Phytochemical screening of flavonoids with their antioxidant activities from rapeseed (Brassica napus L.)

Article abstract of DOI:10.1016/j.phytol.2015.06.014

Abstract Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed, and their protective antioxidant effect on H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as kaempferol-3-O-[(6-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (8), kaempferol-3,7-di-O-β-d-glucopyranoside-4'-O-(6-O-sinapoyl)-β-d-glucopyranoside (9), and kaempferol-3-O-[(3-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (10). The protective effects of all of the isolated compounds on H₂O₂-induced oxidative damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. All of compounds had a protective effect on H₂O₂-induced oxidative damage in ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were used to elucidate the activity differences.

Full text of DOI:10.1016/j.phytol.2015.06.014

Phytochemistry Letters 13 (2015) 239–245
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Phytochemical screening of flavonoids with their antioxidant activities
from rapeseed (Brassica napus L.)
*
Wen Guang Jing, Jiang Fu, Yan Guo, An Liu
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed,
and their protective antioxidant effect on H2O2-induced oxidative damage in human umbilical vein
endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as
Received 27 March 2015
Received in revised form 6 June 2015
Accepted 22 June 2015
Available online xxx
kaempferol-3-O-[(6-O-sinapoyl)-
anoside (8), kaempferol-3,7-di-O-
and kaempferol-3-O-[(3-O-sinapoyl)-
b
-D
-glucopyranosyl-(1 !2)-
-glucopyranoside-4'-O-(6-O-sinapoyl)-
-glucopyranosyl-(1 !2)- -glucopyranoside]-7-O-
b
-
D
-glucopyranoside]-7-O-
-glucopyranoside (9),
-glu-
b-D-glucopyr-
b
-D
b-D
b
-D
b
-D
b-D
Keywords:
copyranoside (10). The protective effects of all of the isolated compounds on H2O2-induced oxidative
damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase
(LDH) were measured. All of compounds had a protective effect on H2O2-induced oxidative damage in
ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were
used to elucidate the activity differences.
Phytochemical screening
Rapeseed meal (Brassica napus L.)
Flavonoid glycosides
LDH activity
SOD activity
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
and glucopyranosyl sinapate from rapeseed were reported, limited
information was available on further study of their antioxidant
Rapeseed (Brassia napus L.) is the most common source of
vegetable oils in Europe. Rapeseed meal, comprised of substantial
available natural resources, is a byproduct of the oil removal
process. Since Marczak et al. (2003) had examined the protein of
the rapeseed meal as a source of new peptides with ACE-inhibitory
activity, it appears to be a good source of other bioactive
compounds, such as polyphenols, phytosterols, tocopherols from
the rapeseed meal, which are important in the prevention and
treatment of many disease (Szydlowska-Czerniak et al., 2011).
Much work has been performed to find interesting constituents as
sources of natural antioxidants from rapeseed meal. Up to now,
phytochemical studies led to the isolation of several phenolic
compounds and peptides hydrolyzed from rapeseed oil and protein
(Harbaum-Piayda et al., 2010). Kuwahara et al., (2004) and
Wakamatsu et al., (2005) had reported a phenolic compound,
named 4-vinyl-2,6-dimethoxyphenol (canolol), exhibited more
potent anti-alkylperoxyl radical activity than well-known anti-
activity (Amarowicz et al., 1995; Amarowicz and Kolodziejczyk,
2001; Amarowicz and Shahidia, 1994).
From this information, we speculated that rapeseed contains
other unknown constituents with remarkable antioxidant activity
and were prompted to undertake further studies to assist in its
medicinal and food applications. Three new flavonoid glycosides
were isolated and identified, their antioxidant activity were also
discussed.
