saha et al (2012) sfn and erucin from fresh frozen broccoli (mnfr) (published)

1906 Mol. Nutr. Food Res. 2012, 56, 1906–1916DOI 10.1002/mnfr.201200225

RESEARCH ARTICLE

Isothiocyanate concentrations and interconversion

of sulforaphane to erucin in human subjects after

consumption of commercial frozen broccoli compared

to fresh broccoli

Shikha Saha1, Wendy Hollands1, Birgit Teucher1, Paul W. Needs1, Arjan Narbad1,Catharine A. Ortori2, David A. Barrett2, John T. Rossiter3, Richard F. Mithen1 and Paul A. Kroon1

1 Institute of Food Research, Norwich Research Park, Norwich, UK2 Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, UK3 Department of Life Sciences, Division of Cell & Molecular Biology, Sir Alexander Fleming Building, Imperial

College, London, UK

Scope: Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin)

is widely considered responsible for the protective effects of broccoli consumption. Broccoli

is typically purchased fresh or frozen and cooked before consumption. We compared the

bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen

broccoli, and investigated the bioconversion of sulforaphane to erucin.

Methods and results: Eighteen healthy volunteers consumed broccoli soups produced from

fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates

quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the

soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to

destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane

appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin

N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human

gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin.

Conclusion: The short period of blanching used to produce commercial frozen broccoli destroys

myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted

to erucin and excreted in urine, and it was shown that human colonic flora were capable of this

conversion.

Keywords:

Broccoli / Erucin / Sulforaphane

Received: April 20, 2012

Revised: August 22, 2012

Accepted: August 28, 2012

1 Introduction

Epidemiological studies indicate that the consumption of

broccoli is associated with the reduction in the risk of de-

generative disease such as cancer [1]. 4-Methylsulphinylbutyl

glucosinolate (glucoraphanin) accumulates in the florets of

broccoli. Following consumption, it is hydrolysed into the

corresponding isothiocyanate (sulforaphane) either by the

Correspondence: Dr. Paul Kroon, Institute of Food Research,

Colney Lane, Norwich NR4 7UA, UK

E-mail: [email protected]

Fax: +44-1603-507723

Abbreviations: ESP, epithiospecifier like proteins; ITC, isothio-

cyanate

plant thioglucosidase myrosinase or by bacterial thioglu-

cosidases in the colon, if the myrosinase has been dena-

tured by cooking [2]. A nitrile derivative of the glucosino-

late may also be produced due to the interaction of plant

“epithiospecifier-like” proteins (ESP). Mild cooking can de-

nature these proteins but leave myrosinase enzymes intact,

maximising the amount of glucosinolate conversion to isoth-

iocyanate. Following absorption, sulforaphane is conjugated

with glutathione and metabolised via the mercapturic acid

pathway, resulting in predominantly N-acetyl-cysteine conju-

gates that are excreted in the urine [3]. Several studies have

quantified the pharmacokinetics of sulforaphane following

the consumption of broccoli with either active or inactive my-

rosinase. Most of these studies have analysed sulforaphane

and its thiol metabolites in plasma and urine through a

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

Mol. Nutr. Food Res. 2012, 56, 1906–1916 1907

cyclocondensation reaction [4, 5]. While this method is ef-

ficient at quantifying total thiols, it is not able to discriminate

between free isothiocyanates and individual thiol metabo-

lites, or between different isothiocyanates. LC-MS/MS meth-

ods have been developed that are able to distinguish be-

tween different metabolites, and have demonstrated that un-

conjugated sulforaphane is found in the plasma [6]. Analyses

through both approaches have shown that between 30 and

80% of sulforaphane is absorbed into the blood stream or

excreted in the urine compared to the level of the parent glu-

cosinolate if broccoli is consumed with active myrosinase,

but this drops to 10% or less if broccoli is consumed with

inactive myrosinase, with a later Tmax associated with colonic

absorption as opposed to absorption through the stomach or

small intestine [7].

