A new indole glycoside from the seeds of Raphanus sativus

Article abstract of DOI:10.1007/s12272-016-0758-0

A new indole glycoside, β-d-glucopyranosyl 2-(methylthio)-1H-indole-3-carboxylate, named raphanuside A (1), as well as eight known compounds, β-d-fructofuranosyl-(2?→?1)-(6-O-sinapoyl)-α-d-glucopyranoside (2), (3-O-sinapoyl)-β-d-fructofuranosyl-(2?→?1)-α-d-glucopyranoside (3), (3-O-sinapoyl)-β-d-fructofuranosyl-(2?→?1)-(6-O-sinapoyl)-α-d-glucopyranoside (4), (3,4-O-disinapoyl)-β-d-fructofuranosyl-(2?→?1)-(6-O-sinapoyl)-α-d-glucopyranoside (5), isorhamnetin 3,4′-di-O-β-d-glucoside (6), isorhamnetin 3-O-β-d-glucoside-7-O-α-l-rhamnoside (7), isorhamnetin 3-O-β-d-glucoside (8) and 3'-O-methyl-(?)-epicatechin 7-O-β-d-glucoside (9) were isolated from the seeds of Raphanus sativus. Furthermore, compounds 1–3 and 6–9, were isolated from this plant for the first time. The structures of compounds 1–9 were identified using 1D and 2D NMR, including ¹H–¹H COSY, HSQC, HMBC and NOESY spectroscopic analyses. The inhibitory activity of these isolated compounds against interleukin-6 (IL-6) production in TNF-α stimulated MG-63 cells was also examined.

Full text of DOI:10.1007/s12272-016-0758-0

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0758-0
RESEARCH ARTICLE
A new indole glycoside from the seeds of Raphanus sativus
Hong-Guang Jin2 Hae Ju Ko1 Md. Anisuzzaman Chowdhury1 Dong-Sung Lee1
Eun-Rhan Woo1
•
•
•
•
Received: 21 April 2015 / Accepted: 10 May 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract A new indole glycoside, b-D-glucopyranosyl
2-(methylthio)-1H-indole-3-carboxylate, named raphanu-
side A (1), as well as eight known compounds, b-D-
fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-a-D-glucopyra-
noside (2), (3-O-sinapoyl)-b-D-fructofuranosyl-(2 ? 1)-a-
D-glucopyranoside (3), (3-O-sinapoyl)-b-D-fructofuranosyl-
(2 ? 1)-(6-O-sinapoyl)-a-D-glucopyranoside (4), (3,4-O-
disinapoyl)-b-D-fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-a-
D-glucopyranoside (5), isorhamnetin 3,40-di-O-b-D-glucoside
(6), isorhamnetin 3-O-b-D-glucoside-7-O-a-L-rhamnoside
(7), isorhamnetin 3-O-b-D-glucoside (8) and 3’-O-methyl-
(-)-epicatechin 7-O-b-D-glucoside (9) were isolated from
the seeds of Raphanus sativus. Furthermore, compounds 1–3
and 6–9, were isolated from this plant for the first time. The
structures of compounds 1–9 were identified using 1D and 2D
NMR, including 1H–1H COSY, HSQC, HMBC and NOESY
spectroscopic analyses. The inhibitory activity of these iso-
lated compounds against interleukin-6 (IL-6) production in
TNF-a stimulated MG-63 cells was also examined.
Introduction
Raphani Semen, the seeds of Raphanus sativus L. (Bras-
sicaceae), are used in oriental medicine as a carminative,
diuretic, expectorant, and laxative, and is reported to
exhibit stomachic, anti-cancer and anti-inflammatory
activity (Duke and Ayensu 1985; Yeung 1985; Chopra
et al. 1986). Previous phytochemical investigations resul-
ted in the isolation of some glucosinolates responsible for
the aforementioned cancer-chemopreventive properties
(Daxenbichler et al. 1991; Nastruzzi et al. 2000; Barillari
et al. 2005; Duan et al. 2007). Glucosinolates and/or their
decomposition products have recently attracted consider-
able interest due to their anticancer properties (Noyan-
Ashraf et al. 2005; Giacoppo et al. 2013). Recently, three
new 4-methylthio-butanyl derivatives were isolated from
R. sativus, and their anti-tumor and anti-neuroinflammatory
activities were investigated (Kim et al. 2014). Although
many biologically active glucosinolates were isolated from
R. sativus, only a few studies of its anti-inflammatory
components have been performed (Kim et al. 2015). In
addition, there has been no investigations into the alkaloid
component of R. sativus.
Keywords Indole glycoside Á IL-6 inhibitory effect Á
Brassicaceae Á Raphanus sativus
In an ongoing investigation into anti-inflammatory
compounds from R. sativus, the methanol extract of its
seeds was investigated. By means of repeated column
chromatography using silica gel, MCI gel, Sephadex LH-
20, and LiChroprep RP-18, a new indole glycoside, b-D-
glucopyranosyl 2-(methylthio)-1H-indole-3-carboxylate,
named raphanuside A (1), was isolated along with eight
known compounds. The structures of the known com-
pounds were identified as b-D-fructofuranosyl-(2 ? 1)-(6-
O-sinapoyl)-a-D-glucopyranoside (2), (3-O-sinapoyl)-b-D-
fructofuranosyl-(2 ? 1)-a-D-glucopyranoside (3), (3-O-si-
napoyl)-b-D-fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-a-D-
Hong-Guang Jin and Hae Ju Ko have equally contributed to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-016-0758-0) contains supplementary
material, which is available to authorized users.