2. Materials and methods
2.1. General procedures
Compound samples were dissolved in dimethyl sulfoxide
(DMSO-d6). 1H, 13C, and 2D NMR spectra (1H–1H COSY, HSQC,
HMBC) were obtained from the analytical center of the Beijing
University of Chemical Technology and recorded at 25 ꢀC on a
Bruker AV 600 NMR spectrometer operated at a 1H frequency of
600 MHz and 13C frequency of 150 MHz (Bruker BioSpin, Rhein-
stetten, Germany). Chemical shifts (1H) were expressed in ppm and
coupling constants (J) were reported in Hz. Thermo Scientific
Orbitrap Velos Pro hybrid ion trap Orbitrap mass spectrometer was
used for determining the accurate molecular weight of the isolated
compounds. Optical rotations were measured using an Atago AP-
300 automatic polarimeter (Atago Co., Tokyo, Japan), and UV
oxidants, such as
a-tocopherol, vitamin C, and b-carotene.
Although (Durkee and Harborne, 1973) few flavonoid glycosides
* Corresponding author at: Institute of Chinese Materia Medica, China Academy
of Chinese Medical Sciences, No.16 Nanxiaojie, Dongzhimen Nei Ave., Beijing
100700, China. Fax: +86 10 64013996.
E-mail address: liuan_508@163.com (A. Liu).
http://dx.doi.org/10.1016/j.phytol.2015.06.014
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

240
W.G. Jing et al. / Phytochemistry Letters 13 (2015) 239–245
spectra were recorded on a Shimadzu SPD-M20AVP UV–vis
photodiode array detector (DAD) (Shimadzu Co., Kyoto, Japan).
An LC 3000 preparative liquid chromatography system (Beijing
ChuangXin TongHeng Science and Technology Co., Ltd., Beijing,
China) was used to carry out separations and purifications. Diaion
HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), Sephadex LH-20
(Pharmacia Biotech AB, Uppsala, Sweden), and silica gel 60 (160–
200 mesh, Qingdao, Qingdao Chemical Work) were used for
column chromatography. Thin-layer chromatography (TLC) was
performed on silica gel F-254 plates (Merck), and spots were
detected by ultraviolet (UV) illumination. GC analysis was
performed on an HP-5 column (30 m, 0.320 mm ID) with the
column temperature at 230 ꢀC.
crude sample preparation were of analytical grade and supplied by
Beijing Chemical Factory (Beijing, China). HPLC-grade methanol
and acetonitrile (Merck KGaA, Darmstadt, Germany) were supplied
by Beijing Chemical Factory (Beijing, China). Deuterated solvents
were supplied by J&K Scientific Ltd., (Beijing, China). Water was
purified by a Milli-Q water purification system (Millipore, Milford,
MA, USA). Dulbecco's modified Eagle’s medium F12 (DMEM-F12)
was obtained from Gibco (Grand Island, NY, USA, batch no:
8119292). Reagent kits for measurement of superoxide dismutase
(SOD), with batch No. 20140108, and lactate dehydrogenase (LDH),
with batch No. 20140307, were provided by Nanjing Jiancheng
Bioengineering Institute (Nanjing, China).
2.3. Extraction and isolation
2.2. Materials, solvents and chemicals
The milled rapeseed meal (20 kg) was defatted three times with
n-hexane and subjected to extraction at reflux with a 60% aqueous
ethanol solution for three times. The obtained extract was then
The defatted rapeseed meal was supplied by Guizhou Great
Wall Oil Co., Ltd. (Guiyang, Guizhou). All organic solvents used for
Table 1
1Ha- and 13Cb-NMR data of compounds 8,9 and 10.