An important question is the fate of the isothiocyanate

or glucosinolate that is not absorbed. One possibility is that

it is not absorbed but excreted in the faeces. Alternatively,

there may be alternative routes to metabolism of sulforaphane

post absorption, and conjugation with proteins has been pro-

posed [4]. A third possibility may be represented by the bio-

conversion of sulforaphane to other isothiocyanates. Recent

studies carried out in humans and rats have reported the in

vivo interconversion of sulforaphane to erucin [8]. The erucin

is the enzymatic hydrolysis product of glucoerucin, mainly

found in rocket salad species, and structurally represents

the reduced analogue of sulforaphane [8]. It was suggested

that the conversion of glucoraphanin/sulforaphane to the re-

duced glucosinolate/isothiocyanate occurred in the liver, and

that there was enterohepatic circulation of glucosinolates so

that hydrolysis occurred via gut microbes [9]. Another study

has confirmed the in vivo interconversion of sulforaphane to

erucin suggesting an inter-subject variability [10].

In the current study, we quantified sulforaphane

metabolism following consumption of soups containing

mildly cooked broccoli (i.e. with active myrosinase, but in-

active ESP) and frozen broccoli with inactivated myrosinase,

and analysed a subset of samples for the presence of erucin

and its thiol conjugates. In addition, we investigated whether

this conversion could be due to gut microbial activity.

2 Materials and methods

2.1 Materials

Sinigrin was obtained from Sigma Chemicals (UK). Sul-

foraphane was obtained from LKT Laboratories, Inc. (Min-

nesota, USA). Methyl-, ethyl- and butyl-isothiocyanates,

cysteine, cysteine-glycine, glutathione and N-acetyl-cysteine

were purchased from Aldrich (UK). DEAE Sephadex A25 and

SP Sephadex C25 were obtained from Amersham Biosciences

(Sweden). The synthesis of cysteine, cysteine-glycine, glu-

tathione and N-acetyl-cysteine conjugates of sulforaphane

was done by an optimized protocol published elsewhere [3].

The synthetic products were purified by preparative HPLC

using a C18 column and the structures of the synthesized ma-

terials were confirmed by 1H NMR (JEOL, EX 270) and LC–

MS–MS (Quattro Ultima tandem mass, Waters Micromass,

Manchester, UK). Sulforaphane-nitrile was synthesized by

the published protocol [11]. All solvents and other chemi-

cals used were of HPLC grade and purity was assessed to be

95% or greater in all compounds. Sulfatase (Type H-1 from

Helix pomatia) was obtained from Sigma Chemicals and pu-

rified before use. Sulfatase (300 mg) was dissolved in ice-cold

water (12 mL), mixed with ice-cold ethanol (12 mL) and

stirred. Following centrifugation (3000 × g, 6 min) the su-

pernatant was mixed with ice-cold ethanol (1.5 × vol), stirred

and centrifuged to obtain a pellet that was dissolved in water

(8 mL). Samples (2 mL) were passed sequentially through

columns of Sephadex A25 and Sephadex C25 to obtain the

purified sulfatase that was stored at −20�C.

2.2 Subjects and study design

Eighteen apparently healthy volunteers aged 20–65 years were

recruited by study scientists to participate in this study, which

was conducted at the Human Nutrition Unit at the Institute

of Food Research. All study participants were assessed for

eligibility on the basis of a health questionnaire and the re-

sults of clinical laboratory tests. All volunteers were screened

for full blood count, fasting glucose, liver function and urea

and electrolytes. The following exclusions applied: smokers;

diagnosed with long-term medical conditions such as asthma

(unless untreated within the past 2 years), heart disease,

gastro-intestinal disease, diabetes, cancer; regular prescribed

medication (except HRT and oral contraceptive); taking di-

etary supplements (unless judged not to affect study out-

come) or antibiotics for greater than 4 weeks before the start

of the study; pregnant; blood donation within 4 months prior

to start of study; BMI less than 18.5 or greater than 35; clin-

ical results at screening judged by the medical advisor to

affect study outcome or be indicative of a health problem.