& Eun-Rhan Woo
wooer@chosun.ac.kr
1
College of Pharmacy, Chosun University, 309 Pilmun-daero,
Dong-gu, Gwangju 501-759, Republic of Korea
2
College of Pharmacy and Life Science, Jiu Jiang University,
320 Xun Yang Road, Jiujiang 33200, China
123

H.-G. Jin et al.
glucopyranoside (4), (3,4-O-disinapoyl)-b-D-fructofura-
nosyl-(2 ? 1)-(6-O-sinapoyl)-a-D-glucopyranoside (5),
resolution electrospray ionization mass spectrometry (HR-
ESI-MS) was performed on a quadrupole time-of-flight (Q-
TOF) mass spectrometer (Synapt HDMS system, Waters,
USA). Nuclear magnetic resonance (NMR) spectra,
including nuclear Overhauser effect spectroscopy
(NOESY), correlation spectroscopy (COSY), heteronuclear
multiple quantum coherence (HMQC), and heteronuclear
multiple bond correlation (HMBC) experiments, were
recorded on a Varian UNITY INOVA 500 NMR spec-
trometer (KBSI-Gwangju center) operating at 500 MHz for
1H-NMR, and 125 MHz for 13C-NMR, with chemical
shifts given in ppm (d). TLC was carried out in a precoated
Kieselgel 60 F254 (art. 5715, Merck) and RP-18 F254s (Art.
15389, Merck) plates. Column chromatography was per-
formed on silica gel 60 (40–63 and 63–200 lm, Merck),
MCI gel CHP20P (75–150 lm, Mitsubishi Chemical Co.),
and Sephadex LH-20 (25–100 lm, Sigma). Low pressure
liquid chromatography was carried out in a Merck
Lichroprep LobarÒ-A RP-18 (240 9 10 mm2) column
with a FMI QSY-0 pump (ISCO).
isorhamnetin 3,40-di-O-b-D-glucoside (6), isorhamnetin
3-O-b-D-glucoside-7-O-a-L-rhamnoside (7), isorhamnetin
3-O-b-D-glucoside (8), and 3’-O-methyl-(-)-epicatechin
7-O-b-D-glucoside (9), by comparison of their spectro-
scopic data with those reported in the literature (Fig. 1).
Furthermore, these nine known compounds, with the
exception of 4 and 5, were isolated from this plant for the
first time. The inhibitory activity of these isolated com-
pounds against IL-6 production in TNF-a stimulated MG-
63 cells was examined.
This paper reports the isolation and structural charac-
terization of these compounds and their inhibitory activities
against IL-6 production.
Materials and methods
General experimental procedure
Optical rotations were measured using an Autopol-IV
polarimeter. Ultraviolet (UV) spectra were obtained on a
Shimadzu UV/Visible Spectrophotometer. Infrared (IR)
spectra were recorded on an IMS 85 (Bruker). High-
Plant materials
The seeds of R. sativus were collected in Andong,
Gyeongbuk province, Republic of Korea, in August 2013
Fig. 1 Structures of
compounds 1–9
OR3
6'
4'
5'
2'O
OH
HO
HO
O
3
3'
6'
OH
O
HO
1'
OH
OH
4
1
2
1'
3'
O
8
2
3a
7a
OR2
5
6
O
5
O
SCH3
OH
N
H
7
3
4
6
OH
OR1
1
R1
R2
H
H
H
X
R3
X
O
2
3
4
5
H
X
X
X
OCH3
OH
H
X
X =
X
OCH3
OCH3
OR3
OH
4a
2'
OH
3
4'
7
R2O
O
2
2
7
R1O
O
8a
1'
4'
OR1
5
4
OH
OH
O
OCH
3
R1
glc
glc
glc
R2
H
rha
H
R3
glc
H
R1
glc
6
7
8
9
H
123

Indole glycoside from seeds of Raphanus sativus
and identified by Dr. J. H. Lee, Professor of the department
of Korean Medicine, Dongguk University. A voucher
specimen (CSU-1050-17) was deposited in the Herbarium
of the College of Pharmacy, Chosun University.
10), 79.0 (C-50), 78.6 (C-30), 74.4 (C-20), 71.3 (C-40), 62.6
(C-60), 14.6 (C-9–SCH3).
b-D-fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-a-D-glu-
copyranoside (2): Amorphous powder; [a]2D0 ? 10.4°
(MeOH; c 0.36); IR mmax (film) cm-1: 3408, 1700, 1632,
1608; FAB-MS, m/z: 571[M ? Na]?; 1H-NMR (500 MHz,
CD3OD) d: 7.63 (1H, d, J = 16.0 Hz, H-700), 6.95 (2H, s,
H-200, 600), 6.46 (1H, d, J = 16.0 Hz, H-800), 5.42 (1H, d,
J = 3.5 Hz, H-10), 4.51 (1H, dd, J = 1.5, 12.0 Hz, Ha-60),
4.25 (1H, dd, J = 6.5, 12.0 Hz, Hb-60), 4.14 (1H, m, H-50),
4.08 (1H, d, J = 8.0 Hz, H-3), 4.06 (1H, dd, J = 8.0,
8.0 Hz, H-4), 3.87 (6H, s, 300, 500-OCH3), 3.76-3.81 (3H, m,
H-5, 6), 3.74 (1H, dd, J = 9.5, 10.0 Hz, H-30), 3.61 (1H, d,
J = 12.5 Hz, Ha-1), 3.62 (1H, d, J = 12.5 Hz, Hb-1), 3.46
(1H, dd, J = 4.0, 9.5 Hz, H-20), 3.33 (1H, dd, J = 9.0,
9.5 Hz, H-40); 13C-NMR (125 MHz, CD3OD) d: 169.2 (C-
900), 149.6 (C-300, 500), 147.4 (C-700), 139.9 (C-400), 126.6 (C-
100), 115.8 (C-800), 107.1 (C-200, 600), 105.3 (C-2), 93.3 (C-
10), 84.0 (C-5), 79.3 (C-3), 76.2 (C-4), 74.8 (C-30), 73.3 (C-
20), 72.2 (C-50), 72.0 (C-40), 65.3 (C-1), 64.4 (C-60), 64.4
(C-6), 57.0 (C-300, 500-OCH3).
