American Journal of Plant Sciences, 2010, 1, 113-118
doi:10.4236/ajps.2010.12015 Published Online December 2010 (
Copyright © 2010 SciRes. AJPS
Purification of a Sinapine-Glucoraphanin Salt
from Broccoli Seeds
Mark A. Berhow1*, Karl Vermillion1, Gulab N. Jham2, Brent Tisserat1, Steven F. Vaughn1
1United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research,
Functional Foods Research, Peoria, USA; 2Universidade Federal de Viçosa, Departamento de Fitopatologia, Viçosa, Brazil.
Email: *
Received August 17th, 2010; revised October 28th, 2010; accepted November 4th, 2010.
A sinapine (sinapoylcholin e)-glucoraphanin salt has been isolated from broccoli seeds and characterized by NMR and
mass spectrometry. This salt extraction method can be used to purify glucoraphanin free from contamination by
Keywords: Broccoli, Brassica, Glucoraphanin, Sinapine, Glucosinolate, Salt
1. Introduction
Broccoli seeds contain several glucosinolates, including
glucoraphanin, glucoiberin, glucoerucin, sinigrin, 4-hy-
droxyglucobrassicin, glucobrassicin, neoglucobrassicin,
and a number of other minor glucosinolates depending
on the hybrid and cultivar [1,2]. Glucoraphanin has at-
tracted attention due to studies linking its degradation
products, including its isothiocyanate form sulphorap-
hane, to the prevention of cancer in humans [3,4]. Due to
the complex mixture of the glucosinolates in the Brassica
species, isolation of large quantities of pure glucose-
nolates for further study and biological characterization
has been difficult, time consuming, and expensive [5-7].
Brassica species also contain significant quantities of
substituted phenolic acids, including sinapine [8-13].
These phenolic acids have been shown to have antioxi-
dant activity [14,15] and may also play a role in the
health promoting activities of a diet high in crucifer
vegetables. Two major UV absorbing compounds were
isolated from broccoli seeds, which decreased during the
course of germination and sprouting. Characterization of
these compounds resulted in the identification of a
unique salt formed from the combination of sinapine and
glucoraphanin, while the second compound was shown
to be the methyl ester of sinapic acid.
2. Results
MeOH extracts prepared from broccoli seeds were sepa-
rated and characterized with a general phenolic HPLC
gradient system monitored at 285 nm for phenolic com-
pounds. The resulting chromatograph showed two major
peaks at 285 nm with retention times of 10 and 33 min-
utes. The two compounds (compounds 1 and 2) were
purified from the MeOH extract using a combination of
flash chromatography and preparative HPLC, yielding
115 mg of compound 1 and 133 mg of compound 2 from
1 kg of defat t ed broccoli seeds.
2.1. Spectral Analysis
Compound 1 appeared pure by HPLC DAD, and the 1H
and 13C NMR spectra were obtained from five milligrams
(Table 1). Initial NMR spectra showed that there were
two components present in the purified compound 1 (in
addition to acetate) that were not fixed in their
stoichiometry. Different batches of the product always
contained both components, but in slightly varying ratios.
The more abundant compound contained two coupled
olefinic protons at 7.70 ppm (1H, d, J = 15.9 Hz, H-7)
and 6.47 ppm (1H, d, H-8) and an aromatic singlet at
6.96 ppm (2H, s, H-2/6), which suggested a symmetri-
cally substituted ring system with an olefinic side chain.
The geometry of the olefinic double bond was deter-
mined to be trans on the basis of the coupling constants.
The NMR spectrum was consistent with a 2,4,6- or a
3,4,5-trioxy-substituted cinnamic acid fragments, in-
cluding the carboxylic carbon at 166.4 ppm (C-9). Two
*Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department o
Agriculture. USDA is an equal opport unity provider and employer.
Purification of a Sinapine-Glucoraphanin Salt from Broccoli Seeds
Copyright © 2010 SciRes. AJPS
methoxy-resonances at 3.90 ppm (6H, s, H-3/5-OMe)
showed correlations to the aromatic ring carbon at 148.1
ppm (C-3/5) in the HMBC NMR spectrum. Since the
substitution pattern must be symmetric, the meth-
oxy-substituents must be at the 3 and 5 positions, or al-
ternately at the 2 and 6 positions, leaving position 4 as a
phenolic moiety. Simulations conducted with ACDLabs
CNMR predictor quickly eliminated the 2,4,6- substitu-
tion pattern and confirmed the 3,4,5- substitution pattern.