Subjects were genotyped for the GSTM1 and GSTT1 alleles

but remained blind to the test results. Genomic DNA was

isolated from whole blood samples (200 �L) using a Gene-

lute genomic DNA mini-prep kit (Sigma-Aldrich). GSTM1

and GSTT1 genotypes were identified by quantitative real-

time PCR (Taqman R©) on a 7500 real-time PCR system (Ap-

plied Biosytems, Warrington, UK). Amplification reactions

(25 �L) contained Taqman R© Universal Master Mix, genomic

DNA (50 ng), forward and reverse primers (500 ng each),

a dye-labelled TaqMan R© probe with 3′TAMRA and 5′-FAM

dye-labels (see Gasper et al. for primer and probe sequencess

[16]) and Amplitaq Gold R© DNA polymerase enzyme (Applied

Biosystems). Following activation of the DNA polymerase (10

min at 95�C), reaction mixtures were subjected to 40 PCR cy-

cles, each consisting of 95�C for 15 s and 60�C for 60 s. The

study was explained to participants and written informed con-

sent was subsequently obtained. The study protocol was ap-

proved by the Human Research Governance Committee at


1908 S. Saha et al. Mol. Nutr. Food Res. 2012, 56, 1906–1916

the Institute of Food Research and Norwich Research Ethics

Committee (LREC 2003/082).

The study was a randomized two-phase crossover design

investigating the bioavailability of phytochemicals from fresh

and processed broccoli in subjects of known GSTM1 geno-

type. Each of the two test phases comprised a 5-day period

of intervention separated by a washout period of at least

1 week. During each period of intervention, subjects followed

a cruciferous vegetable-free diet. To aid compliance, a list of

authorized and prohibited foods was provided to volunteers.

On day 3 of the intervention, subjects arrived at the Human

Nutrition Unit following an overnight fast and an intravenous

catheter was inserted. A baseline blood sample was obtained.

Subjects were given a standard breakfast consisting of two

slices of white toast with spread prior to ingesting a cold broc-

coli soup that had been prepared either from lightly cooked

fresh broccoli (100 g fresh weight) or frozen broccoli (100 g)

(see below). The volunteers were blinded to the intervention

meal. Blood samples (10 mL) were collected into lithium hep-

arin tubes at 15, 30 and 45 min and 1, 1.5, 2, 3, 4, 6, 8, 24 and

48 h after broccoli consumption. Blood was immediately cen-

trifuged at 2000 × g (10 min at 4�C) and samples of the plasma

acidified with HCl. Urine was collected the day before con-

sumption of broccoli (24-h collection) and between 0–2, 2–4,

4–6, 6–8, 8–24 and 24–48 h after consumption. The amount

of urine from each period was measured and subsamples

were acidified with HCl. All plasma and urine samples were

subsequently stored at −80�C until analysis.

2.3 Broccoli cultivation and preparation

Broccoli (cultivar Marathon) was grown at the ADAS Experi-

mental Research Station (Terrington, Norfolk, UK) and har-

vested in September 2005. One half of the harvested broccoli

was used fresh (within 24 h of harvesting from the field)

while the other half was processed at a commercial vegetable

processing factory (Christian Salvesen, Bourne, Lincolnshire,

UK) as follows: blanching (91.2�C × 140 s), blast freezing

(−33�C), storage (−28�C). For both fresh and frozen broccoli,

individual soups were prepared by cooking 100 g broccoli flo-

rets (pre-thawed in case of frozen material) with 150 g water

for 75 s in a microwave oven on full power (700 W), followed

by homogenisation in a domestic food blender. For each of

the fresh and frozen broccoli soups, the individual soup por-

tions were combined, mixed and individual portions (235 g)

frozen in bags. Broccoli soups were kept frozen (−18�C) until

use. For the interventions, soups were thawed overnight in

a fridge and consumed without further processing. Samples

of each soup type were taken for analysis of glucosinolates,

isothiocyanate and nitrile.

