 Hz, H-3″″), 3.11
(1H, dd, J = 9.0, 9.0 Hz, H-4″″), 2.68 (1H, m, H-5″″), 3.43 (2H, m,
H -6″″), 5.08 (1H, d, J = 7.5 Hz, H-1″‴), 3.26 (1H, dd, J = 7.5,
2
9.0 Hz, H-2″‴), 3.29 (1H, dd, J = 9.0, 9.0 Hz, H-3″‴), 3.20 (1H, dd,
J = 9.0, 9.0 Hz, H-4″‴), 3.43 (1H, m, H-5″‴), [3.48 (1H, m,
overlapped), 3.71 (1H, br. d, ca. J = 12 Hz), H
J = 8.5 Hz, H-2‴‴,H-6‴‴), 6.86 (1H, d, J = 8.5 Hz, H-3‴‴,H-5‴‴),
7.48 (1H, d, J = 16.0 Hz, H-7‴‴), 6.12 (1H, d, J = 16.0 Hz, H-8‴‴),
2
11.5 Hz), 3.72 (1H, br. d, ca. J = 12 Hz), H -6″‴], 7.10 (1H, d, 284
J = 2.0 Hz, H-2‴‴), 6.73 (1H, d, J = 8.0 Hz, H-5‴‴), 6.87 (1H, 285
dd, J = 2.0, 8.0 Hz, H-6‴‴), 7.35 (1H, d, J = 16.0 Hz, H-7‴‴), 286
6.18 (1H, d, J = 16.0 Hz, H-8‴‴), 12.60 (1H, br. s, 5-OH), 3.76 287
1
3
3 6
(3H, s, 3‴‴-OCH ); C NMR (125 MHz, DMSO-d ) spectro- 288
2
-6″‴], 7.51 (2H, d,
scopic data, see Table 1; HRESI-TOF-MS: Negative-ion mode 289
−
m/z 1125.2946 [M–H] (calcd for C49
H
57
O
30 1125.2940).
290
1
3
12.75 (1H, br. s, 5-OH); C NMR (125 MHz, DMSO-d
spectroscopic data, see Table 1; HRESI-TOF-MS: Negative-ion
mode m/z 1095.2842 [M–H] (calcd for C48
6
)
7
2.3.8. Apetalumoside B (8)
291
25
Yellow powder. [α]
D
−66.5° (c = 0.81, MeOH); IR νmax 292
(KBr) cm : 3367, 2924, 1698, 1654, 1599, 1515, 1453, 1344, 293
−
−1
H
55
O
29 1095.2834).
1
276, 1177, 1074, 820; UV λmax (MeOH) nm (log ε): 335 (4.46), 294
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
36 2.3.6. Apetalumoside B
5
(6)
6
272 (4.35, sh), 255 (4.46). H NMR (500 MHz, DMSO-d ): δ 6.47 295
25
37
Yellow powder. [α]
D
−37.8° (c = 0.98, MeOH); IR νmax
(1H, br. s, H-6), 6.72 (1H, br. s, H-8), 7.63 (1H, br. s, H-2′), 6.87 296
(1H, d, J = 7.5 Hz, H-5′), 7.57 (1H, br. d, ca. J = 8 Hz, H-6′), 5.66 297
(1H, d, J = 7.0 Hz, H-1″), 3.53 (1H, dd, J = 7.0, 9.0 Hz, H-2″), 3.40 298
(1H, dd, J = 8.5, 9.0 Hz, H-3″), 3.04 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 299
3.17 (1H, m, overlapped, H-5″), [3.49 (1H, m, overlapped), 3.72 300
−
1
38 (KBr) cm : 3339, 2922, 1699, 1654, 1600, 1492, 1452, 1346,
39 1272, 1174, 1073, 818; UV λmax (MeOH) nm (log ε): 336 (4.34),
40 270 (4.28, sh), 253 (4.38). H NMR (500 MHz, DMSO-d
1
6
): δ 6.40
41 (1H, br. s, H-6), 6.61 (1H, br. s, H-8), 7.62 (1H, br. s, H-2′), 6.89
42 (1H, d, J = 8.5 Hz, H-5′), 7.59 (1H, d, ca. J = 9 Hz, H-6′), 5.67
43 (1H, d, J = 7.0 Hz, H-1″), 3.53 (1H, dd, J = 7.0, 8.0 Hz, H-2″),
44 3.51 (1H, dd, J = 8.0, 8.0 Hz, H-3″), 3.25 (1H, dd, J = 8.0, 8.5 Hz,
45 H-4″), 3.32 (1H, m, H-5″), [3.46 (1H, dd, J = 5.5, 11.5 Hz), 3.78
2
(1H, br. d, ca. J = 11 Hz), H -6″], 4.57 (1H, d, J = 7.0 Hz, H-1‴), 301
3.05 (1H, dd, J = 7.0, 9.0 Hz, H-2‴), 3.17 (1H, m, overlapped, 302
H-3‴), 3.17 (1H, m, overlapped, H-4‴), 3.07 (1H, m, H-5‴), 303
[3.49 (1H, m, overlapped), 3.58 (1H, br. d, ca. J = 11 Hz), 304
H -6‴], 4.27 (1H, d, J = 7.5 Hz, H-1″″), 4.42 (1H, dd, J = 7.5, 305
2