Position
8
9
10
1H
13C
1H
13C
1H
13C
2
3
4
5
6
7
8
156.3
133.0
177.5
160.8
99.2
156.1
134.0
177.7
160.9
99.4
156.2
133.0
177.6
160.8
99.3
6.37 (d, 1.9)
6.62 (d, 1.9)
6.46 (br. s)
6.70 (br. s)
6.43 (d, 2.0)
6.80 (d, 2.0)
162.6
94.2
162.9
94.4
162.7
94.3
9
155.8
105.3
120.3
131.0
115.4
160.6
115.4
131.0
156.1
105.8
123.6
130.7
115.6
159.1
115.6
130.7
155.9
105.6
120.6
131.0
115.4
160.3
115.4
131.0
10
10
20
8.01 (d, 8.8)
6.89 (d, 8.8)
8.10 (d, 8.8)
7.18 (d, 8.8)
8.09 (d, 8.8)
6.94 (d, 8.8)
30
40
50
6.89 (d, 8.8)
8.01 (d, 8.8)
12.51 (br. s)
5.60 (d, 7.2)
3.43 (oc)
3.32 (m)
3.25 (o)
3.06 (o)
3.70,3.75 (each, o)
4.69 (d, 7.6)
3.32 (d, 8.4)
3.42 (o)
3.24 (d, 8.1)
3.50 (o)
4.30 (m), 4.23 (d, 10.7)
5.03 (d, 7.6)
3.26 (o)
3.17 (o)
3.27 (o)
3.07 (o)
3.44,3.52 (each, o)
7.18 (d, 8.8)
8.10 (d, 8.8)
12.55 (br. s)
5.48 (d, 7.5)
3.16 (o)
3.22 (m)
3.16 (o)
3.41 (o)
3.67, 3.45 (each, o)
5.06 (d, 7.2)
3.26 (o)
3.42 (o)
3.08 (o)
6.94 (d, 8.8)
8.09 (d, 8.8)
12.65 (br. s)
5.75 (d, 7.3)
3.55 (o)
3.30 (m)
3.10 (o)
3.11 (o)
3.51,3.26 (each, o)
4.81 (d, 7.8)
3.29 (o)
4.96 (t, 9.4)
3.45 (m)
60
5-OH
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
1””
2””
3””
4””
5””
6””
97.9
83.2
76.4
69.6
77.1
60.4
104.4
74.5
76.2
69.6
73.9
63.3
99.7
73.1
77.1
69.6
77.5
60.6
100.7
74.2
76.5
69.5
77.2
60.6
99.7
73.2
76.2
69.9
77.7
60.6
99.8
73.1
76.2
69.4
73.7
62.9
97.8
81.9
76.8
69.7
77.5
60.5
103.6
72.3
77.4
67.6
76.7
60.4
99.7
73.1
76.4
69.6
77.2
60.6
3.27 (o)
3.51 (o)
3.58, 3.35 (each, o)
5.14 (d, 7.5)
3.32 (o)
3.17 (o)
3.35 (o)
3.63, 3.55 (each, o)
5.08 (d, 7.4)
3.26 (o)
3.30 (o)
3.17 (o)
3.34 (o)
4.35,4.29 (each, o)
3.45 (o)
3.71,3.47 (each, o)
Sinapoyl
1
2
166.5
114.1
145.5
124.1
106.0
148.0
138.7
147.9
106.0
56.0
166.6
114.5
145.6
166.2
115.4
145.0
6.24 (d, 15.8)
7.37 (d, 15.8)
123.9
6.77 (s)
147.9
6.54 (d, 15.8)
7.55 (d, 15.8)
124.5
6.99 (s)
148.0
6.95 (d, 15.8)
7.56 (d, 15.8)
3
10
20
106.2
7.03 (s)
106.1
30
40
138.6
148.0
106.2
56.0
138.1
148.0
106.1
56.1
50
60
OCH3
a
1H NMR data were obtained at 600 MHz with dimethyl sulfoxide-d6 (25 ꢀC). Chemical shifts are shown in
d
values relative to solvent peak. Multiplicity and coupling
constant (s) in Hz are in parentheses.
b
13C NMR data were obtained at 150 MHz with dimethyl sulfoxide-d6 (25 ꢀC). Chemical shifts are shown in
Overlapped signals were indicated by “o”.
d values relative to solvent peak.
