Thermal degradation of sulforaphane in aqueous solution

Article abstract of DOI:10.1021/jf990082e

Sulforaphane, a cancer chemopreventive agent identified from broccoli, was degraded in an aqueous solution at 50 and 100 °C. The reaction mixtures were extracted with methylene chloride and analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Dimethyl disulfide, S- methyl methylthiosulfinate, S-methyl methylthiosulfonate, methyl (methylthio)methyl disulfide, 1,2,4-trithiolane, 4-isothiocyanato-1- (methylthio)-1-butene, and 3-butenyl isothiocyanate were identified as volatile decomposition products. After methylene chloride extraction, the aqueous layer was dried and silica gel column chromatography was used to separate and purify the nonvolatile decomposition products. The major thermal degradation compound was determined by ¹H NMR, ¹³C NMR, and FAB-MS as N,N'-di(4-methylsulfinyl)butyl thiourea. A possible mechanism for the formation of these products is proposed.

Full text of DOI:10.1021/jf990082e

J. Agric. Food Chem. 1999, 47, 3121−3123
3121
Th er m a l Degr a d a tion of Su lfor a p h a n e in Aqu eou s Solu tion
Yi J in, Mingfu Wang, Robert T. Rosen, and Chi-Tang Ho*
Department of Food Science and Center for Advanced Food Technology, Cook College, Rutgers University,
65 Dudley Road, New Brunswick, New J ersey 08901-8520
Sulforaphane, a cancer chemopreventive agent identified from broccoli, was degraded in an aqueous
solution at 50 and 100 °C. The reaction mixtures were extracted with methylene chloride and
analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS).
Dimethyl disulfide, S-methyl methylthiosulfinate, S-methyl methylthiosulfonate, methyl (meth-
ylthio)methyl disulfide, 1,2,4-trithiolane, 4-isothiocyanato-1-(methylthio)-1-butene, and 3-butenyl
isothiocyanate were identified as volatile decomposition products. After methylene chloride
extraction, the aqueous layer was dried and silica gel column chromatography was used to separate
and purify the nonvolatile decomposition products. The major thermal degradation compound was
determined by 1H NMR, 13C NMR, and FAB-MS as N,N′-di(4-methylsulfinyl)butyl thiourea. A
possible mechanism for the formation of these products is proposed.′′′
Keyw or d s: Sulforaphane; isothiocyante; thermal decomposition; N,N′-di(methylsulfinyl)butyl
thiourea
INTRODUCTION
at different temperatures, to identify the thermal
degradation products from sulforaphane, and to discuss
the possible formation mechanism for these degradation
products.
Epidemiological studies provide consistent evidence
that individuals who consume high quantities of cru-
ciferous vegetables experience a lower risk of developing
several types of cancer (Zhang et al., 1992). Although
the exact mechanism for this is not clear, the phy-
tochemicals or secondary metabolites found in these
vegetables cause much interest and are suggested to be
responsible for this activity. Sulforaphane [(-)-1-isothio-
cyanato-(4R)-(methylsulfinyl)butane], one phytochemi-
cal produced by enzyme hydrolysis of glucoraphanin (a
glucosinolate in broccoli, cabbage, etc.), has received the
most attention for its cancer chemopreventive activity
(Zhang et al., 1994). Sulforaphane is a potent inducer
of phase II enzymes, including quinone reductase and
glutathione S-transferase, which protect against car-
cinogens and other toxic electrophiles. Because sul-
foraphane is monofunctional, it does not influence the
phase I enzymes (Zhang et al., 1994).