8.0 Hz, H-2″″), 2.77 (1H, dd, J = 8.0, 9.0 Hz, H-3″″), 3.12 (1H, dd, 306
J = 9.0, 9.5 Hz, H-4″″), 2.70 (1H, m, H-5″″), 3.44 (2H, m, 307
overlapped, H -6″″), 5.10 (1H, d, J = 7.0 Hz, H-1″‴), 3.28 (1H, dd, 308
2
J = 7.0, 8.5 Hz, H-2″‴), 3.29 (1H, dd, J = 8.5, 9.0 Hz, H-3″‴), 3.17 309
(1H, m, overlapped, H-4″‴), 3.44 (1H, m, overlapped, H-5″‴), 310
2
[3.49 (1H, m, overlapped), 3.72 (1H, br. d, ca. J = 11 Hz), H -6″‴], 311
7.35 (1H, br. s, H-2‴‴), 6.87 (1H, d, J = 7.5 Hz, H-5‴‴), 7.13 (1H, 312
br. d, ca. J = 8 Hz, H-6‴‴), 7.50 (1H, d, J = 15.5 Hz, H-7‴‴), 6.25 313
46 (1H, br. d, ca. J = 12 Hz), H
2
-6″], 4.66 (1H, d, J = 7.0 Hz, H-1‴),
47 3.16 (1H, dd, J = 7.0, 8.5 Hz, H-2‴), 3.26 (1H, dd, J = 8.5, 9.0 Hz,
48 H-3‴), 3.26 (1H, dd, J = 9.0, 9.0 Hz, H-4‴), 3.49 (1H, m, H-5‴),
49 [4.18 (1H, br. d, ca. J = 12 Hz), 4.22 (1H, dd, J = 4.5, 12.0 Hz),
50
51 9.0 Hz, H-2″″), 2.91 (1H, dd, J = 9.0, 9.0 Hz, H-3″″), 2.99 (1H, dd,
52
J = 9.0, 9.0 Hz, H-4″″), 2.85 (1H, m, H-5″″), [3.39 (1H, dd, J = 4.5,
53 11.0 Hz), 3.55 (1H, br. d, ca. J = 11 Hz), H -6″″], 5.04 (1H, d,
54 J = 7.0 Hz, H-1″‴), 3.27 (1H, dd, J = 7.0, 9.0 Hz, H-2″‴), 3.32
2
H -6‴], 4.00 (1H, d, J = 7.5 Hz, H-1″″), 2.81 (1H, dd, J = 7.5,
2
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
5
3
3
3
3
14 (1H, d, J = 15.5 Hz, H-8‴‴), 12.79 (1H, br. s, 5-OH), 3.90 (3H, s,
reaction mixture was dealt, then analyzed by CH
3
CN–H
2
O 371
1
3
3 6
15 3‴‴-OCH ); C NMR (125 MHz, DMSO-d ) spectroscopic data,
(70:30, v/v; flow rate 1.0 mL/min) using the same column 372
and detector as reference [9]. As result, D-glusose from 1–10 373
presented in the aqueous was carried out by comparison of its 374
retention time and optical rotation with that of authentic 375
16 see Table 1; HRESI-TOF-MS: Negative-ion mode m/z 1125.2963
−
17 [M–H] (calcd for C49
57
H O30 1125.2940).
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
18 2.3.9. Apetalumoside C (9)
samples, t
R
8.8 min (positive).
376
25
19
Yellow powder. [α]
D
−42.1° (c = 0.99, MeOH); IR νmax
−
1
20 (KBr) cm : 3366, 2923, 1702, 1655, 1601, 1492, 1449, 1347,
2.5. TG accumulation inhibitory effects in HepG2 cells
377
21 1277, 1178, 1073, 814; UV λmax (MeOH) nm (log ε): 331 (4.41),
22 267 (4.36, sh), 244 (4.35). H NMR (500 MHz, DMSO-d
1
6
): δ 6.40
Triglyceride accumulation inhibitory effects were screened 378
by free fatty acid induced hepatic cell line method as described 379
previously [9]. Briefly, HepG2 cells in high glucose Minimum 380
Essential Medium (MEM) was supplemented with 10% FBS and 3Q 8 41
0.2 mM oleic acid sodium salt, together with sample DMSO 382
solution (final concentration of DMSO was less than 0.1%). After 383
48 hour incubation, the amount of intracellular triglycerides was 384
determined with the Triglycerides kit (BioSino Bio-technology 385
and Science Inc., China) after cell lysis. Orlistat was used as 386
positive control.