To investigate the effects of cooking broccoli on the po-

tential for sulforaphane production, a large batch (20 kg) of

broccoli was purchased from a local supermarket, washed in

water and cut into small florets (3–4 cm). Individual sam-

ples (200 g fresh weight) were subjected to steaming (florets

cooked in pre-heated domestic steamer), or boiling (florets

added to pan of boiling water with enough water to just cover

florets) or microwave cooking (in a bowl with 50 g water on

700 watts full power) for 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5 or

7 min. Immediately after the cooking period had ended, the

broccoli tissue was cooled using dry ice and frozen (−20�C).

After freeze-drying and milling, samples were analysed

for glucosinolates, isothiocyanate and nitrile as described

below.

2.4 Analysis of glucosinolates, isothiocyanate,

and nitrile in broccoli

Broccoli tissue and soups were freeze-dried and powdered

(domestic food mixer) prior to analysis. All samples were

extracted and analysed in triplicate. Broccoli glucosinolates

were measured using a method that converts the glucosi-

nolates to the equivalent desulfoglucosinolates [12]. Briefly,

Samples (100–200 mg) of broccoli powder were extracted with

hot aqueous methanol (70% vol/vol, 5 mL) following the ad-

dition of internal standard (sinigrin). Samples were mixed

by vortexing and incubated at 70�C for 20–30 min with oc-

casional mixing. Extracts were allowed to cool and a sample

(3 mL) of the supernatant was applied to an ion exchange

column that was subsequently washed with water (2 × 0.5

mL) and then 0.02 M sodium acetate (2 × 0.5 mL). The

columns were then layered with purified sulfatase (75 �L)

and incubated at RTP overnight. The desulfoglucosinolates

were eluted by sequential application of 0.5, 0.5, and 0.25 mL

water and analysed using HPLC as described below.

Glucosinolate breakdown products (isothiocyanate and ni-

trile) were measured in samples of broccoli powder that had

been dissolved in buffer to facilitate the action of endoge-

nous enzymes (myrosinase) and cofactors (ESP). Samples

(40 mg) of freeze-dried broccoli powder were thoroughly

mixed with phosphate-buffered saline (Invitrogen, Paisley,

UK). The tubes were sealed and vortex mixed. The samples

were incubated on the heating block at 37�C for 2 h, with

vortex mixing every 15 min to ensure optimal hydrolysis. The

tubes were centrifuged (13 000 × g, 4�C for 30 min) and the

supernatants were removed and filtered (0.2 �m) and anal-

ysed by HPLC as described below.

Desulfoglucosinolates were analysed using a Waters

Spherisorb ODS2 (250 × 4.6 mm id, 5 �m particle size)

column connected to a model 1100 HPLC system (Agilent

Technologies, Waldbronn, Germany) comprising of a binary

pump, degasser, cooled autosampler, column oven and diode

array detector. Samples were eluted at 1.0 mL/min with a

gradient of increasing ACN using water (solvent A) and ACN

(solvent B). The gradient started at 0% solution B increas-

ing over 25 min to 50% B and finally re-equilibrated to 0%

B for 13 min. Desulfoglucosinolates were monitored at 229.

Glucoraphanin was detected in the broccoli and soups but

glucoerucin was not detected in the broccoli and soups. Glu-

coraphanin was quantified using absorbance at 229 nm by


Mol. Nutr. Food Res. 2012, 56, 1906–1916 1909

comparison with the internal standard (sinigrin) peak area

ratio and with the correction factor (1.13) of the desulfoglu-

coraphanin [12]. Glucosinolate breakdown products (isothio-

cyanate and nitrile) were analysed using a Phenomenex Luna

C-18(2) (150 × 3 mm id, 3 �m particle size) column con-

nected to an Agilent 1100 HPLC system as described above

but with an additional on-line mass spectrometric detector

(Agilent Technologies). Samples were eluted at 0.3 mL/min

with a gradient of increasing ACN using 0.1% aq. formic acid

(solvent A) and 0.1% formic acid in ACN (solvent B). The gra-

dient started at 0% solution B increasing over 30 min to 30%

B and finally re-equilibrated to 0% B for 10 min. Sulforaphane

was monitored using absorbance at 235 nm, and were quanti-

fied by comparison to external standard curves (linear regres-

sion coefficients >0.99). Sulforaphane-nitrile was monitored

using MS in full scan positive ion mode with electrospray ion-

isation and quantified using selected ion monitoring (m/z =

146) and quantified by external standard curve of authentic

sulforaphane-nitrile (R2> 0.99).

