Acta Chromatographica 20(2008)3, 489–496

DOI: 10.1556/AChrom.20.2008.3.14

Isolation of Glucosinolates from Broccoli Seeds

by Use of High-Speed Countercurrent
Chromatography

Q. DU*, L. ZHANG, H. CUI, K. WANG, L. XIU AND L. SHENG

Institute of Food and Biological Engineering, Zhejiang Gongshang University,

Hangzhou, 310035, China
E-mail: qizhendu@163.com

Summary. Cleavage of glucosinolates with myrosinase yields thioglycosidic compounds

which have cancer chemoprevention activity. In this paper, glucosinolates in an extract
(2.0 g) of broccoli seeds (Brassica oleracea var. italica) were separated by high-speed coun-
tercurrent chromatography (HSCCC) with the solvent system n-butanol–acetonitrile–
10% ammonium sulfate solution 1:0.5:2.2 (v/v) to yield five glucosinolate compounds af-
ter desalting and decolorizing by MCI column chromatography. The five compounds,
7-methylsulfinylheptyl glucosinolate (22.4 mg), 4-pentenyl glucosinolate (33.6 mg),
3-butenyl glucosinolate (24.0 mg), 4-methylsulfinylbutyl glucosinolate (161.4 mg), and
3-methylsulfinylpropyl glucosinolate (29.6 mg), were identified by ESI-MS, 1H NMR and
13C NMR. The purity of the products was >98%, and 7-methylsulfinylheptyl glucosi-

nolate was obtained from broccoli seeds for the first time.

Key Words: broccoli seeds, glucosinolates, isolation, high-speed countercurrent chroma-

tography

Introduction
Accurate analysis of the levels of glucosinolates in different vegetables is
important because of the growing awareness of scientists and consumers of
glucosinolates, which play a role in cancer prevention. Lack of pure glu-
cosinolates currently limits quantitative analysis worldwide. Broccoli is a
typical Brassica plant rich in glucosinolates [1–4] and is an excellent material
for preparing several glucosinolate monomers. Flash chromatography and
preparative HPLC have been used to separate the glucosinolates in an ex-
tract of broccoli seeds [5, 6]. This yielded mainly pure 4-methylsulfinylbutyl

0231–2522 © 2008 Akadémiai Kiadó, Budapest

490 Q. Du et al.

glucosinolate (glucoraphanin) and was unsuitable for simultaneous isola-

tion of other glucosinolate monomers.
High-speed countercurrent chromatography (HSCCC) is an all-liquid
chromatographic system, working without solid support, in which separa-
tion is based on partitioning of the analytes between two immiscible liquid
phases [7]. Irreversible adsorption and artifact formation is thus minimized.
Application of HSCCC in natural product chemistry is steadily increasing,
because of this advantage and the excellent recoveries achieved [8]. This
paper reports a rapid method for isolation and purification of the five glu-
cosinolate monomers 7-methylsulfinylheptyl glucosinolate, 4-methylsul-
finylbutyl glucosinolate, 3-methylsulfinylpropyl glucosinolate, 4-pentenyl
glucosinolate, and 3-butenyl glucosinolate (Fig. 1) from broccoli seeds by
use of HSCCC.

Fig. 1. Structures of the glucosinolate compounds isolated from broccoli seeds:

1, 3-butenyl glucosinolate; 2, 4-pentenyl glucosinolate; 3, 7-methylsulfinylheptyl
glucosinolate; 4, 4-methylsulfinylbutyl glucosinolate; 5, 3-methylsulfinylpropyl
glucosinolate
HSCCC Isolation of Glucosinolates from Broccoli Seeds 491

Experimental

Materials

Broccoli seeds (Brassica oleracea var. italica; 1 kg) were provided by Hang-
zhou Seeds Company (Hangzhou, China). All solvents for extraction and
separation (analytical grade) were purchased from Hangzhou Huadong
Chemicals, China. Tetramethyl ammonium bromide (TMAB) was obtained
from Sigma Chemical Company, Shanghai, China. MCI gel (Mitsubishi
Chemical, Japan) was purchased from Greenherbs Science and Technology,
Beijing, China.

Extraction and Pre-Separation

Broccoli seeds (1 kg) were marinated in 3 L methanol for 2 h at 70°C to inac-

tivate endogenous myrosinase and to prepare the sample for extraction of
the glucosinolates. After filtration of the extract solution, the broccoli resi-
dues were pulverized and extracted again with 5 L methanol for 2 h at
70°C. The two extracts were combined and evaporated to dryness, and the
residue was defatted with 1 L of diethyl ether. The residue was then lyophi-
lized to yield 137 g crude extract which was subjected to HSCCC isolation
of the glucosinolates.