23 (1H, br. s, H-6), 6.64 (1H, br. s, H-8), 8.04 (2H, d, J = 8.0 Hz,
24 H-2′,6′), 6.90 (2H, d, J = 8.0 Hz, H-3′,5′), 5.56 (1H, d, J = 7.0 Hz,
25 H-1″), 3.49 (1H, dd, J = 7.0, 8.5 Hz, H-2″), 3.46 (1H, dd, J = 8.5,
26 8.5 Hz, H-3″), 3.21 (1H, dd, J = 8.5, 8.5 Hz, H-4″), 3.31 (1H, m,
27 H-5″), [3.42 (1H, dd, J = 5.0, 12.0 Hz), 3.77 (1H, br. d, ca.
28 J = 12 Hz), H -6″], 4.67 (1H, d, J = 7.5 Hz, H-1‴), 3.14 (1H, dd,
2
29 J = 7.5, 9.0 Hz, H-2‴), 3.24 (1H, dd, J = 9.0, 9.0 Hz, H-3‴), 3.20
30 (1H, dd, J = 9.0, 9.0 Hz, H-4‴), 3.48 (1H, m, H-5‴), [4.19 (1H,
OOF
31 dd, J = 5.0, 11.5 Hz), 4.29 (1H, br. d, ca. J = 12 Hz), H
2
-6‴], 3.97
387
32 (1H, d, J = 7.5 Hz, H-1″″), 2.79 (1H, dd, J = 7.5, 8.5 Hz, H-2″″),
33 2.89 (1H, dd, J = 8.5, 9.0 Hz, H-3″″), 2.97 (1H, dd, J = 9.0,
34 9.0 Hz, H-4″″), 2.84 (1H, m, H-5″″), [3.36 (1H, dd, J = 5.0,
2.5.1 . Statistical analysis
388
Values are expressed as mean ± S.D. All the grouped data 389
were statistically performed with SPSS 11.0. Significant differ- 390
ences between means were evaluated by one-way analysis of 391
variance (ANOVA) and Tukey's Studentized range test was used 392
for post hoc evaluations. P b 0.05 was considered to indicate 393
2
35 11.5 Hz), 3.54 (1H, br. d, ca. J = 12 Hz), H -6″″], 5.04 (1H, d,
36 J = 7.0 Hz, H-1″‴), 3.26 (1H, dd, J = 7.0, 8.5 Hz, H-2″‴), 3.30
37 (1H, dd, J = 8.5, 8.5 Hz, H-3″‴), 3.19 (1H, dd, J = 8.5, 9.0 Hz,
38 H-4″‴), 3.46 (1H, m, H-5″‴), [3.48 (1H, dd, J = 5.0, 12.0 Hz),
39 3.72 (1H, br. d, ca. J = 12 Hz), H
40 6.69 (1H, d, J = 8.0 Hz, H-5‴‴), 6.76 (1H, br. d, ca. J = 8 Hz,
41
H-6‴‴), 7.30 (1H, d, J = 16.0 Hz, H-7‴‴), 6.01 (1H, d, J = 16.0 Hz,
42
H-8‴‴), 12.62 (1H, br. s, 5-OH); 13C NMR (125 MHz, DMSO-d
43
spectroscopic data, see Table 1; HRESI-TOF-MS: Negative-ion
mode m/z 1095.2831 [M–H]− (calcd for C48
29 1095.2834).
2
-6″‴], 6.89 (1H, br. s, H-2‴‴),
statistical significance.
394
3
3. Results and discussion
395
3
6
)
3
The seeds of L. apetalum were crashed and refluxed with 396
50% EtOH for 3 times. The solvent was evaporated under reduced 397
pressure to yield the 50% EtOH extract. Then, the extract was 398
344
55
H O
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
45 2.3.10.
46 glucopyranoside (10)
quercetin
3-O-(2,6-di-O-β-D-glucopyranosyl)-β-D-
partitioned in a CHCl
and H O partitions. The H
macroporous resin CC and successively eluted with H
2
95% EtOH. The 95% EtOH eluate from D101 CC was isolated by 402
Silica gel, ODS, Sephadex LH-20 CC, and finally preparative 403
HPLC to give nine new flavonoid glycosides, named as 404
apetalumosides A (1), B –B (2–8), and C (9), together with a 405
1 7
known one, quercetin 3-O-(2,6-di-O-β-D-glucopyranosyl)-β-D- 406
glucopyranoside. The structures of the new isolates were 407
3
–H
2
O mixture (1:1, v/v) to give CHCl
O layer was subjected to D101 400
O and 401
3
399
2
2
2
5
47
Yellow powder. [α]
D
2
−16.1° (c = 0.97, H O); IR νmax (KBr)
48 cm− : 3394, 2923, 1656, 1607, 1503, 1449, 1360, 1301, 1200,
1
49 1075, 817; UV λmax (MeOH) nm (log ε): 355 (4.19), 267 (4.23,
50 sh), 256 (4.29). H NMR (500 MHz, DMSO-d
1
6
): δ 6.18 (1H, d,
51 J = 1.5 Hz, H-6), 6.38 (1H, d, J = 1.5 Hz, H-8), 7.56 (1H, d,
52 J = 1.5 Hz, H-2′), 6.87 (1H, d, J = 8.5 Hz, H-5′), 7.59 (1H, dd,
53 J = 1.5, 8.5 Hz, H-6′), 5.64 (1H, d, J = 7.0 Hz, H-1″), 3.55
54 (1H, dd, J = 7.0, 9.0 Hz, H-2″), 3.48 (1H, dd, J = 9.0, 9.0 Hz,
55 H-3″), 3.27 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.31 (1H, m, H-5″),
56 [3.45 (1H, dd, J = 5.0, 11.5 Hz), 3.80 (1H, br. d, ca. J = 12 Hz),
shown in Fig. 1.