Extraction and isolation
The air-dried seeds of R. sativus (19.1 kg) were pulverized
and extracted with MeOH three times for 4 h at 80 °C. The
resultant MeOH extract (800.5 g) was suspended in water
(3 9 2.0 L) and then partitioned sequentially with equal
volumes of n-hexane, dichloromethane, ethyl acetate, and
n-butanol. Each fraction was evaporated in vacuo to yield
the residues of n-hexane (195.4 g), CH2Cl2 (10.3 g),
EtOAc (7.3 g), n-BuOH (60.6 g) and water (400.3 g)
extracts. The n-BuOH soluble fraction (22.2 g) was sub-
jected to column chromatography (CC) over a Diaion HP
20 column and eluted with a H2O/MeOH gradient system
(100:0 ? 0:100). Fractions were combined based on their
TLC pattern to yield subfractions designated B1-B6.
Fraction B3 (4.38 g) was further purified by MCI gel CC
(MeOH/H2O, 1:3 ? 1:1) to yield five subfractions (B31-
B35). Subfraction B31 (0.62 g), containing 1 and 9, was
again purified by silica gel CC (CHCl3/MeOH, 5:1 ? 1:1)
and finally by Sephadex LH 20 CC (MeOH/H2O, 1:10) to
yield 1 (6.5 mg) and 9 (5.6 mg). In addition, subfraction
B33 (1.87 g) was purified by silica gel CC (CHCl3/MeOH/
H2O, 3:1:0.2 ? 2:1:0.2) and finally by Lichroprep RP-18
CC (MeOH/H2O, 1:4 ? 1:1) to yield 6 (32.4 mg), 7
(20.8 mg) and 8 (11.2 mg). Fraction B4 (5.4 g) was sub-
jected to MCI gel CC eluting with a MeOH/H2O
(45:55 ? 65:35) to yield four subfractions (B41-B44).
Subfraction B41 (1.04 g) was then purified by silica gel CC
(3-O-sinapoyl)-b-D-fructofuranosyl-(2 ? 1)-a-D-glu-
copyranoside (3): Amorphous powder; [a]2D0 -28.0°
(MeOH; c 0.72); IR mmax (film) cm-1: 3408, 1700, 1632,
1608; FAB-MS, m/z: 571[M ? Na]?;1H-NMR (500 MHz,
CD3OD) d: 7.71 (1H, d, J = 16.0 Hz, H-700), 6.97 (2H, s,
H-200, 600), 6.45 (1H, d, J = 16.0 Hz, H-800), 5.47 (1H, d,
J = 7.5 Hz, H-3), 5.44 (1H, d, J = 3.5 Hz, H-10), 4.38
(1H, t, J = 8.0 Hz, H-4), 3.92-3.96 (2H, m, H-5, 50), 3.89
(6H, s, 300, 500-OCH3), 3.76-3.87 (4H, m, H-6, 60), 3.67 (1H,
dd, J = 9.5, 10.0 Hz, H-30), 3.66 (1H, d, J = 12.0 Hz, Ha-
1), 3.58 (1H, d, J = 12.0 Hz, Hb-1), 3.44 (1H, dd, J = 4.0,
9.5 Hz, H-20), 3.40 (1H, dd, J = 9.0, 9.5 Hz, H-40); 13C-
NMR (125 MHz, CD3OD) d: 168.3 (C-900), 149.6 (C-300,
500), 148.0 (C-700), 139.8 (C-400), 126.8 (C-100), 115.7 (C-800),
107.2 (C-200, 600), 104.9 (C-2), 93.5 (C-10), 84.3 (C-5), 79.8
(C-3), 75.1 (C-30), 74.7 (C-4), 74.0 (C-50), 73.3 (C-20), 71.3
(C-40), 65.5 (C-1), 63.1 (C-6), 62.5 (C-6), 57.1 (C-300-
OCH3), 57.0 (C-500-OCH3).
(CHCl3/MeOH/H2O, 7:1:0.2 ? 3:1:0.2) to yield
2
(41.5 mg). Subfraction B42 and B43 (3.39 g) were purified
by repeated Lichroprep RP 18 CC (MeOH/H2O,
1:2 ? 1:1) to yield 3 (38.8 mg) and 4 (2.49 g). Fraction
B5 (0.83 g) was purified by silica gel CC (CHCl3/MeOH,
10:1 ? 3:1) to yield 5 (375.4 mg).