c

W.G. Jing et al. / Phytochemistry Letters 13 (2015) 239–245
241
concentrated under reduced pressure in a rotary evaporator to
remove the ethanol, yielding the crude sample extract. The residue
was dissolved in water and extracted with ethyl acetate for three
times, yielding the EtOAc extract (190 g). The water solution was
separated by commercially available AB-8 column chromatogra-
phy using H2O as the initial eluent, followed by 30%, 60%, and 90%
aqueous ethanol. Each solution was evaporated in vacuo to yield
the H2O fraction (523 g), 30% fraction (180 g), 60% fraction (200 g)
and 90% fraction (9.6 g). The EtOAc extract was pulverized and
chromatographed on a silica gel column (column volume 1500 mL)
using varying ratios of petroleum ether–ethyl acetate (PE/EtOAc),
PE–acetone and chloroform–methanol (CHCl3/MeOH) as the
eluting solvents to afford 1–135 fractions.
acetonitrile (91:9). Similarly, compounds 9 (32 mg) and 10 (19 mg)
were purified by semi-preparative scale HPLC eluted with H2O-
acetonitrile (89:11) from the 45% subfraction.
Kaempferol-3-O-[(6-O-sinapoyl)-
b
-D-glucopyranosyl-(1 !2)-
b
-D-glucopyranoside]-7-O-b-D-glucopyranoside (8). Pale yellow
powder; ½a 2D0
ꢃ2.7 (c 0.100, MeOH); UV lmax (MeOH): 225, 267,
ꢂ
328 nm; 1H and 13C NMR data: see Table 1; ESI/Orbit Trap MS (m/z):
[M + Na]+ 1001.2541 calcd. for C44H50O25Na: found, 1001.2559.
Kaempferol-3,7-di-O-b
-D-glucopyranoside-40-O-(6-O-sina-
poyl)-
b
-D-glucopyranoside (9). Pale yellow powder; ½a D20
ꢃ3.2 (c
ꢂ
0.100, MeOH); UV lmax (MeOH): 243, 267, 324 nm; 1H and 13C NMR
data: see Table 1; ESI/Orbit Trap MS (m/z): [M + Na]+
1001.2541 calcd for C44H50O25Na: found, 1001.2532.
Fractions 72-83 were further subjected to Sephadex LH-
20 column chromatography elution with 90% aqueous methanol
to give compound 1 (60 mg). Fractions 115–127 were chromato-
Kaempferol-3-O-[(3-O-sinapoyl)-
b
-D-glucopyranosyl-(1 !2)-
b
-D-glucopyranoside]-7-O-b-D-glucopyranoside (10). Pale yellow
powder; ½a 2D0
ꢃ5.1 (c 0.100, MeOH); UV lmax (MeOH): 234, 266,
ꢂ
graphed on an ODS C18 gel column (20 mm ꢁ 250 mm, 5
mm) and
367 nm; 1H and 13C NMR data: see Table 1; ESI/Orbit Trap MS (m/z):
eluted with MeOH–H2O (6:4) to afford compound 2 (35 mg),
compound 3 (15 mg) and compound 4 (27 mg). The 60% fraction
from the macroporous resin was preliminarily separated on an MCI
gel column to yield 10%, 30%, 45%, 60% and 100% subfractions. The
30% aqueous methanol subfraction was separated on a Sephadex
[M + Na]+ 1001.2541 calcd. for C44H50O25Na: found, 1001.2551.
2.4. Acid hydrolysis of compounds 8, 9 and 10
Compounds 8, 9 and 10, 2 mg each in a mixture of 3.0 M
trifluoroacetic acid (2 mL), were separately hydrolyzed for 3 h at
110 ꢀC. In each case, the reaction mixture was extracted with ethyl
LH-20 column chromatography to yield
compounds 5 (21 mg), 6 (13 mg), 7 (120 mg) and 8 (55 mg) and
further purified on a semi-preparative HPLC eluted with H2O–
a crude sample of
Fig. 2. Structures and significant HMBC and 1H-1H COSY correlations of compounds 8, 9 and 10.