408
Apetalumoside A (1) was obtained as a yellow powder with 409
2
5
negative optical rotation [[α]
the molecular formula, C34
D
−26.5° (c = 0.33, MeOH)]. It had 410
57
2
H -6″], 4.61 (1H, d, J = 7.0 Hz, H-1‴), 3.08 (1H, dd, J = 7.0,
H
42
O
22 as deduced from the negative- 411
−
58 8.0 Hz, H-2‴), 3.18 (1H, dd, J = 8.0, 8.0 Hz, H-3‴), 3.16 (1H, dd,
59 J = 8.0, 8.0 Hz, H-4‴), 3.14 (1H, m, H-5‴), [3.49 (1H, dd, J = 4.5,
60 11.0 Hz), 3.56 (1H, br. d, ca. J = 11 Hz), H
61 J = 7.0 Hz, H-1″″), 2.82 (1H, dd, J = 7.0, 8.0 Hz, H-2″″), 2.95
62 (1H, dd, J = 8.0, 9.0 Hz, H-3″″), 3.00 (1H, dd, J = 9.0, 9.0 Hz,
63 H-4″″), 2.83 (1H, m, H-5″″), [3.38 (1H, dd, J = 4.5, 12.0 Hz),
ion HRESI-TOF-MS (m/z 801.2114 [M–H] , calcd for C34
H
41
O22 412
801.2095). The IR spectrum showed absorption bands ascribable 413
2
-6‴], 4.02 (1H, d,
UNCORRECTED PR
64 3.55 (1H, br. d, ca. J = 12 Hz), H
2
-6″″],12.63 (1H, br. s, 5-OH);
) spectroscopic data, see
1
3
65
C NMR (125 MHz, DMSO-d
6
66 Table 1; HRESI-TOF-MS: Negative-ion mode m/z 787.1952
67 [M–H] (calcd for C33
−
H
39
O
22 787.1938).
3
68 2.4. Acid hydrolysis of 1–10
3
69
70
A solution of ten new compounds (1–10, each 1.5 mg) in 1 M
HCl (1 mL) was heated under reflux for 3 h, respectively. The
3
Fig. 1. The compounds (1–10) obtained from the seeds of L. apetalum.
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

6
P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
−
1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
14
15
16
17
18
19
to hydroxyl (3443 cm ), aromatic ring (1655, 1604, 1507,
J = 2.0 Hz, H-2′), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 12.70 457
(1H, br. s, 5-OH)], three β-D-glucopyranosyl [δ 4.01 (1H, d, 458
J = 7.5 Hz, H-1″″), 4.66 (1H, d, J = 7.5 Hz, H-1‴), 5.64 (1H, d, 459
−
1
−1
1457 cm ), and O-glycosidic linkage (1073 cm ). On acid
hydrolysis and identification with HPLC analysis, the presence of
D-glucose was determined [9]. The H and C NMR (DMSO-d
1
13
6
,
J = 7.0 Hz, H-1″)], together with a trans-feruloyl group [δ
(3H, s, 3″‴-OCH ), 6.19 (1H, d, J = 16.0 Hz, H-8″‴), 6.73 (1H, d, 461
J = 8.0 Hz, H-5″‴), 6.88 (1H, dd, J = 1.5, 8.0 Hz, H-6″‴), 7.11 462
(1H, d, J = 1.5 Hz, H-2″‴), 7.36 (1H, d, J = 16.0 Hz, H-7″‴); δ 463
166.4 (C-9″‴)]. In the HMBC spectra, long-range correlations 464
between δ 5.64 (H-1″) and δ 132.8 (C-3), δ 4.66 (H-1‴) and δ 465
83.1 (C-2″), δ 4.01 (H-1″″) and δ 67.8 (C-6″), δ 4.22 (2H, m, 466
-6‴) and δ 166.4 (C-9″‴) were observed. Finally, the structure 467
of 2 was elucidated as shown in Fig. 1. 468
Apetalumoside B (3) was obtained as yellow powder 469
−39.1° (c = 0.69, H O)]. The same molecular formula, 470
25, as 2 was determined from the negative-ion HRESI- 471
TOF-MS. Treated it with 1 M HCl, and D-glucose was yielded [9]. 472
H
3.76 460
Table 1) spectra of 1, which were assigned by various NMR
3
1
1
experiments including
H H COSY, HSQC, HMBC, and HSQC-
20 TOCSY spectra (Fig. 2) revealed that 1 had an isorhamnetin
21 aglycon [δ 3.86 (3H, s, 3′-OCH ), 6.19 (1H, br. s, H-6), 6.43
22 (1H, br. s, H-8), 6.92 (1H, d, J = 8.5 Hz, H-5′), 7.61 (1H, dd,
23 J = 1.5, 8.5 Hz, H-6′), 7.79 (1H, d, J = 1.5 Hz, H-2′), 12.60
24 (1H, br. s, 5-OH)], and three β-D-glucopyranosyl groups [δ 4.02
25 (1H, d, J = 7.5 Hz, H-1″″), 4.63 (1H, d, J = 7.5 Hz, H-1‴), 5.69
C
3
H
C
H
C
H
C
H
H
2
C
1
13
26 (1H, d, J = 7.0 Hz, H-1″)]. The assignment of H and C NMR
27 data for three β-D-glucopyranosyls were achieved by HSQC-
2
2
5
[[α]
D
2
28 TOCSY experiments. According to the correlations between δ
29 98.3 (C-1″) and δ 3.24 (H-4″), 3.49 (H-3″), 3.54 (H-2″), 5.69
30 (H-1″); δ 3.43, 3.81 (H -6″) and δ 67.7 (C-6″), 69.3 (C-4″),