Other signals included in this more abundant fragment
included two strongly coupled methylene protons 4.68
ppm (2H, m, H-10) and 3.79 ppm (2H, m, H-11). The
protons at 4.68 ppm (H-10 ) showed a weak corr elation in
the HMBC NMR spectrum to the carboxylic carbon C-9
while the protons at 3.79 ppm (H-11) showed a 1:1:1
splitting pattern, characteristic of coupling to a spin 1
quadrupolar nucleus such as nitrogen. This implied that
the methylene at 4.68 ppm would have an ester linkage to
the rest of the fragment, while there was probably a ni-
trogen atom attached to the methylene at 3.79 ppm. In
addition, the singlet at 3 .27 ppm (9H, s, N-Me) showed a
correlation to the methylene carbon C-11 attached to the
nitrogen atom in the HMBC NMR spectrum. This sug-
gested a quaternary ammonium salt with three methyl
substituents. The remaining position on the quaternary
nitrogen was th e –CH2-CH2- branch linked by an ester to
the substituted cinnamic acid fragment. This compound
was identified as sinapine. The NMR data for this com-
pound compared well with those shown for (E)-sina-
poylcholin e 4-O-ß-glucopyranosi de [1 6] .
The minor component contained a hexose sugar. In-
spection of the HSQC NMR spectrum indicated shifts
typical of an anomeric proton 4.85 ppm (1H, H-1’), but
in the 13C dimension of the HSQC spectrum the C-1’
carbon (82.2 ppm) did not have a shift typical of an
anomeric carbon (normally 95-104 ppm). This suggested
this fragment was not an O-glycoside and had some other
linkage to the rest of the molecule. Overlap in the COSY
NMR spectra made interpretation difficult, but the HSQC
NMR spectrum showed the presence of a 4-carbon me-
thylene chain (C-1,2,3,4), and an isolated methyl group
(C-5). The carbon atom (C-4) at one end of this chain
had a shift of 53.0 ppm, which is somewhat upfield for
an oxygen-bearing carbon. It is more characteristic of a
nitrogen- or sulfur-containing moiety attached to the
carbon. Several simulations with ACDLab software sug-
gested a sulfoxide would account for the 13C NMR shifts
for the methylene at C-4 and the isolated methyl at C-5.
The protons at 2.75 ppm (2H, m, H-1) on the other end of
this chain showed a weak correlation in the HMBC NMR
spectrum to the carbon at 159.4 ppm (the oddly-named
C-0 of glucosinolate nomenclature). This carbon at 159.4
ppm showed another HMBC NMR correlation from the
“anomeric” proton H-1’ of the sugar fragment. The NMR
analysis, along with the mass spectrometry data, which
supported the presence of a sulfate group, indicated this
compound was glucoraphanin [16].
Full spectral assignments of the observed 1H and 13C
NMR shifts indicated that purified compound 1 was a
mixture of sinapine and gluco raphanin (Figure 1) .
Table 1. NMR spectroscopic data (CD3OD , 1H 500 MHz,
13C 125 MHz) for compound 1.
NMR Spectroscopic data
Carbon # 1H Shift
1H Splitting,
coupling (Hz)
13C Shift
2.82, 2.91
3.62, 3.86
d, 15.9
d, 15.9
m; m
t, 8.8
dd, 6.3, 12.0; m
21.8; 177.4
*Multiplicity not determined due to spectral overlap with acetate and solvent
peaks. **Che mical shifts () are in ppm from TMS.
Figure 1. Structure of compound 1: a sinapine-glucorap-
hanin salt, for glucosinolate numbering system see [16].
Purification of a Sinapine-Glucoraphanin Salt from Broccoli Seeds
Copyright © 2010 SciRes. AJPS
The salt was contaminated with a small amount of
acetate, likely due to the purification method, which
contained acetic acid. This was probably acquired during
the freeze-drying step to obtain the purified salt. The
complete NMR spectra obtained for compound 1 agreed
very closely with that published for a similar compound,
Boreavan A purified and characterized from Boreava
orientalis, which is a sinapine and sinigrin salt [17,18].