242
W.G. Jing et al. / Phytochemistry Letters 13 (2015) 239–245
acetate (3 ꢁ 2 mL) and the water soluble mixture was reduced in
3. Result and discussion
vacuo to dryness. The residue was dissolved in anhydrous pyridine
(100
added. After heating for 1 h at 60 ꢀC, the reaction mixture was
treated with 150 L of HMDS–TMCS (1:1) and incubated for 0.5 h
m
L), and 0.06 M L-cysteine methyl ester hydrochloride was
3.1. Structural elucidation of compounds 8, 9 and 10
m
Compound 8 was obtained as pale amorphous powder and
exhibited a quasi-molecular ion peak at m/z 1001.2559 [M + Na]+,
consistent with a molecular formula of C44H50O25Na from the HR
ESI–MS. Acid hydrolysis of the compound afforded D-glucose as
the sugar residues, which was confirmed by GC using authentic
at 60 ꢀC. Finally, the reaction mixture was centrifuged, and the
supernatant was analyzed by gas chromatography. The sugars
were identified by comparison of the retention times with those of
authentic samples. The derivative of D-glucose was detected at a
retention time tR of 12.04 min.
samples. A typical signal at
d 12.51 ascribable to 5-OH was
observed in the 1H NMR spectrum, indicating a free hydroxyl group
at the C-5. Signals due to an aromatic AA'BB' system with the
expected coupling pattern were also observed, and the assignment
of 1H and 13C NMR spectra data, supported by HSQC and HMBC
correlations, confirmed the aglycone as kaempferol. Moreover,
2.5. Effect of the isolated compounds on hydrogen peroxide-induced
oxidative damage in human umbilical vein endothelial cells (ECV-304)
ECV-304 cells were isolated from the vein of normal human
umbilical cord as previously reported (Martin et al., 1993). The
cells were cultured in DMEM-F12 supplemented with 10% heat-
inactivated fetal bovine serum, 100 units/mL penicillin and
100 units/mL streptomycin at 37 ꢀC under 5% CO2 and 95% air.
Cells digested by trypsin and cultured in vitro were transferred to
each well of a 24-well microplate with fresh DMEM culture
medium and no serum, and randomly divided into three groups:
peaks corresponding to three anomeric protons at
5.03 (d, 7.6) and 4.69 (d, 7.6), with corresponding anomeric carbon
signals at 97.9, 99.7 and 104.4 based on the HSQC spectrum,
d 5.60 (d, 7.2),
d
suggested a tri-glycoside nature of this compound. The diaxial
coupling constants between the anomeric protons and H-2 in the
1H NMR indicated that all of the glucosyl units were
b
-linked
(Agrawal, 1992). In addition, the presence of two methoxy groups
at 3.72 (each, 3H, s), two phenyl protons at 6.77 (each,1H, s), and
the trans olefinic protons at
6.24 and 7.37 (each, d, 15.8) in the 1H
blank control group, model group treated with 100
mg/mL of H2O2
d
d
for 12 h, and drug group successively treated with H2O2 and test
compounds for 12 h. After 12 h, the supernatant was collected and
used for the detection of SOD and LDH activity using commer-
cially available kits. SOD was detected at 550 nm, while LDH
release represents cell viability and was determined at 440 nm.
Each experiment was carried out at least six times, and all of the
results were expressed as the mean ꢄ S.D. Comparison between
experiment groups was performed by ANOVA followed by Tukey’s
multiple range tests using SPSS 19.0 (SPSS, Inc., Chicago, IL). P
values less than 0.05 were considered to be significant.
d
NMR clearly revealed the existence of a sinapoyl group (Jung et al.,
2009).