(3-O-sinapoyl)-b-D-fructofuranosyl-(2 ? 1)-(6-O-si-
napoyl)-a-D-glucopyranoside (4): Yellowish amorphous
Raphanuside A (1): Yellowish amorphous powder; [a]D20
?31.0° (MeOH; c 0.26); HR-ESI–MS (negative mode) m/
z: 368.0797[M–H]- (calcd for C16H18NO7S, 368.0804); IR
mmax (film) cm-1: 3480, 3400, 1620, 1490, 850; UV
(MeOH) kmax nm: 320, 253, 272; 1H-NMR (500 MHz,
CD3OD) d: 7.98 (1H, m, H-4), 7.39 (1H, m, H-7), 7.13
(2H, m, H-5, 6), 5.75 (1H, d, J = 7.5 Hz, H-10), 3.87 (1H,
dd, J = 2.0, 12.0 Hz, H-60a), 3.72 (1H, dd, J = 5.0,
12.0 Hz, H-60b), 3.62 (1H, t, J = 8.5 Hz, H-20), 3.50 (1H,
t, J = 8.5 Hz, H-30), 3.42-3.46 (2H, m, H-40, 50), 2.66 (3H,
s, H-9-SCH3); 13C-NMR (125 MHz, CD3OD) d: 165.8 (C-
8), 148.2 (C-2), 138.5 (C-7a), 128.7 (C-3a), 123.1 (C-6),
122.9 (C-5), 121.6 (C-4), 111.8 (C-7), 103.9 (C-3), 95.5 (C-
powder; [a]D20 -94.0° (MeOH; c 0.6); IR mmax (film) cm-1
:
3396, 1704, 1632, 1606; FAB-MS, m/z: 777[M ? Na]?;
1H-NMR (600 MHz, CD3OD) d: 7.67 (1H, d, J = 15.6 Hz,
H-700), 7.59 (1H, d, J = 15.6 Hz, H-7000), 6.93 (2H, s, H-200,
600), 6.88 (2H, s, H-2000, 6000), 6.47 (1H, d, J = 15.6 Hz,
H-800), 6.46 (1H, d, J = 15.6 Hz, H-8000), 5.53 (1H, d,
J = 3.6 Hz, H-10), 5.51 (1H, d, J = 7.8 Hz, H-3), 4.68
(1H, dd, J = 1.8, 12.0 Hz, Ha-60), 4.51 (1H, t, J = 7.8 Hz,
H-4), 4.29 (1H, ddd, J = 1.8, 7.2, 9.6 Hz, H-50), 4.20 (1H,
dd, J = 7.2, 12.0 Hz, Hb-60), 3.97 (1H, ddd, J = 3.0, 6.6,
7.8 Hz, H-5), 3.90 (1H, dd, J = 7.2, 12.0 Hz, Hb-6), 3.87
(6H, s, 300, 500-OCH3), 3.84 (6H, s, 3000, 5000-OCH3), 3.82
123

H.-G. Jin et al.
(1H, dd, J = 1.8, 12.0 Hz, Ha-6), 3.67 (1H, dd, J = 9.0,
9.6 Hz, H-30), 3.61 (1H, d, J = 12.6 Hz, Ha-1), 3.59 (1H,
d, J = 12.6 Hz, Hb-1), 3.48 (1H, dd, J = 3.6, 9.6 Hz,
H-2000, 3000), 3.18-3.24 (3H, m, H-200, 300, 4000), 3.10-3.12 (2H,
m, H-400, 500); 13C-NMR (125 MHz, DMSO-d6) d: 177.4
(C-4), 164.8 (C-7), 161.2 (C-5), 156.5 (C-8a), 155.6 (C-2),
148.4 (C-40), 148.0 (C-30), 133.4 (C-3), 123.7 (C-10), 121.4
(C-60), 114.5 (C-50), 113.5 (C-20), 103.9 (C-4a), 100.7 (C-
100), 99.5 (C-1000), 98.9 (C-6), 93.8 (C-8), 77.5 (C-500), 77.1
(C-5000), 76.8 (C-3000), 76.4 (C-300), 74.3 (C-200), 73.1 (C-
2000), 69.8 (C-400), 69.5 (C-4000), 60.6 (C-600), 60.5 (C-6000),
55.6 (C-30-OCH3).
13
H-20), 3.31(1H, dd, J = 9.0, 9.6 Hz, H-40); C-NMR
(150 MHz, CD3OD) d: 169.2 (C-9000), 168.6 (C-900), 149.6
(C-300, 500), 149.5 (C-3000, 5000), 148.0 (C-700), 147.4 (C-7000),
139.8 (C-400), 139.6 (C-4000), 126.7 (C-100), 126.6 (C-1000),
115.9 (C-800), 115.6 (C-800), 107.2 (C-200, 600), 107.0 (C-2000,
6000), 104.9 (C-2), 92.8 (C-10), 84.5 (C-5), 79.4 (C-3), 75.2
(C-30), 74.3 (C-4), 73.3 (C-20), 72.6 (C-50), 72.1 (C-40),
65.8 (C-1), 65.8 (C-60), 64.0 (C-6), 57.0 (C-300, 500-OCH3),
56.9 (C-3000, 5000-OCH3).