C
43 48
C H O
H
1
13
H
2
C
6
The H and C NMR (DMSO-d , Table 1) spectra, which were 473
1
1
31 76.2 (C-5″) observed in HSQC-TOCSY spectra, the NMR data
32 for inside β-D-glucopyranosyl were assigned. Meanwhile, the
assigned by various NMR experiments including H H COSY, 474
HSQC, HMBC, and HSQC-TOCSY spectra (Fig. 2) for 3 suggested 475
the presence of same moieties including quercetin, three 476
β-D-glucopyranosyls, and trans-feruloyl as 2. The difference 477
between 2 and 3 was on the linkage position of the trans- 478
feruloyl group, which was determined by HMBC experiment. In 479
33 correlations observed between δ
H
4.63 (H-1‴) and δ
3.49, 3.56
60.7 (C-6‴), 69.6 (C-4‴), 76.7 (C-5‴); δ 103.1
2.80 (H-2″″), 2.94 (H-3″″), 2.98 (H-4″″), 4.02
-6″″) and δ 60.8 (C-6″″), 69.7 (C-4″″),
C
69.6
34 (H-4‴), 74.1 (H-2‴), 76.5 (C-3‴), 103.3 (C-1‴); δ
H
35 (H
36 (C-1″″) and δ
37
(H-1″″); δ
38
39
40
41
42
43
2
-6‴) and δ
C
C
H
4
H
3.37, 3.55 (H
2
C
HMBC spectra, long-range correlations between δ
and δ 132.6 (C-3), δ 4.56 (H-1‴) and δ 81.8 (C-2″), δ
(H-1″″) and δ 66.6 (C-6″), δ 4.42 (1H, dd, J = 8.0, 8.5 Hz, H-2″″) 482
and δ 165.5 (C-9″‴) were found. On the other hand, to assign 483
H
5.65 (H-1″) 480
4
76.5 (C-5″″) in HSQC-TOCSY experiment suggested the chemical
shifts of two outer β-D-glucopyranosyl groups. The connectivities
of oligoglycoside moieties to the aglycon part were characterized
by a HMBC experiment on 1, in which long-range correlations
C
H
C
H
4.28 481
4
C
H
4
C
4
the badly overlapped protons in sugar chemical shift range, 484
HSQC-TOCSY experiment was developed. In the HSQC-TOCSY 485
spectra, the correlations between the following proton and 486
4
were found between δ
H
5.69 (H-1″) and δ
C
H
132.7 (C-3), δ 4.63
4
4
4
4
4
4
4
(H-1‴) and δ 81.5 (C-2″), δ
C
H
4.02 (H-1″″) and δ
C
67.7 (C-6″).
44 Consequently, the structure of apetalumoside A was deter-
45 mined as isorhamnetin 3-O-(2,6-di-O-β-D-glucopyranosyl)-β-
46 D-glucopyranoside (1). Though its name is the same as that in
47 Ref. [10], their structures are not the same as each other. Then,
48 compound 1 in this paper is a new one.
carbon pairs were observed: δ
3.16 (H-5″), 3.39 (H-3″), 3.52 (H-2″), 5.65 (H-1″); δ
(H-6b″) and δ 66.6 (C-6″), 69.3 (C-4″), 78.0 (C-5″); δ
(C-1‴) and δ 3.05 (H-2‴), 3.13 (H-4‴), 3.17 (H-3‴), 4.56 (H-1‴); 490
3.08 (H-5‴) and δ 60.7 (C-6‴), 69.6 (C-4‴), 74.2 (C-2‴), 76.4 491
(C-3‴), 76.6 (C-5‴); δ 100.0 (C-1″″) and δ 2.68 (H-5″″), 2.84 492
(H-3″″), 3.13 (H-4″″), 4.28 (H-1″″), 4.42 (H-2″″); δ 2.68 (H-5″″) 493
and δ 60.1 (C-6″″), 69.6 (C-4″″), 73.6 (C-2″″), 74.1 (C-3″″), 76.2 494
(C-5″″). Finally, the structure of 3 was determined.
Apetalumoside B (4) was yellow powder [[α]
50
(c = 0.96, MeOH)]. It had the molecular formula, C39H O27 as 497
C
98.0 (C-1″) and δ
H
3.03 (H-4″), 487
3.70 488
103.8 489
H
C
C
H
δ
H
C
49
50
51
52
53
54
55
56 J = 1.5 Hz, H-8), 6.86 (1H, d, J = 8.5 Hz, H-5′), 7.55 (1H, d,
Apetalumoside B
1
(2) was a yellow powder with negative
C
H
25
4
optical rotation [α]
D
−47.7° (c = 0.70, MeOH)]. Its molecular
H
4
48
formula C43H O
25 was determined by negative-ion HRESI-TOF-
C
−
4
MS (m/z 963.2395 [M–H] (calcd for C43
H
47
O
25 963.2412).
495
1
25
4
Treated 2 with 1.0 M HCl, and D-glucose was detected [9]. The H
and 13C NMR (DMSO-d
, Table 1) spectra indicated the presence
of quercetin moiety [δ 6.14 (1H, d, J = 1.5 Hz, H-6), 6.26 (1H, d,
3
D
−52.6° 496
4
6
4
deduced from the negative-ion HRESI-TOF-MS (m/z 949.2481 498
−
4
[M–H] , calcd for C39
49
H O27 949.2467). On acid hydrolysis and 499
Fig. 2. The main 1H 1H COSY and HMBC correlations of 1–3.