2.5 Analysis of sulforaphane and sulforaphane

conjugates in plasma and urine

Plasma and urine were analysed for sulforaphane and its

glutathione, cysteine-glycine, cysteine and N-acetyl-cysteine

conjugates using a novel, validated method that has been

described recently [6]. Each sulforaphane conjugate was syn-

thesized and purified for use in this study as described previ-

ously [3].

2.6 Analyses of erucin conjugate in urine

Erucin N-acetyl-cysteine conjugate was synthesized by anal-

ogy with the procedure of Kassanhun et al. [3] for

sulforaphane–N-acetyl-cysteine. Hence erucin was added to a

solution of N-acetyl-cysteine, pre-adjusted to pH 7.8, in aque-

ous ethanol, it was purified by reversed-phase HPLC. Urine

samples (1 mL) were acidified to pH 4 with formic acid,

centrifuged at 13 000 × g for 10 min, filtered by (0.45 �m)

polypropylene syringe filter and 100 �L samples were injected

directly onto the HPLC column.

Erucin N-acetyl-cysteine was analysed using a Phe-

nomenex Luna C-18(2) (150 × 3 mm id, 3 �m particle size)

column connected to an Agilent 1100 HPLC system as de-

scribed above with an additional on-line mass spectromet-

ric detector (LC/MSD SL, Agilent Technologies) was added.

Samples were eluted at 0.3 mL/min with a gradient of in-

creasing ACN using 0.1% aq. formic acid (solvent A) and

0.1% formic acid in ACN (solvent B). The gradient started at

0% solution B increasing over 12 min to 50% B and finally re-

equilibrated to 0% B for 8 min. Erucin N-acetyl-cysteine was

monitored using MS in full scan positive ion mode with elec-

trospray ionisation. Identification was performed on the ba-

sis of retention time and spectra matching respect to spiking

synthetic standard in urine. Quantification was performed by

selected ion monitoring (m/z = 325) mode and spiking stan-

dard calibration curve of authentic erucin N-acetyl-cysteine.

(R2> 0.99).

2.7 Bioconversion of glucoraphanin by gut bacteria

The ability of the human gut microbiota to transform specific

glucosinolates was examined in a simple batch fermentation

model. The faecal inoculum was obtained from a healthy vol-

unteer who had not taken any antibiotics or pre- or probiotics

in the previous 2 months. The freshly voided faecal mate-

rial was homogenised (10% w/v) in 0.1M PBS (pH 7.0). One

millilitre of this slurry was then used to inoculate a vessel con-

taining 9 mL of pre-sterilised and pre-reduced basal growth

medium (peptone water 2 g/L, yeast extract 2 g/L, NaCl

0.1 g/L, K2HPO4 0.04 g/L, MgSO4.7H2O 0.01 g/L,

CaCl2.6H2O 0.01 g/L, NaHCO3 2 g/L, Tween-80 2 mL, hemin

0.02 g/L, vitamin K1 10 �L, cysteine HCl 0.5 g/L, bile salts 0.5

g/L, pH 7.0). The incubation was performed at 37�C under

oxygen-free atmosphere (10% H2; 10% CO2; 80% N2) using

an anaerobic cabinet (Don Whitley Scientific, Shipley, West

Yorkshire, UK). Glucosinolates were added to a final concen-

tration of 3 �g/mL. Samples (1 mL) were removed at intervals,

centrifuged at 12 000 × g for 5 min and the supernatants were

filter sterilised and stored at −20�C until further analysis. The

glucosinolate concentrations were analysed by HPLC using

pure glucosinolate standards glucoraphanin and glucoerucin.