13
In the C NMR spectrum, a carbon signal due to C-200 of a
glucose unit shifted downfield at
d 83.2. In comparison with
kaempferol-3-glucoside (Agrawal, 1992), it was proved the
attachment of an additional glucose unit at this position and
suggested a glucopyranosyl-(1 !2)-
This was also further supported by the HMBC correlation peak
observed between the anomeric proton (H-1000,
4.69) of a glucose
unit and the carbon at
83.2 assigned to C-200 of the kaempferol
inner 3-glucosyl moiety. Additionally, according to the HMBC
spectrum, the signals of glucosyl anomeric protons at
5.60 (H-100)
b-D-glucopyranoside moiety.
d
d
d
Fig. 1. Structures of the known flavonoids 1–7.

W.G. Jing et al. / Phytochemistry Letters 13 (2015) 239–245
243
Table 2
Evaluation on LDH and SOD activity in the supernatant of ECV-304 cells of the isolated compounds (mean ꢄ s).
Compounds
Concentration (
m
g/
LDH (U/L)
SOD (U/mL)
mL)
Blank control group
Model control group
–
–
121.9 ꢄ 10.1
25.90 ꢄ 1.62
323.8 ꢄ 6.8a,# 19.60 ꢄ 0.73#
154.5 ꢄ 14.6** 24.81 ꢄ 0.42**
Kaempferol (1)
Astragalin (2)
200
100
162.8 ꢄ 6.4**
23.90 ꢄ 0.29**
200
100
245.9 ꢄ 7.4**
314.1 ꢄ 11.5
22.96 ꢄ 0.34**
20.44 ꢄ 0.48*
Kaempferol-7-O-
b-D
-glucopyranoside (3)
200
100
171.2 ꢄ 2.6**
276.0 ꢄ 9.0**
23.91 ꢄ 0.55**
22.00 ꢄ 0.46**
Isorhamnetin-3-O-
b
-D-glucopyranoside (4)
200
100
270.2 ꢄ 6.1**
317.8 ꢄ 8.7
22.65 ꢄ 0.36**
20.13 ꢄ 0.27*
Kaempferol-3,7,40-tir-O-
b-D
-glucopyranoside (5)
200
100
239.8 ꢄ 3.8**
21.80 ꢄ 0.48**
293.8 ꢄ 29.3** 19.77 ꢄ 0.30
Kaempferol-3-O-
b-D
-glucopyranosyl-(1 !2)-
b-D
-glucopyranoside-7-O-
b
-D-glucopyranoside (6)
200
100
198.4 ꢄ 3.2**
21.90 ꢄ 0.33**
257.9 ꢄ 13.7** 20.36 ꢄ 0.28
Kaempferol-3-O-[(2-O-sinapoyl)-
(7)
b
-
D
-glucopyranosyl-(1 !2)-
b
-
D
-glucopyranoside]-7-O-
b
-
D
-glucopyranoside 200
125.2 ꢄ 4.9**
177.7 ꢄ 18.4**
24.22 ꢄ 0.35**
23.42 ꢄ 0.73**
100
-glucopyranoside 200
100
Kaempferol-3-O-[(6-O-sinapoyl)-
(8)
b
-
D
-glucopyranosyl-(1 !2)-
b
-
D
-glucopyranoside]-7-O-
b
-
D
245.2 ꢄ 17.2** 21.56 ꢄ 0.42**
273.8 ꢄ 16.12** 20.02 ꢄ 1.71
Kaempferol-3,7-di-O-
b-D
-glucopyranoside-40-O-(6-O-sinapoyl)-
b-D
-glucopyranoside (9)
200
100
239.8 ꢄ 7.3**
268.0 ꢄ 8.7**
19.97 ꢄ 0.77
18.80 ꢄ 0.32
Kaempferol-3-O-[(3-O-sinapoyl)-
(10)
b-D
-glucopyranosyl-(1 !2)-
b-D
-glucopyranoside]-7-O-
b
-D
-glucopyranoside 200
196.6 ꢄ 4.9**
21.50 ꢄ 1.51**
100
229.6 ꢄ 11.6** 21.98 ꢄ 0.41**
a
Compared with blank control group.
#
*
P < 0.01, compared with model control group.