Due to the importance of this compound, various
methods, including high-performance liquid chromatog-
raphy (HPLC) (Zhang et al., 1992), gas chromatography
(GC), GC/mass spectrometry (GC/MS) (Spencer and
Daxenbichler, 1980; Chiang et al., 1998), and paired-
ion chromatography (Fahey et al., 1997) have been used
to analyze sulforaphane in Brassica vegetables. How-
ever, due to its instability, sulforaphane is difficult to
analyze quantitatively. Recently, sulforaphane was
found to decompose to other compounds due to the high-
temperature conditions in the injection ports of GC or
GC/MS equipment (Chiang et al., 1998). Although many
studies about this compound have been published, no
information is available on the stability and chemical
reaction of sulforaphane under normal cooking condi-
tions. The purpose of this study is to investigate the
thermal stability of sulforaphane in aqueous solution
EXPERIMENTAL PROCEDURES
Ma ter ia l. TLC plates (250 µm thickness, 2-25 µm particle
size), tridecane, and silica gel (130-270 mesh) were purchased
from Aldrich Chemical Co. (Milwaukee, WI) and used for
chromatography. All solvents used for chromatography were
of analytical grade quality and purchased from Fisher Scien-
tific (Springfield, NJ ). Sulforaphane was purchased from LKT
Laboratories, Inc. (St. Paul, MN). Its purity was checked by
nuclear magnetic resonance (NMR) and thin-layer chroma-
tography (TLC) methods, and it was stored below 0 °C.
NMR a n d F a st At om Bom ba r d m en t Ma ss Sp ect r o-
scop y (F AB-MS). 1H NMR and 13C NMR spectra were
obtained on a Varian Gemini-200 instrument (Varian Inc., Palo
Alto, CA) at 200 and 50 MHz. CH3OH-d4 was used as a solvent,
and chemical shifts were expressed in parts per million (δ).
13C NMR multiplicity was determined by attached proton test
(APT) experiment. FAB mass spectra were recorded on a
Finnigan MAT-90 instrument (Finnigan Inc., San J ose, CA).
Th er m a l Decom p osition of Su lfor a p h a n e. Preparation
of the Model System. A sample of 30-60 mg of sulforaphane
was added to 5-6 mL of distilled water. The mixture was
heated in a 50 mL flask fitted with a condenser at different
temperatures for 1 h in an oil bath. The reaction solution was
then cooled to room temperature and extracted with 2 × 5 mL
of methylene chloride. The methylene chloride phase was then
dried using anhydrous sodium sulfate and concentrated to 0.2
mL by a stream of nitrogen gas after spiking with 4 µL of 1000
ppm tridecane as the internal standard. The concentrated
sample was subjected to GC and GC/MS analysis directly.
Purification of the Major Degradation Products in Aqueous
Solution. The aqueous phase was evaporated to dryness by a
rotary evaporator with the water bath at 45 °C. The residue
was subjected to silica gel column (30 g) chromatography. The
solvent system used was chloroform/methanol (6:1). The frac-
tions containing the major degradation products were collected,
and the purity of the isolated compound was monitored by TLC
(the solvent used was chloroform/methanol 4:1). The solvents
were removed under reduced pressure by a rotary evaporator
at a temperature of 45 °C. Approximately 25 mg of colorless
* Corresponding author [fax (732) 932-8004; e-mail ho@aesop.
rutgers.edu].
10.1021/jf990082e CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/28/1999

3122 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Jin et al.
Ta ble 1. Ma ss Sp ectr a l Da ta of Decom p osition P r od u cts of Su lfor a p h a n e
compound
RIa
MS spectral data m/z (relative intensity)
identification
dimethyl disulfide
S-methyl methylthiosulfinate
S-methyl methylthiosulfonate
1355 96 [(M + 2)+, 10], 94 (M+, 100), 79 (64), 64 (16), 61 (24), 48 (24), 47 (42), 46 (54), 45 (78)
3272 112 [(M + 2)+, 1], 110 (M+, 16), 95 (11), 64 (64), 63 (26), 47 (64), 46 (19), 45 (100)
3535 128 [(M + 2)+, 2], 126 (M+, 40), 111 (4), 81 (66), 79 (52), 64 (40), 63 (64), 48 (26), 47 (100),
45 (98), 46 (46)
ref 1a
ref 1
ref 1
methyl (methylthio)methyl disulfide 4230 142 [(M + 2)+,1], 140 (M+, 8), 93 (4), 79 (8), 61 (100), 45 (50)
1,2,4-trithiolane
ref 1
ref 1
ref 1
4438 126 [(M + 2)+, 12], 124 (M+, 60), 78 (78), 60 (24), 59 (18), 46 (40), 45 (100), 44 (18)
4-isothiocyanato-1-(methylthio)-1- 6452 161 [(M + 2)+, 1], 159 (M+, 12), 87 (80), 85 (20), 72 (74), 61 (20), 59 (16), 53 (20), 47 (30),
butene
3-butenyl isothiocyanate
46 (20), 45 (100)
3205 113 (M+, 47), 72 (100), 55 (23), 39 (48)
a
b
RI, retention index on DB-1 column. Reference 1, Wiley 138.