Apparatus

The high-speed countercurrent chromatograph used in this study was con-

structed at the Institute of Food and Biological Engineering, Zhejiang Gong-
shang University, Hangzhou, China. The apparatus was equipped with a
1200-mL column with six layers of coils made from 5.0 mm i.d. polytetra-
fluoroethylene (PTFE) tubing. A K-1800 Wellchrom preparative HPLC
pump (Knauer, Germany), a 100-mL sample loop made of 3 mm i.d. PTFE
tubing, a 210 UV-detector (Elite, Dalian, China), and a B-684 collector
(Büchi, Switzerland) with 15-mL tube racks were used to establish the isola-
tion system.

High-Speed Countercurrent Chromatographic Isolation Procedure

HSCCC was performed with a two-phase solvent system comprising n-

butanol–acetonitrile–10% ammonium sulfate solution 1:0.5:2.2 (v/v), which
was selected after experimental partition of the crude extract in a series of
492 Q. Du et al.

solvent systems prepared from n-butanol, acetonitrile, 10% ammonium sul-

fate solution in different volume ratios. After thorough equilibration of the
solvents in a separatory funnel, the two resulting phases were separated
shortly before use. The multilayer coil column was completely filled with
the aqueous lower phase as the stationary phase. The apparatus was then
rotated at 700 rpm. The sample solution was prepared by dissolving 2.0 g
crude extract in 50 mL organic mobile phase. Injection into the HSCCC sys-
tem was achieved by use of the PTFE sample loop, followed by of the
pumping mobile phase at a flow rate of 3.0 mL min−1. The HSCCC elution
mode of mobile phase was head to tail. The effluent was monitored by UV
detection at 233 nm and collected by use of the fraction collector, 15 mL for
each fraction. When the separation was complete the solvents in the column
were pushed out and the retention of the stationary phase was measured to
be 55%.

TLC Analysis

To estimate their purity the compounds from HSCCC separation were ana-
lyzed by TLC on Merck aluminum foil plates coated with silica gel GF254;
the mobile phase was chloroform–methanol–water 15:15:1. Spots were de-
tected by treatment with a 10% solution of sulfuric acid in ethanol and heat-
ing at 110°C.

Desalting and Decolorizing by MCI Column Chromatography

The compounds from HSCCC separation were dissolved in water and the
solutions were subjected to MCI column chromatography, and eluted with
pure water and 50% methanol. The solutions in 50% methanol were evapo-
rated under vacuum to yield pure glucosinolate compounds.

HPLC Analysis

HPLC was performed with Waters (Milford, MA, USA) equipment – a 2695
pump, a 717 autosampler, and a 996 diode-array detector. Compounds
were separated on a 300 mm × 3.9 mm i.d., 10-μm particle, C18 Bondapak
column (Waters) fitted with a C18 guard column. The mobile phase was
0.005 M TMAB dissolved in 2% (v/v) methanol–water. The flow rate was
1.5 mL min−1 and the separations were monitored at 233 nm. Waters Mil-
lennium HPLC 2010 software was used to process chromatographic data.
HSCCC Isolation of Glucosinolates from Broccoli Seeds 493

ESI-MS and NMR

All ESI-MS experiments were performed with a Bruker (Bremen, Germany)

Esquire LC–MS ion-trap multiple mass spectrometer in negative-ionization
mode, with analysis of ions up to m/z 2200. 1H and 13C NMR spectra were
recorded in CD3OD with a Bruker (Karlsruhe, Germany) Avance 500, at
500 MHz for 1H NMR and 125 MHz for 13C NMR. ESI-MS analysis of com-
pounds 1, 2, 3, 4, and 5 from HSCCC separation gave pseudomolecular ions
m/z (neg.) a: 371, 385, 477, 435, and 421, respectively. 1H and 13C NMR data
are listed in Table I.

Table I. Chemical shifts (δ, ppm) from 13C and 1H NMR of glucosinolates 1–5

Compound Compound Compound

Carbon Compound 1 Compound 2
3 4 5
no. 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H

3.74, 3.63, 3.69 3.63 3.66

1 62.5 62.1 62.8 62.6 61.2
3.76 4.12 3.90 3.87 3.69
2 79.5 3.56 82.5 3.16 79.5 3.62 82.1 3.56 81.3 3.38
3 71.1 3.54 71.0 3.09 71.3 3.50 71.2 3.47 70.1 3.20
4 82.1 3.57 79.4 3.18 74.4 3.61 79.3 3.56 78.3 3.34
5 74.1 3.54 74.1 3.16 73.1 3.52 74.1 3.47 73.1 3.18
6 83.6 5.04 83.3 5.01 82.4 4.83 83.4 5.09 82.8 5.10
7 161.6 156.5 163.6 162.5 161.6
8 33.0 2.65 35.8 3.04 35.8 2.84 32.9 2.80 31.1 2.83
9 32.9 2.41 32.3 2.32 23.2 2.67 22.8 1.89 20.7 2.49
10 138.2 5.85 32.4 2.65 32.6 2.28 26.9 2.21 52.9 3.00
5.05,
11 116.0 138.8 5.88 27.3 1.79 54.1 2.93 38.1 2.72
4.93
5.12,
12 116.5 25.7 1.85 38.4 2.68
5.08
13 23.0 2.81
14 54.4 2.86
15 38.1 2.68