P < 0.05, compared with model control group.
P < 0.01 compared with model control group.
**
and
d
5.03 (H-1000) were correlated with C-3 (
d
133.0) and C-7 (
d
and 40-positions. This was further supported by HMBC correlation
162.6) of the aglycone, respectively, indicating the presence of a
3,7-diglucosyl residue. Similarly, the location of the sinapoyl group
was confirmed by an HMBC experiment, where correlation peaks
peaks between the anomeric protons at
the corresponding aglycone carbons at
d
d
5.48, 5.06 and 5.14 with
134.0 (C-3), 159.1 (C-40)
and 162.9 (C-7), respectively. Furthermore, a comparative analysis
were observed between proton signals at
glucose unit and the ester carbonyl at 166.5 of the sinapoyl unit,
proving that the attachment of the sinapoyl unit was at C-6000. These
results were consistent with a downfield shift of C-6000(
-C) at
63.3 and an upfield shift of C-5000
-C) at 73.9 of the kaempferol
d
4.30, 4.23 (H-6000) of a
of the 13C NMR chemical shifts of the sugar units with those of
d
compound 5 showed a significant d 2.2 ppm downfield shift for C-
6 of a glucose unit, suggesting the sinapoyl moiety was attached at
a
d
this position. This was confirmed from a HMBC experiment where
(b
the signals at
with the ester carbonyl (
long-range correlations were observed between the signals at
3.35, 3.58 (H-6000) and C-5000
77.7). Meanwhile, the anomeric
proton (
5.06) assigned to the 40-O-glucose moiety was also
correlated with C-5000
(d 77.7), which confirmed that the linkage of
d
3.35, 3.58 (H-6000) in the 1H NMR were correlated
outer 3-glucosyl moiety, and further corroborated by HMBC
analyses: correlation peaks were observed between the anomeric
d 166.6) of the sinapoyl unit. In addition,
d
proton H-1000
(
d
4.69) and C-5000
(
d
73.9). From the above evidence,
the structure of compound 8 was established as kaempferol-3-O-
[(6-sinapoyl)- -D-glucopyrano-
-D-glucopyranosyl-(1 !2)-O-
side]-7-O- -D-glucopyranoside.
(d
d
b
b
b
the sinapoyl unit was at the 6-position of the 40-O-glucose moiety.
From these results, the structure of compound 9 was concluded to
Compound 9 afforded D-glucose as the sugar residues upon acid
hydrolysis. It showed a quasi-molecular ion peak at m/z 1001.2532
[M + Na]+, consistent with a molecular formula of C44H50O25Na
based on HR ESI–MS. The 1H NMR spectrum revealed the presence
of three anomeric protons at 5.48 (d, 7.5), 5.06 (d, 7.2) and 5.14 (d,
7.5), indicating three glucose units. A characteristic sinapoyl
moiety was also observed in the 1H NMR, which gave two methoxy
be kaempferol-40-O-(6-sinapoyl)-
b-D-glucopyranoside-3,7- di-O-
b-D-glucopyranoside.
Compound 10 was obtained as a yellow powder, and the HR
ESI–MS spectrum showed an [M + Na]+ ion at m/z 1001.2551, which
was attributable to a molecular formula of C44H50O25. Acid
hydrolysis gave kaempferol and D-glucose with comparison to
authentic samples. The 1H NMR of compound 10 showed three
groups at
1H, s) and trans olefinic protons at
Comparative analysis of the 13C NMR data of the aglycone carbons
in compound 9 and kaempferol-3,7,40-tri-O-
-D-glucoside (5)
proved that the glycosylation sites of compound 9 were at the 3-, 7-
d
3.75 (each, 3H, s), two phenyl protons at
d 6.99 (each,
d
6.54 (d, 15.8) and 7.55 (d, 15.8).