oil was obtained. The pure sample was subjected to NMR and
MS analysis directly after the purifying procedure.
GC a n d GC/MS An a lysis. A Varian Model 3400 gas
chromatograph equipped with a flame ionization detector and
a fused silica capillary column [DB-1, 60 m × 0.32 mm (i.d.),
1.0 µM film thickness, J &W Scientific] was used. The oven
temperature was programmed from 40 to 220 °C at an increase
rate of 3 °C/min. The temperatures of the detector and injector
were maintained at 280 and 210 °C, respectively. The flow rate
of the helium carrier gas was 1 mL/min, and the split ratio
was 10:1. GC/MS analysis was performed on an HP Model
5980 coupled with an HP 5971 mass selective detector. The
capillary column and temperature program were the same as
for the GC analysis. Mass spectra were obtained by electron
ionization at 70 eV, and mass scan was from 33 to 300.
Compound quantification was based on the GC/FID data. The
concentration of the compound was calculated by using the
following equation: A × 0.004/IS × M, where A is the area
count of the compound, IS is the area count of the internal
standard, and M is the mass of sulforaphane (mg). The
identification of volatile compounds was based on the mass
spectra obtained from the GC/MS.
F igu r e 1. Possible pathways for the formation of volatile
compounds generated from thermal degradation of sul-
foraphane [1, dimethyl disulfide; 2, S-methyl methylthiosul-
finate; 3, S-methyl methylthiosulfonate; 4, methyl (methylthio)-
methyl disulfide; 6, 4-isothiocyanato-1-(methylthio)-1-butene].
Ta ble 2. Qu a n tita tion of Th er m a l Degr a d a tion P r od u cts
fr om Su lfor a p h a n e a t Tw o Differ en t Tem p er a tu r es
RESULTS AND DISCUSSION
50 °C
100 °C
(ppm)
compound
dimethyl disulfide
S-methyl methylthiosulfinate
S-methyl methylthiosulfonate
methyl (methylthio)methyl disulfide
1,2,4-trithiolane
(ppm)a
Vola tile Com p ou n d s Gen er a ted fr om th e Th er -
m a l Degr a d a tion of Su lfor a p h a n e. After thermal
degradation of sulforaphane in aqueous solution, the
resulting mixture was extracted with methylene chlo-
ride and analyzed by GC and GC/MS. Table 1 lists
volatile compounds identified from the methylene chlo-
ride extracts. Their structures were tentatively deter-
mined by comparing their mass spectral data with
reference (Table 1). Among them, 3-butenyl isothiocy-
anate has been reported to be a thermal degradation
product of sulforaphane caused by the high temperature
of the injection ports of GC and GC/MS (Chiang et al.,
1998). Other compounds were reported for the first time
as degradation products of sulforaphane.
2.1
17.2
26.6
4.9
309.0
184.8
174.0
38.7
6.5
23.0
4-isothiocyanato-1-(methylthio)-1-butene
13.2
134.9
a
Micrograms per gram of sulforaphane
concentrations of volatile decomposition products at 100
°C were much higher than those at 50 °C, suggesting
that higher temperature accelerates the rate of degra-
dation. The differences of the concentrations at the two
temperatures are shown in Table 2.