Isorhamnetin 3-O-b-D-glucoside-7-O-a-L-rhamnoside
(7): Yellowish amorphous powder; [a]2D0 -42.0° (MeOH;
c 0.30); IR mmax (film) cm-1: 3480, 3100, 1645, 1610; ESI–
MS, m/z: 623[M-H]-; 1H-NMR (500 MHz, CD3OD) d:
7.93 (1H, d, J = 2.0 Hz, H-20), 7.62 (1H, dd, J = 2.0,
8.5 Hz, H-60), 6.88 (1H, d, J = 8.5 Hz, H-50), 6.75 (1H, d,
J = 2.0 Hz, H-8), 6.45 (1H, d, J = 2.0 Hz, H-6), 5.57 (1H,
s, H-1000), 5.45 (1H, d, J = 7.5 Hz, H-100), 4.02 (1H, m,
H-2000), 3.94 (3H, s, 30-OCH3), 3.83 (1H, dd, J = 3.5,
10.0 Hz, H-5000), 3.74 (1H, dd, J = 2.5, 12.0 Hz, H-600),
3.57 (1H, dd, J = 5.0, 12.0 Hz, H-600), 3.56 (1H, m, H-3000),
3.43-3.49 (4H, m, H-200, 300, 400, 4000), 3.25 (1H, m, H-500),
1.25 (3H, d, J = 6.0 Hz, H-6000); 13C-NMR (125 MHz,
CD3OD) d: 179.6 (C-4), 163.6 (C-7), 163.0 (C-5), 159.5
(C-2), 158.1 (C-8a), 152.6 (C-40), 148.9 (C-30), 135.5 (C-
3), 124.3 (C-50), 122.2 (C-10), 116.5 (C-60), 114.4 (C-20),
107.6 (C-4a), 103.5 (C-100), 100.7 (C-6), 99.9 (C-1000), 95.6
(C-8), 78.7 (C-500), 78.2 (C-300), 76.1 (C-200), 73.7 (C-4000),
72.2 (C-5000), 71.8 (C-2000), 71.6 (C-400), 71.4 (C-3000), 62.6
(C-600), 56.8 (C-30-OCH3), 18.2 (C-6000).
(3,4-O-disinapoyl)-b-D-fructofuranosyl-(2 ? 1)-(6-O-
sinapoyl)-a-D-glucopyranoside (5): Yellow amorphous
powder; [a]D20 -28.0° (MeOH; c 0.2); IR mmax (film) cm-1
:
3400, 1704, 1640, 1600; FAB-MS, m/z: 983[M ? Na]?;
1H-NMR (500 MHz, CD3OD) d: 7.71 (1H, d, J = 15.5 Hz,
H-700), 7.56 (1H, d, J = 15.5 Hz, H-70000), 7.46 (1H, d,
J = 15.5 Hz, H-7000), 6.91 (2H, s, H-200, 600), 6.81 (2H, s,
H-20000, 60000), 6.76 (2H, s, H-2000, 6000), 6.48 (1H, d,
J = 15.5 Hz, H-80000), 6.46 (1H, d, J = 15.5 Hz, H-800),
6.26 (1H, d, J = 15.5 Hz, H-8000), 5.85 (1H, d, J = 8.0 Hz,
H-3), 5.71 (1H, t, J = 8.0 Hz, H-4), 5.57 (1H, d,
J = 3.5 Hz, H-10), 4.75 (1H, dd, J = 1.5, 12.0 Hz, Ha-60),
4.38 (1H, ddd, J = 1.5, 6.0, 9.5 Hz, H-50), 4.21 (1H, dd,
J = 6.0, 12.0 Hz, Hb-60), 4.20 (1H, m, H-5), 3.99 (1H, dd,
J = 7.0, 12.5 Hz, Hb-6), 3.94 (1H, dd, J = 4.5, 12.5 Hz,
Ha-6), 3.84 (12H, s, 300, 500, 30000, 50000-OCH3), 3.78 (6H, s,
3000, 5000-OCH3), 3.71 (1H, m, H-30), 3.65 (2H, d,
J = 3.5 Hz, H-1), 3.51 (1H, dd, J = 4.0, 10.0 Hz, H-20),
3.30 (1H, m, H-40); 13C-NMR (125 MHz, CD3OD) d:
169.4 (C-90000), 168.2 (C-9000), 167.9 (C-900), 149.5 (C-3000,
5000), 149.5000(C-300, 500), 149.4 (C-30000, 50000), 148.5 (C-700),
148.4 (C-70000), 147.2 (C-70000), 139.9 (C-400), 139.8 (C-4000),
139.6 (C-4 ), 126.7 (C-10000), 126.6 (C-100), 126.5 (C-1000),
116.2 (C-80000), 115.3 (C-800), 114.8 (C-8000), 107.2 (C-20000,
60000), 107.2 (C-200, 600), 107.0 (C-2000, 6000), 105.5 (C-2), 93.0
(C-10), 83.0 (C-5), 77.1 (C-3), 76.6 (C-4), 75.1 (C-30), 73.2
(C-20), 73.0 (C-50), 72.3 (C-40), 66.1 (C-60), 65.2 (C-1),
64.2 (C-6), 57.0 (C-300, 500, 30000, 50000-OCH3), 56.9 (C-3000,
5000-OCH3).