The NMR assignment for the sinapine fragment agreed
closely with the similar (E)-sinapoylcholine 4-O-ß-glu-
copyranoside [19] and the NMR assignment for the glu-
coraphanin fragment agreed closely with the published
spectra of the ammonium salt of glucoraphanin [16].
Compound 1 was infused under the conditions de-
scribed in the materials and methods section into an ESI
mass spectrometer in both the positive and negative
modes. The positive ESI mass spectrum yielded a single
major ion at m/z 310, which corresponds to the [M]+ ion
of sinapine. In the presence of formic acid, the spectrum
showed m/z ions at 354, 663, and 973, corresponding to
[M]+, [2M]+ and [3M]+ adducts with formic acid, respec-
tively. The negative ESI mass spectrum yielded two ma-
jor ions at m/z 436 and 873, with a minor ion at m/z 895.
These correspond to [M]-, [2M+H]-, and the [2M+Na]-
ions of glucoraphanin. Electrospray mass spectrometry
acquired in the negative mode of pure glucoraphanin
resulting in similar spectra. From this data it was appar-
ent that compound 1 was the salt of sinapine and glu-
coraphanin, contaminated with a small amount of acetate.
After assignment of the sinapine fragment of com-
pound 1 it was obvious that compound 2 was a derivative
of sinapic acid. Compound 2 was identified by NMR
spectroscopy (Table 2) and mass spectrometry and found
to be the methyl ester of sinapic acid (Figure 2). The 1H
and 13C NMR spectrum compared closely with the pub-
lished spectra of methyl-sinapate [20].
The spectral analysis of the sinapoylcholine-glucora-
phanin salt was as follows—Isolated as light yellow
crystals; mp 184-187; UV (MeOH) max (log e), 322
(3.60) nm; IR (KBr, disc) max ( cm-1): 3036, 3010,
2940, 2845, 1706, 1632, 1595, 1515, 1457, 1427, 1338,
1279 (sh), 1254, 1232 (sh), 1153, 1114, 1055; LREIMS
positive mode m/z 310.2 [M]+ and negative mode m/z
436.4 [M]-; HREIMS positive mode m/z 310.1657 [M]+
(calcd. for C16H24NO5, 310.1654); negative mode m/z
436.0424 [M]- (calcd. for C12H22NO10S3, 436.0406). For
1H and 13C NMR spectroscopic data, see Table 1.
3. Discussion
Glucosinolates have previously been shown to be con-
verted enzymatically or chemically to a number of deg-
radation products, such as isothiocyanates, nitriles, and
thiocyanates, once the plant tissue that they are contained
in is damaged or consumed [21]. These degradation
products have been shown to have a wide array of bio-
logical activities both in vivo and in vitro [1,3,4]. The
isothiocyanate degradation product of glucoraphanin is
sulforaphane which has been shown in extensive studies
to have a number of interestin g cancer chemo-preventive
activities in both in vivo and in vitro studies [3,4]. Broc-
coli and related species have fairly high levels of glu-
coraphanin in their seeds and sprouts and are excellent
sources of these compounds for purification and study.
While conversion of glucoraphanin to sulforaphane is
fairly simple, the purification of glucoraphanin is com-
plicated by the presence of other glucosinolates in the
seeds especially glucoiberin, which co-elutes in most
chromatographic procedures.
A recently published method using counter current
chromatography resulted in the purification of 61 grams
of glucoraphanin from 500 grams of crude broccoli seed
extract [22]. The authors did not state how many grams
of broccoli seed were used to obtain the 500 grams of
extract. The chromatographic isolation of the sinapine
glucoraphanin salt is more straight-forward and provides
Table 2. NMR spectroscopic data (CD3OD, 1H 500 MHz,
13C 125 MHz) for compound 2.
NMR Spectroscopic data
Carbon # 1H Shift
1H Splitting,
coupling (Hz)
13C Shift
d, 15.9
d, 15.9
Figure 2. Structure of compound 2: sinapic acid methyl
Purification of a Sinapine-Glucoraphanin Salt from Broccoli Seeds
Copyright © 2010 SciRes. AJPS
a good separation even with very crude chromatographic
methods such as flash chromatography. The purified salt
is free from contamination by glucoiberin. The purified
sinapine-glucoraphanin salt can be passed through an
anion exchange column to remove the sinapine and eluted
with potassium sulfate if the potassium salt is needed.