The mechanisms for the formation of these volatile
compounds from sulforaphane are proposed as shown
in Figure 1. 4-Methylthio-4-hydroxybutyl isothiocyanate
(compound B), which could be formed by the transaction
of oxygen from sulfur to carbon via an epoxide (com-
pound A), is an important intermediate for generating
the methylthio radical and is the source of the meth-
ylthio group in those decomposition compounds. 4-Isothio-
cyanato-1-(methylthio)-1-butene can be produced by
dehydration of 4-methylthio-4-hydroxybutyl isothiocy-
anate. The methylsulfinyl radical can be produced
directly from sulforaphane. Different combinations of
methylsulfinyl radical and methylthio radical will lead
to different adducts.
Non vola tile Com p ou n d fr om Th er m a l Degr a d a -
tion . After thermal reaction, the aqueous phase was
subjected to silica gel column chromatography. Only one
main compound was obtained as a colorless oil. It
exhibited prominent quasi-molecular ion peaks in the
positive FAB mass spectrum at m/z 313 [M + 1]+ and
335 [M + Na]+ about 2 times the molecular weight of
sulforaphane. In the 1H NMR (CD3OD), it showed
signals at δ 1.78 (8H, m, -CH2CH2-), 2.65 (6H, s, CH3),
2.87 (4H, m-CH2SO), and 3.53 (4H, m, CH2 N), whereas
in the 13C NMR (CD3OD), it showed signals at δ 24.0
(CH2), 32.3 (CH2), 41.2 (-CH2N-), 47.2 (CH3S-), and
57.4 (-SCH2-). These NMR data were similar to those
of sulforaphane (Kore et al., 1993; Zhang et al., 1992),
suggesting this compound is a dimer of sulforaphane.
Sulforaphane is an isothiocyanate compound. Recently
one of these compounds, allyl isothiocyanate, was
reported to convert to diallylthiourea in aqueous solu-
tion (Chen and Ho, 1998). This compound was, there-
Tem p er a tu r e In flu en ce on th e Gen er a tion of
Vola tile Com p ou n d s fr om Su lfor a p h a n e. Two dif-
ferent temperatures have been used in the experiment.
Temperature was found to have a great effect on the
concentrations of the volatile compounds generated. The

Sulforaphane Degradation
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3123
compound needs to be addressed, and the generation of
the thiourea compound from foods containing isothio-
cyanate compounds needs to be studied further.
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F igu r e 2. Possible pathways for the formation of N,N′-di-
(methylsulfinyl)butyl thiourea from sulforaphane.
fore, tentatively determined as (methylsulfinyl)butyl
thiourea. After reexamination of the NMR and FAB-
MS, it is uncertain whether the carbon signal for the
thiourea group was observed in the 13C NMR spectrum.
To clarify this, diallylthiourea was synthesized (Chen
and Ho, 1998). We did not observe the carbon signal
for thiourea group in its 13C NMR spectrum either. It
is possible that the low sensitivity of the NMR instru-
ment does not allow us to observe this weak signal. The
major degradation product was elucidated as N,N′-di-
(methylsulfinyl)butyl thiourea (Figure 2).
The proposed mechanism for the formation of this
compound is shown in Figure 2. A similar mechanism
has been proposed by Kawakishi and Namiki (1969) and
Chen and Ho (1998) for the formation of diallylthiourea
from allyl isothiocyanate. Sulforaphane is first hydro-
lyzed to an amine, and the resulting amine reacts with
sulforaphane to generate this thiourea compound.
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Con clu sion . Our study demonstrates that sul-
foraphane, a cancer chemopreventive agent, is an
unstable compound. It will degrade under cooking
conditions, and the major degradation product is a
thiourea compound. The bioactivity of this thiourea
Received for review J anuary 29, 1999. Revised manuscript
received J une 25, 1999. Accepted J uly 7, 1999.
J F990082E