Isorhamnetin 3-O-b-D-glucoside (8): Yellowish amor-
phous powder; [a]D20 -21.7° (MeOH; c 0.06); IR mmax (film)
cm-1: 3480, 3100, 1645, 1610; ESI–MS, m/z: 477[M-H]-;
1H-NMR (500 MHz, DMSO-d6) d: 7.93 (1H, d,
J = 2.0 Hz, H-20), 7.45 (1H, dd, J = 2.0, 8.5 Hz, H-60),
6.89 (1H, d, J = 8.5 Hz, H-50), 6.30 (1H, d, J = 1.5 Hz,
H-8), 6.09 (1H, d, J = 1.5 Hz, H-6), 5.54 (1H, d,
J = 7.5 Hz, H-100), 3.83 (3H, s, 30-OCH3), 3.58 (1H, dd,
J = 2.5, 12.0 Hz, H-600), 3.38 (1H, dd, J = 5.0, 12.0 Hz,
H-600), 3.21-3.23 (2H, m, H-200, 300), 3.10-3.12 (2H, m,
H-400, 500); 13C-NMR (125 MHz, DMSO-d6) d: 176.7 (C-4),
161.1 (C-5, 7), 156.7 (C-8a), 155.6 (C-2), 149.4 (C-40),
146.8 (C-30), 132.7 (C-3), 121.8 (C-60), 121.1 (C-10), 115.2
(C-50), 113.4 (C-20), 102.8 (C-4a), 100.9 (C-100), 99.5 (C-6),
94.0 (C-8), 77.4 (C-500), 76.4 (C-300), 74.3 (C-200), 69.8 (C-
400), 60.5 (C-600), 55.6 (C-30-OCH3).
Isorhamnetin 3,40-di-O-b-D-glucoside (6): Yellowish
amorphous powder; [a]2D0 -85.0° (MeOH; c 0.38); IR mmax
(film) cm-1: 3480, 3100, 1645, 1610; ESI–MS, m/z:
1
639[M-H]-; H-NMR (500 MHz, DMSO-d6) d: 7.96 (1H,
d, J = 2.0 Hz, H-20), 7.53 (1H, dd, J = 2.0, 8.5 Hz, H-60),
7.23 (1H, d, J = 8.5 Hz, H-50), 6.44 (1H, d, J = 1.5 Hz,
H-8), 6.21 (1H, d, J = 1.5 Hz, H-6), 5.58 (1H, d,
J = 7.5 Hz, H-100), 5.06 (1H, d, J = 7.5 Hz, H-1000), 3.84
(3H, s, 30-OCH3), 3.68 (1H, dd, J = 1.5, 12.0 Hz, H-600),
3.58 (1H, dd, J = 2.0, 12.0 Hz, H-6000), 3.46 (1H, dd,
J = 5.0, 12.0 Hz, H-600), 3.39 (1H, dd, J = 5.5, 12.0 Hz,
H-6000), 3.36 (1H, t, J = 9.5 Hz, H-5000), 3.29-3.30 (2H, m,
30-O-methyl-(-)-epicatechin 7-O-b-D-glucoside (9):
Colorless powder; [a]2D0 -77.3° (EtOH; c 0.42); IR mmax
(film) cm-1: 3400, 1704, 1632, 1600; ESI–MS, m/z:
1
465[M-H]-; H-NMR (500 MHz, DMSO-d6) d: 7.04 (1H,
d, J = 2.0 Hz, H-20), 6.83 (1H, dd, J = 2.0, 8.0 Hz, H-60),
6.72 (1H, d, J = 8.0 Hz, H-50), 6.10 (1H, d, J = 2.0 Hz,
H-8), 6.02 (1H, d, J = 2.0 Hz, H-6), 4.82 (1H, s, H-2),
4.70 (1H, d, J = 7.5 Hz, H-100), 4.05 (1H, m, H-3), 3.76
123

Indole glycoside from seeds of Raphanus sativus
1
(3H, s, 30-OCH3), 3. 08 (1H, dd, J = 2.0, 12.0 Hz, H-600),
3.65 (1H, dd, J = 5.0, 12.0 Hz, H-600), 3.12-3.43 (4H, m,
H-200, 300, 400, 500), 2.73 (1H, dd, J = 4.5, 17.0 Hz, H-4),
2.54 (1H, dd, J = 2.5, 17.0 Hz, H-4); 13C-NMR
(125 MHz, DMSO-d6) d: 156.6 (C-7), 156.5 (C-5), 155.7
(C-8a), 146.9 (C-30), 145.8 (C-40), 130.4 (C-10), 119.7 (0C0 -
60), 114.7 (C-50), 111.6 (C-20), 101.5 (C-4a), 100.7 (C-1 ),
96.4 (C-8), 95.1 (C-6), 78.3 (C-2), 76.9 (C-300), 76.6 (C-500),
73.2 (C-200), 69.6 (C-400), 64.6 (C-3), 60.3 (C-600), 55.6 (C-
30-OCH3), 30.7 (C-4).
(1620 cm-1) groups were observed. The H-NMR spec-
trum of 1 showed four aromatic protons at dH 7.98 (1H, m,
H-4), 7.39 (1H, m, H-7) and 7.13 (2H, m, H-5,6), a glu-
cosyl anomeric proton at dH 5.75 (1H, d, J = 7.5 Hz,
H-10), thiomethyl protons at dH 2.66 (3H, s, –SCH3) and
characteristic signals attributable to the glucose moiety.
1
Furthermore, in the H-NMR spectrum measured in pyr-
idine-d5 instead of CD3OD, the -NH group was identi-
fied at dH 12.85 (br. s) and confirmed by its disappearance
in a D2O exchange experiment. In the 13C-NMR spectrum,
13 carbon signals appeared in addition to those of the sugar
unit, including one carbonyl carbon at dC 165.8 (C-8), four
aromatic methine carbons at dC 123.1 (C-6), 122.9 (C-5),
121.6 (C-4) and 111.8 (C-7), four quaternary carbons at dC
148.2 (C-2), 138.5 (C-7a), 128.7 (C-3a) and 103.9 (C-3),
one thiomethyl carbon at dC 14.6 (–SCH3). These spectral
data indicated that 1 was to an indole 3-carboxylate ester.