Further development and refinement of this purification
technique promises to increase the yields and may pro-
vide an alternative method for the production of pure
glucoraphanin than the conventional chromatographic
isolation of the acid, or for the sodium or potassium salts.
Sinapine also has biological activity [13-15], and the two
compounds together may have interesting biological ac-
tivities as an enhanced antioxidant and for th e prevention
of chronic diseases.
4. Experimental
4.1. Chemicals
All chemicals were of analytical reagent grade and pur-
chased from national distribution venders. Solvents for
chromatography were purchased from EMD-Merck
(Gibbstown, New Jersey, USA). Water was purified us-
ing a ELGA Pure Lab Ultra system from Veolia Water
Solutions and Technologies (Woodbridge, Illinois,
4.2. Sample Preparation and Extraction
Broccoli seeds (Brassica oleracea L. var. botrytis cv.
‘Liberty’) were obtained from Sakata Seed America
(Morgan Hill, CA). One kg of seeds were ground to a
fine powder in a commercial coffee grinder and defatted
overnight in four Soxhlet extractors (250 g each) with
hexane for 24 hours. The hexane was then removed and
the samples were dried in a fumehood for 24 hours. The
defatted samples were then extracted in four Soxhlet ex-
tractors with MeOH for 72 hours. The pooled MeOH
extracts from the original 1 kg of ground seed samples
was evaporated to dryness in a rotary evaporator and
resuspended in approximately 10 0 mL 50:50 M e OH: H 2O.
4.3. Isolation and Purification
A Büchi (Newcastle, DE) Sepacore flash chromatogra-
phy system with dual C-605 pump modules, C-615 pump
manager, C-660 fraction collector, C-635 UV photometer,
with SepacoreRecord chromatography software was used.
A Büchi C-670 Cartridger system to load 40 × 150 mm
flash columns with approximately 90 grams of prepara-
tive C-18 reverse-phase bulk packing material (125Å,
55-105 µ, Waters Corp, Milford, MA). The columns
were installed in the flash chromatography system and
equilibrated with 20% MeOH and 0.5% HOAc in water
for five minutes at a flow rate of 30 mL per minute. After
samples (20 mL) were injected, the column was devel-
oped with a binary gradient to 50% MeOH over 30 min-
utes. The eluant was monitored at 237 nm and fractions
based on absorbance were collected in the fraction col-
lector by the software program. This was repeated 5
times to purify the entire extracted sample. Three major
broad UV-absorbing peaks (fractions A, B, and C) were
collected. The fractions were evaluated by analytical
HPLC. Fraction A contained a single major UV absorb-
ing peak with a retention time of 10 minutes (compound
1), and fraction C contained a single major UV absorbing
peak at 33 minutes (compound 2). Fraction B contained a
mixture of several peaks including those found in Frac-
tion A and C. Fractions A and C were evaporated to dry-
ness with nitrogen gas and resuspended in 30 mL of 1:1
mix of MeOH and water.
Fractions A and C were further purified using a Shi-
madzu (Columbia, MD) preparative HPLC system was
used with dual 8A pumps, SIL 10vp autoinjector, SPD
M10Avp photodiode array detector, SCL 10Avp system
controller all operating under the Shimadzu Class VP
operating system. Ten mL sample aliquots in 1:1 MeOH:
water were injected on a Phenomenex (Torrance, CA)
Luna C-18(2) semi-preparative reversed-phase column
(10 µ, 100Å, 250 50 mm). The column was pre-
equilibrated with a solvent system consisting of 10%
MeOH and 90% water (containing 1% HOAc) at a flow
rate of 50 mL per minute and the eluant was monitored at
237 nm. The column was developed to 100% MeOH
over 50 minutes. The major UV absorbing peak in each
fraction was collected, pooled, allowed to evaporate for
removal of the MeOH, then freeze-dried to remove the
remaining water to afford approximately 115 mg of pure
compound 1 and 133 mg of pure compound 2.
4.4. HPLC Analysis
General phenolic HPLC analysis was conducted on a
Shimadzu LC-20 HPLC system (LC-20AT quaternary
pump, DGU-20A5 degasser, SIL-20A HT autosampler,
and a SPD M20A photodiode array detector, running
under Shimadzu LCSolution version 1.22 chromatogra-
phy software, Columbia, MD, USA). The column used
was an Inertsil ODS- 3 reversed-phase C-18 column (5 µ,
250 4.6 mm, with a Metaguard column, from Varian).