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
7
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
18 identification with HPLC analysis, the presence of D-glucose
δ
H
2.68 (H-5″″) and δ
C
60.1 (C-6″″), 69.6 (C-4″″), 74.0 (C-3″″), 561
1
13
19 was determined [9]. The H and C NMR (DMSO-d
6
, Table 1)
76.3 (C-5″″), then the NMR data for 3- and 6″-O-β-D- 562
glucopyranosyl were assigned, respectively. On the other 563
hand, the NMR data for 7-O-β-D-glucopyranosyl were decided 564
by the correlations found between δ 73.1 (C-2″‴) and δ 3.20 565
C H
(H-4″‴), 3.26 (H-2″‴), 3.29 (H-3″‴), 3.43 (H-5″‴), 5.08 (H-1″‴) 566
in HSQC-TOCSY spectra and the correlation observed between 567
20 spectra of 4, which were assigned by various NMR experiments
1
1
21 including
H
H COSY, HSQC, and HMBC spectra (Fig. 3)
22 revealed it had the same moiety, quercetin 3-O-(2,6-di-O-β-D-
23 glucopyranosyl)-β-D-glucopyranosyl [δ 4.00 (1H, d, J = 7.5 Hz,
24 H-1″″), 4.62 (1H, d, J = 7.5 Hz, H-1‴), 5.64 (1H, d, J = 7.5 Hz,
25 H-1″), 6.43 (1H, br. s, H-6), 6.73 (1H, br. s, H-8), 6.89 (1H, d,
26 J = 8.5 Hz, H-5′), 7.60 (2H, m, H-2′ and 6′), 12.64 (1H, br. s,
27 5-OH)] as 2 and 3. Meanwhile, another β-D-glucopyranosyl
28 [δ 5.09 (1H, d, J = 7.0 Hz, H-1″‴)] was found in 4. Comparison
1
1
δ
H
3.43 (H-5″‴) and δ
last, the NMR data for 2″-O-β-D-glucopyranosyl were deduced 569
by the correlations between δ 104.0 (C-1‴) and δ 3.07 (H-2‴), 570
3.16 (H-3‴ and 4‴), 4.56 (H-1‴); δ 3.08 (H-5‴) and δ 60.6 571
H
3.71 (H-6b″‴) in H H COSY spectra. At 568
C
H
H
C
1
13
29 of the H and C NMR data of 6–8 positions in 4 [δ
30 6.73 (H-8); δ 94.4 (C-8), 99.2 (C-6), 162.7 (C-7)] with those
31 in 3 [δ 6.17 (H-6), 6.38 (H-8); δ 93.8 (C-8), 98.9 (C-6),
H
6.43 (H-6),
(C-6‴), 69.5 (C-4‴), 74.2 (C-2‴), 76.4 (C-3‴), 76.6 (C-5‴) 572
observed from HSQC-TOCSY experiment. Finally, acid hydrolysis 573
of 5 with 1.0 M HCl, and D-glucose was yielded [9]. On the basis 574
of above mentioned evidence, the structure of apetalumoside B
(5) was determined.
C
H
C
32 164.6 (C-7)] revealed a glycoside substitution shift around the
33 7-position. Furthermore, in the HMBC experiment, long-
4
575
576
34 range correlation was observed between δ
H
5.09 (H-1″‴)
5
Apetalumoside B (6) was a yellow powder. Its molecular 577
OOF
35 and δ 162.7 (C-7). Consequently, the structure of 4 was
C
formula C48
MS (m/z 1111.2780 [M–H] , calcd for C48
was treated with 1.0 M HCl to give D-glucose [9]. The proton and 580
carbon signals in H and C NMR spectra (Table 1) were very 581
similar to those of 5, except for the signals due to the substituent 582
group, trans-caffeoyl [δ
(1H, d, J = 8.0 Hz, H-5‴‴), 6.78 (1H, br. d, ca. J = 8 Hz, H-6‴‴), 584
6.89 (1H, br. s, H-2‴‴), 7.30 (1H, d, J = 16.0 Hz, H-7‴‴); δ 166.4 585
(C-9‴‴)] on the oligoglycoside moieties. The linkage position of 586
trans-caffeoyl was elucidated to be 6‴ by the HMBC determina- 587
tion (Fig. 3), which showed long-range correlation between 588
56
H O30 was determined by negative-ion HRESI-TOF- 578
−
36 identified as quercetin 3-O-(2,6-di-O-β-D-glucopyranosyl)-
37 β-D-glucopyranosyl-7-O-β-D-glucopyranoside.