The signals from the sugar unit appeared at dH 5.75 (1H, d,
J = 7.5 Hz, H-10), 3.62 (1H, t, J = 8.5 Hz, H-20), 3.50
(1H, t, J = 8.5 Hz, H-30), 3.42-3.46 (2H, m, H-40, H-50),
3.87 (1H, dd, J = 2.0, 12.0 Hz, H-60a), 3.72 (1H, dd,
J = 5.0, 12.0 Hz, H-60b) [dC 95.5 (C-10), 74.4 (C-20), 78.6
(C-30), 71.3 (C-40), 79.0 (C-50), 62.6 (C-60)] which strongly
supported the presence of D-glucopyranose (Ishimaru et al.
1987; Jin et al. 2014). In addition, acid hydrolysis of 1 in
refluxing 1 N-HCl/MeOH afforded (?)-D-glucose which
was detected by direct comparison with an authentic
sample using co-TLC (Kim et al. 2004). The coupling
constant (J = 7.5 Hz) of the anomeric proton of D-glucose
indicated it to be in the b-form (Ishimaru et al. 1987). The
glycosidic linkage was established by an HMBC experi-
ment and comparison of the reported NMR data (Jin et al.
2011), with the correlation between H-10 and C-8 sug-
gesting that glucose was attached to C-8 of indole 3-car-
boxylate moiety. Furthermore, long-range correlations
were observed between the following protons and carbons:
H-4 and C-7a; H-5 and C-3a; H-6 and C-7a; H0-1 and C-8;
–SCH3 and C-3 (Fig. 2). In the 1H-1H COSY spectrum, the
aromatic proton of H-4 showed couplings with H-5, H-6
and H-7. Accordingly, the structure of 1 was proposed to
be b-D-glucopyranosyl 2-(methylthio)-1H-indole-3-car-
boxylate, and named, raphanuside A.
Bioassay of IL-6
An IL-6 bioassay was carried out using a slight modifica-
tion of an established method (Kim et al. 2003; Liu et al.
2006). Briefly, 500 lL of the MG-63 cells (3 9 104 cells/
mL) in Dulbecco0s modified Eagle0s medium (DMEM)
containing 10 % fatal bovine serum (FBS) were dispensed
into a 24-well plate; the culture was incubated for 24 h at
37 °C. Then, 5 lL of TNF-a (10 ng/mL), 5 lL of BAY
11-7085 (10 ng/mL) and 5 lL of DMSO with or without
the compounds (100 lg/mL) were added. After incubation
at 37 °C with 5 % CO2 for 24 h, the medium was stored at
-20 °C until measurement. The IL-6 content of the med-
ium was measured in an enzyme-linked immunosorbent
assay (ELISA) procedure. The 96-well plates were coated
with 100 lL of purified rat anti-human IL-6 monoclonal
antibody in 0.1 M NaHCO3 (pH 9.6) by overnight incu-
bation at 4 °C. The wells were blocked with 200 lL of 3 %
BSA in PBS for 2 h at room temperature and then incu-
bated with 100 lL of specific antibody for 2 h at rt.
Horseradish peroxidase (HRP) conjugated rabbit anti-goat
IgG (100 lL, 1:1000 dilution) was added to each well and
incubated for 2 h at rt. TMB (3,30,5,50-tetramethyl-ben-
zidine) substrate solution (100 lL) was added and incu-
bated for 10 min at rt. The color reaction was stopped by
addition of 50 lL of 0.4 N HCl and the optical density was
measured at 450 nm using a Microplate Reader (Molecular
Devices Co., Ltd., U.S.A.).
Results and discussion
Compound 2 was obtained as an amorphous powder,
[a]2D0 ? 10.4° (MeOH; c 0.36). The positive mode FAB-
MS spectrum showed a quasi-molecular ion peaks at m/z
571[M ? Na]?, consistent with a molecular formula of
Repeated column chromatography of the n-BuOH soluble
fraction of the seeds of R. sativus yielded a new indole
glycoside, named raphanuside A (1), as well as eight
known phenolic compounds 2–9 (Fig. 1).
1
C23H32O15. The H-NMR spectrum of 2 indicated a trans
Compound 1 was obtained as a yellowish amorphous
powder, [a]2D0 ?31.0° (MeOH; c 0.26). Its molecular for-
mula was determined to be C16H18O7NS by HR-ESI–MS
(negative mode) m/z: 368.0797 [M-H]- (calcd for C16-
H18O7NS, 368.0804). In the IR spectrum, absorption bands
for the imino (3480, 3420, 3300 cm-1) and carbonyl
olefinic protons at [dH 7.63 (1H, d, J = 16.0 Hz, H-700),
6.46 (1H, d, J = 16.0 Hz, H-800)], a 1,3,4,5-tetrasubstituted
aromatic ring protons at [dH 6.95 (2H, s, H-200, 600)] and two
1
methoxy groups at dH 3.87 (6H, s). Furthermore, the H
and 13C NMR spectrum showed a characteristic a-
anomeric resonance at dH 5.42 with a small coupling
123

H.-G. Jin et al.