The initial conditions were 20% MeOH and 80% 0.01 M
H3PO4 in water, at a flow rate of 1 mL per minute. The
eluant was monitored at 285 nm on the PDA. After injec-
tion (typically 15 µL), the column was held at the initial
conditions for 2 minutes, then developed to 100% MeOH
in a linear gradient over 50 minutes.
For the glucosinolate analysis, a modification of a
HPLC method developed by Betz and Fox was used [23]
which gives excellent resolution of glucosinolates with-
Purification of a Sinapine-Glucoraphanin Salt from Broccoli Seeds
Copyright © 2010 SciRes. AJPS
out peak tailing, due to good ion pairing from th e solvent.
The extract was run on a Shimadzu (Columbia, MD)
HPLC System (two LC 20AD pumps; SIL 20A autoin-
jector; DGU 20As degasser; SPD-20A UV-VIS detector;
and a CBM-20A communication BUS module) using the
Shimadzu EZStart Version 7.3 software. The column was
a C-18 Inertsil reversed-phase column (250 mm 4.6
mm; RP C-18, ODS-3, 5 µ; with a Metaguard guard
column; Varian, Torrance, CA). The glucosinolates were
detected by monitoring at 237 nm. The initial mobile
phase conditions were 12% MeOH/88% aqueous 0.005
M tetrabutylammonium bisulfate (TBS) at a flow rate of
1 mL/minute. After injection of 15 µL of sample, the
binary gradient was developed to 70% MeOH/30%
aqueous 0.005 M TBS for 30 minute, and then to 50%
MeOH/50% aque ous 0.005 M TBS over another 20 min-
4.5. HPLC-MS Analysis
To obtain the molecular weights of the compounds found
in the seed extracts, aliquots were injected on a ion-trap
LC-MS. Samples were run on a ThermoFinnigan LCQ
DECA XP Plus LC-MS system with a Surveyor HPLC
system (autoinjector, pump, degasser and PDA detector)
and a nitrogen generator all running under the Xcaliber
1.3 software system. The MS was run with the ESI probe
in either the positive or the negative mode. The column
was a 3 mm 150 mm Inertsil reversed-phase C-18,
ODS-3, 3 µ, column (Varian, Torrance, CA) with a Me-
taguard guard column. Solvent A was 0.25% HOAc in
H2O and Solvent B was 0.25% HOAc in MeOH. The
source inlet temperature was set at 250, the sheath gas
rate was set at 80 arbitrary units and th e sweep (auxiliary)
gas rate was set at 30 arbitrary units. The mass spec-
trometer was optimized for the detection of compounds
in the extracts by using the autotune feature of the soft-
ware while infusing a solution of the purified compound s
in MeOH at a flow rate of 25 µL/minute with the eluant
of the column (50:50 solvent A and B) at a flow rate of
100 µL/minute and tuning on the most prominent ion.
Under these conditions the spray vo ltage was 4.0 kV and
the capillary voltage was 40 V (positive mode) an d -47 V
(negative mode). An aliquot of the samples (typically 15
µL) was then run on the LC. The initial HPLC conditions
were 12% MeOH/H2O (containing 0.25% HOAc) at a
flow rate of 0.3 mL per minute, then the column was
developed to 34% MeOH/H2O (containing 0.25% HOAc)
over 20 minutes and then to 50% MeOH/H2O (contain-
ing 0.25% HOAc) over an additional 20 minutes. The
eluant was also monitored at 237 and 285 nm on the
4.6. NMR Analysis
1H, COSY, DEPT, and 13C NMR spectra were obtained
on a Bruker (Billerica, MA, USA) Avance 500 NMR
spectrometer equipped with a 5 mm inverse broadband
Z-gradient probe (13C NMR, 125 MHz; 1H, 500 MHz).
NMR spectra were recorded in CD3OD and referenced to
solvent resonances (13C, 49.0 ppm, 1H, 3.30 ppm). The
data was analyzed using the Advanced Chemistry De-
velopment, Inc., SpecManager 1D Processor and the
HNMR and CNMR Predictor software suite version
12.01 (Toro nt o , ONT.)
5. Acknowledgements
The authors would like to thank Ray Holloway and San-
dra Duval for their technical assistance, Dr. Sherald H.
Gordon for providing the IR spectra, and Dr. Brian
Moser for melting point determination.
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