H
55
O
30 1111.2784). 6 579
1
13
5
38
39
40
41
42
43
44
Apetalumoside B
[[α]
D
4
(5) was with negative optical rotation
−19.8° (c = 0.52, H O)]. Its molecular formula C48
was determined by negative-ion HRESI-TOF-MS (m/z 1095.2842
25
5
2
56 29
H O
5
H
6.01 (1H, d, J = 16.0 Hz, H-8‴‴), 6.71 583
−
1
13
5
55 6
[M–H] , C48H O29 1095.2834). Its H and C NMR (DMSO-d ,
5
Table 1) spectra displayed a quercetin 3-O-(2,6-di-O-β-D-
glucopyranosyl)-β-D-glucopyranosyl-7-O-β-D-glucopyranosyl [δ
4.26 (1H, d, J = 8.0 Hz, H-1″″), 4.56 (1H, d, J = 8.0 Hz, H-1‴), 5.08
45 (1H, d, J = 7.5 Hz, H-1″‴), 5.60 (1H, d, J = 7.5 Hz, H-1″), 6.46
46 (1H, d, J = 1.5 Hz, H-6), 6.71 (1H, J = 1.5 Hz, H-8), 6.85 (1H, d,
47 J = 8.0 Hz, H-5′), 7.55 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 7.62 (1H,
48 J = 2.0 Hz, H-2′), 12.75 (1H, br. s, 5-OH)], together with a trans-
C
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
δ
H
[4.18 (1H, br. d, ca. J = 12 Hz), 4.22 (1H, dd, J = 4.5, 589
12.0 Hz), H -6‴)] and δ 166.4 (C-9‴‴). Meanwhile, to assign 590
2
C
the badly overlapped protons in sugar chemical shift range, 591
HSQC-TOCSY experiment was determined. In the HSQC-TOCSY 592
spectra, the correlations between the following proton and 593
49 p-coumaroyl [δ
50 J = 8.5 Hz, H-3‴‴,5‴‴), 7.48 (1H, d, J = 16.0 Hz, H-7‴‴), 7.51
51 (2H, d, J = 8.5 Hz, H-2‴‴,6‴‴); δ 165.4 (C-9‴‴)]. Furthermore,
52 according to the long-range correlations between δ 4.42 (1H,
53 dd, J = 8.0, 9.0 Hz, H-2″″) and δ 165.4 (C-9‴‴) observed in the
H
6.12 (1H, d, J = 16.0 Hz, H-8‴‴), 6.86 (2H, d,
C
carbon pairs were observed: δ
3.51 (H-3″), 3.53 (H-2″), 5.67 (H-1″); δ
(H-4″), 3.32 (H-5″), 3.46, 3.78 (H-6″); δ
3.16 (H-2‴), 3.26 (H-3‴ and 4‴), 4.66 (H-1‴); δ
C
97.9 (C-1″) and δ
67.8 (C-6″) and δ
104.5 (C-1‴) and δ
4.18, 4.22 597
63.3 (C-6‴), 69.5 (C-4‴), 73.9 (C-5‴); δ 103.2 598
2.81 (H-2″″), 2.91 (H-3″″), 2.99 (H-4″″), 4.00 599
60.7 (C-6″″), 69.6 (C-4″″), 73.1 600
(C-2″″), 76.2 (H-3″″), 76.4 (H-5″″), 103.2 (H-1″″); δ 99.7 (C-1″‴) 601
and δ 3.20 (H-4″‴), 3.27 (H-2″‴), 3.32 (H-3″‴), 5.04 (H-1″‴); δ 602
3.72 (H-6b″‴) and δ 60.5 (C-6″‴), 69.5 (C-4″‴), 77.0 (C-5″‴). 603
H
3.25 (H-4″), 594
3.25 595
596
H
C
H
C
C
H
54 HMBC experiment (Fig. 3), the linkage position of trans-p-
55 coumaroyl was elucidated to be 2″″. And in the HSQC-TOCSY
56 spectra, the correlations between the following proton and
H
(H
(C-1″″) and δ
(H-1″″); δ
2
-6‴) and δ
C
C
H
57 carbon pairs were observed clearly: δ
58 (H-4″), 3.40 (H-3″), 3.53 (H-2″), 5.60 (H-1″); δ
59
3.04 (H-4″), 3.16 (H-5″), 3.48, 3.71 (H-6″); δ
60 and δ 2.80 (H-3″″), 3.11 (H-4″″), 4.26 (H-1″″), 4.42 (H-2″″);
C
98.0 (C-1″) and δ
66.6 (C-6″) and
100.0 (C-1″″)
H
3.04
H
2.99 (H-4″″) and δ
C
C
C
δ
H
C
H
H
H
C
UNCORRECTED PR
Fig. 3. The main 1H 1H COSY and HMBC correlations of 4–6.
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

8
P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
Fig. 4. The main 1H 1H COSY and HMBC correlations of 7–9.
6
04
05
Consequently, the structure of apetalumoside B
identified.
5
(6) was
and C-9‴‴, H-1″″ and C-6″, H-1″‴ and C-7 (Fig. 4). The acid 646
hydrolysis experiment gave D-glucose as the sugar moiety [9]. 647
On the basis of above mentioned evidence, the structure of 9 648
6
6
06
07
08
09
10 Acid hydrolysis result indicated the presence of D-glucose in
Both apetalumosides B
6
(7) and B
7
(8) were with negative
25
25
6
optical rotation [7: [α]
D
−41.6° (c = 0.99, H
2
O); 8: [α]
D
−66.5°
was determined.
649
6
(c = 0.81, MeOH)], respectively. The same molecular formula,
49 58
C H O30, was deduced from the negative-ion HRESI-TOF-MS.