O
Table 1 Inhibitory effect of compounds 1–9 against IL-6 production
in TNF-a stimulated MG 63 cells
HO
O
OH
OH
O
Treatment
IL-6 (pg/mL)
Inhibition (%)
SCH3
OH
N
H
None
19.7 3.7
–
TNF-a
250.6 3.4
–
Fig. 2 Key HMBC (H ? C) correlations of compound 1
BAY 11-7085
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
30.2 2.1**
215.0 6.8*
142.8 3.8**
138.3 3.9**
133.8 5.8**
107.6 10.5**
177.5 5.0**
172.0 4.4**
182.8 5.4**
193.5 4.3**
87.9**
14.0*
42.9**
44.7**
46.6**
57.1**
29.0**
31.2**
26.9**
22.6**
constant (d, J = 3. 5 Hz, H-10), together with 12 oxy-
genated carbon signals in the range dC 105.3–64.4. These
data implied that compound 2 had a disaccharide moiety
composed of a pentose and hexose unit. On the basis of
chemical evidence, detailed analysis by 1H–1H COSY,
HMQC, and HMBC experiments, and comparison of
spectroscopic data with literature reports, compound 2 was
identified as b-D-fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-
a-D-glucopyranoside, sibiricose A1, which was previously
isolated from the roots of Polygala sibirica (Ikeya et al.
1991; Miyase et al. 1999).
MG-63 cells (3 9 104) were incubated for 24 h. Cultures were
incubated with or without compounds (100 lg/mL) for 30 min and
then stimulated with TNF-a (10 ng/mL) for 24 h. IL-6 in the super-
natant was measured by ELISA as described in Materials and
Methods. Results are expressed as the mean SE from three dif-
ferent experiments. BAY 11-7085 was used as a positive control.
* P \ 0.05 or ** P \ 0.01 compared with TNF-a treated value
The structures of seven other compounds were identified
as
(3-O-sinapoyl)-b-D-fructofuranosyl-(2 ? 1)-a-D-glu-
copyranoside (3) (Bashir et al. 1993; Miyase et al. 1999), (3-
O-sinapoyl)-b-D-fructofuranosyl-(2 ? 1)-(6-O-sinapoyl)-
a-D-glucopyranoside (4) (Bashir et al. 1993; Kim et al.
2014), (3,4-O-disinapoyl)-b-D-fructofuranosyl-(2 ? 1)-
(6-O-sinapoyl)-a-D-glucopyranoside (5) (De Tommasi
et al. 1993; Kim et al. 2014), isorhamnetin 3,40-di-O-b-D-
glucoside (6) (Nørbæk et al. 1999; Park et al. 2007),
isorhamnetin 3-O-b-D-glucoside-7-O-a-L-rhamnoside (7)
(Park et al. 2007), isorhamnetin 3-O-b-D-glucoside (8)
(Woo et al. 1983) and 3’-O-methyl-(-)-epicatechin 7-O-b-
D-glucoside (9) (Korver and Wilkins 1971; Tschesche et al.
1980; Morimoto et al. 1985), by comparing their spectro-
scopic data with literature reports.
macrophages, phorbol 12-myristate 13-acetate (PMA) or
phytohemagglutinin (PHA) stimulated human peripheral
blood mononuclear cell (Comalada et al. 2006), activated
human astrocytes (Sharma et al. 2007), activated human mast
cell line HMC-1 (Min et al. 2007), nasal mucosal fibroblasts
and A549 bronchial epithelial cells (Kim et al. 2006).
In our study, the inhibitory activity of the isolated
compounds (1–9) against IL-6 production in TNF-a stim-
ulated MG-63 cells was examined. Among them, com-
pounds 2–5 showed moderate inhibitory activity against
IL-6 production in TNF-a stimulated MG-63 cells, while
raphanuside A (1), and compounds 6–9 showed negligible
activity (Table 1). Recently, Kim et al. reported the inhi-
bitory effects of phenylpropanoid sucrosides from the seeds
of R. sativus on NO production in LPS-activated murine
microglia BV-2 cells (Kim et al. 2015).
Interleukin-6 (IL-6) is a cytokine that can facilitate
autoimmune phenomena, amplify acute inflammation and
promote evolution into a chronic inflammatory state(Fonseca
et al. 2009). Dysregulation of IL-6 production has been
implicated in a variety of inflammatory and autoimmune
disease states, including rheumatoid arthritis, cardiac myx-
oma, Castleman0s disease and mesangial proliferative
glomerulonephritis (Hirano et al. 1990). The proinflamma-
tory cytokines IL-1 and TNF-a markedly stimulate the pro-
duction of IL-6 (Van Damme et al. 1987). Identification of
plant-derived compounds, such as phenolic compounds, able
to selectively interfere with the production and/or function of
cytokines could offer an important alternative for the treat-
ment of many inflammatory diseases (Calixto et al. 2004;
Comalada et al. 2006). It has been observed that several fla-
vonoids are able to decrease the expression of different pro-
inflammatory cytokines/chemokines, including TNF-a, IL-
1b, IL-6, IL-8, and MCP-1, in many cell types such as
lipopolysaccharide (LPS)-activated mouse primary
In conclusion, this paper reports the isolation, charac-
terization and inhibitory activity of nine isolates, including
one new compound and eight known compounds, from the
seeds of R. sativus.
Acknowledgments This work was supported by
a
Grant
(12172MFDS989) from the Ministry of Food and Drug Safety, Govt.
of Republic of Korea in 2013–2014. We thank Dr. K. D. Park at the
Korea Basic Science Institute for Gwangju Center for help in
obtaining the NMR spectra.
Compliance with ethical standards
Conflict of interest All authors declare that they have no conflict of
interest
123

Indole glycoside from seeds of Raphanus sativus
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