glucose moieties from three anomeric proton signals at
7.3), 4.81 (d, 7.8) and 5.08 (d, 7.4), with the corresponding anomeric
carbon signals at
97.8 (C-100), 103.6 (C-1000) and 99.7 (C-1000),
respectively. The 1H NMR showed that all glucosyl units were
d 5.75 (d,
b
d

244
W.G. Jing et al. / Phytochemistry Letters 13 (2015) 239–245
b
-linked to other glucose units or to the aglycone because the
glucopyranoside (5) at the level of 100 mg/mL was a little weaker
coupling constants between the anomeric protons and H-2 were
always approximately 7–8 Hz (Agrawal, 1992). In the 13C NMR
spectrum, chemical shifts in the aglycone at C-3 and C-7 due to
glycosylation relative to kaempferol (1) indicated that the sugar
units were attached at these positions. The downfield-shifted
than compound 9, which could be explained by an additional
substitution of a sinapoyl group at the 6000-position. However, no
significant difference was noted at the level of 200 mg/mL.
Similarly, the substitution of a sinapoyl group at the C-2000 (7)
and C-3000 (10) led to an increase in antioxidant activities compared
to compound 6, but the additional substitution of a sinapoyl group
at the C-6000 (8) showed a decrease in both LDH and SOD activities.
This decrease may be due to the substitution position of the
sinapoyl group. Jung et al. (2009) had also demonstrated that the
sinapic acid moiety of kaempferol derivatives may play a crucial
role in increasing antioxidant activity, but it was noteworthy that
the compounds studied in the report were all substituted at the 200-
position. Thus, position of the substituted sinapoyl group in
kaempferol derivatives could be considered as another important
factor that influences their antioxidant activity.
carbon signal at
d
81.9, attributed to the C-200 of the inner glucose
-D-glucopyranoside moiety,
unit, suggested a glucosyl (1 !2)-
b
which was similar to that of compound 7. These results were
consistent with the HMBC correlations shown in Fig. 2, where the
signals of the glucosyl anomeric protons at
4.81 were correlated with those of the kaempferol C-3 (
and C-7 ( 81.9).
162.7), and the inner glucose C-200
The two methoxy groups at 3.80 (each, 3H, s), two phenyl
protons at 7.03 (each, 1H, s), and a trans olefinic protons at
d
5.75, 5.08 and
d
133.0)
d
(d
d
d
d
6.95 and 7.56 (each, d, 15.8) proved the presence of a sinapoyl
moiety. In the 13C NMR spectrum, a carbon signal due to the C-4000
(
b-C) of a glucose unit shifted upfield and appeared at
d
Acknowledgement
67.6 revealing that the sinapoyl moiety might be substituted at
C-3000 or C-5000 of the glucose unit. In the HMBC analysis, long-range
We gratefully acknowledge financial support from National
Science and Technology Major Projects for “Major New Drugs
Innovation and Development” (2013ZX09301307-01).
correlations were observed between a triplet signal at
and the ester carbonyl at 166.2, which proved that the sinapoyl
unit was attached at this position. The observation of long-range
correlations between the anomeric proton at
4.81 (H-1000) and the
signal at
3.29 by 1H–1H COSY analysis proved that the signal at
3.29 could be assigned to H-2000. In addition, a 1H-1H COSY
correlation between the signal at
3.29 (H-2000) and the signal at
4.96 (t, 9.4) was also detected, which revealed that the triplet signal
at
4.96 could be assigned to H-3000. Additionally, the anomeric
proton at
4.81 (H-1000) was correlated with the carbon signal at
77.4 in the HMBC spectrum, which demonstrated that the signal at
77.4 should be assigned to C-3000. Therefore, the sinapoyl group in
d 4.96 (t, 9.4)
d
d
Appendix A. Supplementary data
d
d
Supplementary data associated with this article can be found, in
d
d
the
online
version,
at
http://dx.doi.org/10.1016/j.
phytol.2015.06.014.
d
d
d
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-D-
b
-D-glucopyranoside (3) was slightly
b-D-glucopyranoside (4) and this
b

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