The structure of 3-O-(2,6-di-O-β-D-glucopyranosyl)-β-D- 650
glucopyranoside (10) was elucidated by chemical and spectro- 651
6
1
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
scopic methods (UV, IR, NMR, and HRESI-TOF-MS) and its H 652
1
1
13
11 them [9]. The 1D and 2D [( H H COSY, HSQC, HMBC and
12 HSQC-TOCSY (Fig. 4))] spectra revealed that both of them
13 owned quercetin 3-O-(2,6-di-O-β-D-glucopyranosyl)-β-D-
14 glucopyranosyl-7-O-β-D-glucopyranosyl like compounds
15 4–6. On the other hand, there was a trans-feruloyl group in
16 them [7: δ 3.76 (3H, s, 3‴‴-OCH ), 6.18 (1H, d, J = 16.0 Hz,
H 3
17 H-8‴‴), 6.73 (1H, d, J = 8.0 Hz, H-5‴‴), 6.87 (1H, dd, J = 2.0,
18 8.0 Hz, H-6‴‴), 7.10 (1H, d, J = 2.0 Hz, H-2‴‴), 7.35 (1H, d,
19 J = 16.0 Hz, H-7‴‴); δ
20 OCH ), 6.25 (1H, d, J = 15.5 Hz, H-8‴‴), 6.87 (1H, d, J = 7.5 Hz,
21 H-5‴‴), 7.13 (1H, br. d, ca. J = 8 Hz, H-6‴‴), 7.35 (1H, br. s,
22 H-2‴‴), 7.50 (1H, d, J = 15.5 Hz, H-7‴‴); δ 165.5 (C-9‴‴)]. In
23 the HMBC spectra, long-range correlation between δ [4.20
24 (1H, br. d, ca. J =12 Hz), 4.25 (1H, dd, J = 4.5, 12.0 Hz), H -6‴]
166.4 (C-9″‴) was observed for 7; meanwhile, long-range
correlation between δ 4.42 (1H, dd, J = 7.5, 8.0 Hz, H-2″″) and
165.5 (C-9″‴) was found for 8. Finally, the structures of 7 and 8
and C NMR (DMSO-d
6
, Table 1) data were reported for the 653
first time.
654
TG accumulation inhibitory effects were tested. Compared 655
with normal HepG2 cells group, intracellular triglyceride 656
accumulation was significantly increased after free fatty acid 657
incubation for 48 h, which could be down-regulated by 0.5 μM 658
orlistat, a lipase inhibitor. The results showed that compounds 659
1–3 and 10 significantly inhibited TG accumulation at 10 μM 660
(Table 2). We can deduce that the glucosylation of 7-hydroxyl 661
group would reduce the inhibitory effect on TG accumulation. 662
C H
166.4 (C-9‴‴); 8: δ 3.90 (3H, s, 3‴‴-
3
C
H
Acknowledgements
663
2
25
26
27
28
29
and δ
C
This research was supported by Program for New Century 664
Excellent Talents in University (NCET-12-1069), Program for 665
Tianjin Innovative Research Team in University (TD12-5033), 666
and Program for Important Drug Develop of MOST, China 667
H
δ
C
were clarified as shown in Fig. 1.
Apetalumoside C (9) was isolated as a yellow powder
(2013ZX09402201).
668
30 having a negative optical rotation [[α]2
5
−42.1° (c = 0.99,
D
31 MeOH)]. The molecular formula of 9 was determined to be
32
29 by negative-ion HRESI-TOF-MS (m/z 1095.2831
33 [M–H] , calcd for C48
34 NMR (DMSO-d , Table 1) and various 2D NMR experiments
showed signals assignable to a kaempferol part [δ 6.40 (1H, br. s,
48 56
C H O
Table 2
t2:1
t2:2
−
29 1095.2834). The 1H and
13
H
55
O
C
Inhibitory effects of 1–10 on TG accumulation in HepG2 cells.
6
TG relative concentration
TG relative concentration
t2:3
35
36
37
38
39
40
41
42
43
44
(%)
(%)
H-6), 6.64 (1H, br. s, H-8), 6.90 (2H, d, J = 8.0 Hz, H-3′,5′), 8.04
(2H, d, J = 8.0 Hz, H-2′,6′), 12.62 (1H, br. s, 5-OH)], four β-D-
glucopyranosyl moieties [δ 3.97 (1H, d, J = 7.5 Hz, H-1″″), 4.67
(1H, d, J = 7.5 Hz, H-1‴), 5.04 (1H, d, J = 7.0 Hz, H-1″‴), 5.56
(1H, d, J = 7.0 Hz, H-1″)], together with a trans-caffeoyl [δ 6.01
H
(1H, d, J = 16.0 Hz, H-8‴‴), 6.69 (1H, d, J = 8.0 Hz, H-5‴‴), 6.76
(1H, br. d, ca. J = 8 Hz, H-6‴‴), 6.89 (1H, br. s, H-2‴‴), 7.30 (1H,
Normal
Control
Orlistat
1
2
3
4
47.5 ± 0.4**
5
6
7
8
9
89.3 ± 3.8
96.4 ± 1.1
96.2 ± 2.8
87.4 ± 3.3
86.4 ± 3.5
81.2 ± 1.3**
t2:4
t2:5
t2:6
t2:7
t2:8
t2:9
t2:10
100.0 ± 0.9
74.5 ± 1.4**
80.6 ± 3.9**
77.7 ± 1.9**
83.4 ± 1.7**
95.4 ± 2.4
10
TG relative concentration: percentage of control group, which set as 100%. Values t2:11
represent the mean ± SD of three determinations. **P b 0.01 (Differences t2:12
between compound-treated group and control group). N = 6. Final concentra- t2:13
C
d, J = 16.0 Hz, H-7‴‴); δ 166.3 (C-9‴‴)]. The HMBC experiment
on 9 showed long-range correlations between the following
45 proton and carbon pairs: H-1″ and C-3, H-1‴ and C-2″, H-6‴
tion of 1–10 was 10 μM and Oristat was 0.5 μM.
t2:14
Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007

P. Shi et al. / Fitoterapia xxx (2015) xxx–xxx
9
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Please cite this article as: Shi P, et al, New bioactive flavonoid glycosides isolated from the seeds of Lepidium apetalum Willd,
Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.007