Results and Discussion

With the solvent system n-butanol–acetonitrile–10% ammonium sulfate so-
lution 1:0.5:2.2 (v/v) separation of 2 g crude extract was performed at a flow
rate of 3 mL min−1. The chromatogram (Fig. 2) contained seven peaks (A–G).
The fractions corresponding to peaks A and B furnished more than two
spots in TLC analysis whereas the fractions corresponding to peaks C–G
gave one spot. The fractions corresponding to peaks C (886–1050 mL),
D (1231–1575 mL), E (1726–1980 mL), F (2101–2460 mL), and G (2671–
494 Q. Du et al.

3210 mL) were separately combined and evaporated to dryness at 70°C un-
der vacuum. The residues were extracted with methanol (2 × 5 mL) The
methanol solutions were combined and evaporated to yield 46.1 mg com-
ponent I, 28.3 mg component II, 26.7 mg component III, 246.1 mg compo-
nent IV, and 56.1 mg component V. Components I–V were desalted and de-
colorized by MCI column chromatography to yield 33.6, 24.0, 22.4, 161.4,
and 29.6 mg of compounds 1–5, respectively. The purities of compounds 1–
5 were >98% (by HPLC; Fig. 3). Results from ESI-MS, 1H NMR (500 Hz),
and 13C NMR (125 Hz) analysis of compounds 1, 2, 4, and 5 were consistent
with 4-pentenyl glucosinolate (glucobrassicanapin), 3-butenyl glucosinolate
(gluconapin), 4-methylsulfinylbutyl glucosinolate (glucoraphanin), and 3-
methylsulfinylpropyl glucosinolate (glucoiberin) , respectively [9–11]. By
comparison of the 1H NMR and 13C NMR spectra of compound 3 with those
of 4-methylsulfinylbutyl glucosinolate (4) (Table I), compound 3 was identi-
fied as 7-methylsulfinylheptyl glucosinolate (glucoibarin), which was ob-
tained from broccoli seeds for the first time. ESI-MS of compound 3 in nega-
tive-ion mode gave a pseudomolecular ion at m/z 477 [M−], confirming that
3 was glucoibarin.

Fig. 2. HSCCC chromatogram obtained from 2 g crude extract of broccoli seeds. Solvent
system: n-butanol–acetonitrile–10% ammonium sulfate solution, 1:0.5:2.2 (v/v); mobile
phase, upper phase; flow rate, 3 mL min−1; the sample was dissolved in 50 mL upper
phase; retention of the stationary phase was 55%. Peak elution was monitored at 233 nm
by use of a variable-wavelength detector
HSCCC Isolation of Glucosinolates from Broccoli Seeds 495

Fig. 3. HPLC analysis (233 nm) of the crude extract of the broccoli seeds and the
products from HSCCC: (a) crude extract; (b) 3-methylsulfinylpropyl glucosinolate;
(c) 4-methylsulfinylbutyl glucosinolate; (d) 3-butenyl glucosinolate; (e) 4-pentenyl
glucosinolate; (f) 7-methylsulfinylheptyl glucosinolate

An early method of isolation of glucosinolates from broccoli seeds was

flash chromatography which yielded impure components except for 4-
methylsulfinylbutyl glucosinolate, the most abundant glucosinolate in broc-
coli seeds [5]. Recently, solid phase extraction (SPE) and preparative HPLC
have been used for the isolation of glucoraphanin from broccoli seeds. This
method was tedious and inefficient, however, because one preparative
HPLC injection yielded only 17.6 mg 4-methylsulfinylbutyl glucosinolate
from 98 mg semi-purified crude sample from SPE [6]. Our HSCCC method
496 Q. Du et al.

enables loading of 2 g crude sample and yields five glucosinolates. This has
the advantages of simultaneous recovery of several glucosinolate mono-
mers and high sample loading.

Conclusion
HSCCC enabled excellent separation of the glucosinolates of broccoli seeds,
yielding five pure glucosinolate monomers – 7-methylsulfinylheptyl glu-
cosinolate, 4-methylsulfinylbutyl glucosinolate, 3-methylsulfinylpropyl glu-
cosinolate, 4-pentenyl glucosinolate, and 3-butenyl glucosinolate after de-
salting and decolorizing by MCI column chromatography. This method can
be used for rapid isolation of glucosinolate monomers from broccoli seeds,
and is possibly suitable for preparation of the glucosinolates from other
plant materials.

Acknowledgments
The authors thank the Department of Zhejiang Science and Technology for
financial support (2005C12037 and 2004C12005).

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