Glucosinolates were isolated and purified following the pro-

cedure of Thies [13]. Rocket seed (Eruca sativa) was ground

to a fine powder (50 g) in a coffee grinder and then defat-

ted with petroleum ether (40–60�C fraction, 7 × 200 mL).

The defatted seed powder was extracted with boiling 80%

methanol for 20 min, filtered and the extraction repeated.

The combined filtrates were evaporated under reduced pres-

sure using a rotary evaporator. The residue was taken up in

water and treated with an equimolar mixture of barium and

lead acetate (0.5 M). The precipitate was removed by centrifu-

gation and added to a DEAE Sephadex A25 column (1.4 g)

pre-equilibriated with 6M imadazole formate. Following re-

peated washes with water, the column was cleaned with a

mixture of formic acid: isopropanol: water (3:2:5) and rinsed

with water. Glucosinolates were eluted with 0.5 M potassium

sulphate. Excess potassium sulphate was removed by mixing

the aqueous eluate with ethanol in a 1:1 ratio and removing

the precipitate by centrifugation. The supernatant was evap-

orated to near dryness and taken up in methanol and allowed

to stand at −20�C for 20 min and then centrifuged to re-

move the precipitate and the supernatant evaporated to give

a syrupy residue. The glucoerucin was then further purified

by chromatography on a Sephadex G10 equilibriated with wa-

ter. Glucoerucin elution was monitored at an absorbance of

230 nm and fractions freeze-dried to give the purified product

(400 mg). Purity was assessed by HPLC using a pure sinigrin

standard (Apin Chemicals UK).



Glucoraphanin was prepared following the procedure of

Iori et al. [14]. Hydrolysis products (1 mL) were extracted (X2)

with dichloromethane (1 mL) and the combined extracts dried

with anhydrous magnesium sulphate. The dichloromethane

extract was concentrated to 200 �L and analysed by GC-MS

as previously described [11].

2.8 Data analysis

Statistical analyses were performed using the R data analysis

software (R Development Core Team (2006). R: A language

and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria. ISBN 3–900051-07–0,

URL http://www.R-project.org.). Repeated Measures models

were used to analyse the data. For urine, “Total sulforaphane”

was the response and “Volunteer” was included as a “Random

Effect”. Plasma data were treated similarly but using different

response variables (Cmax, Tmax, AUC). For all models, re-

gression diagnostics were checked to determine if data trans-

formation, outlier omissions, or alternative non-parametric

models were required. All results from the models were con-

sidered significant if p < 0.05.

3 Results

3.1 Subjects

A total of 24 volunteers were genotyped in order to obtain

almost equal numbers of GSTM1 null (n = 10) and positive

(n = 8) subjects. In this rather limited sample (n = 24), the

prevalence of the GSTM1 null genotype was 66.6%, which is

within the range for Caucasians [15]. Eighteen subjects (16

females, two males) completed the entire study without re-

porting adverse events, and had the following anthropometric

characteristics (mean ± SD: age 45.1 ± 11.0 years; height

1.70 ± 0.07 m; weight 70.5 ± 12.1 kg; body mass index

25.7 ± 3.9 kg m–2).

3.2 Glucosinolates, sulforaphane, and sulforaphane

nitrile in soup

Analysis of the soups for glucosinolates, sulforaphane and

nitrile revealed that the soup made from lightly cooked fresh

broccoli contained significant quantities of sulforaphane

(4.16 mg per serving) but no glucosinolates; these data are

consistent with lightly cooked fresh broccoli retaining suffi-

cient myrosinase to hydrolyse all the glucoraphanin, part of

which is converted to sulforaphane. In contrast, the soups

made from commercially frozen broccoli contained signifi-

cant amounts of glucosinolates (18.6 mg per serving) but no

detectable sulforaphane, indicating complete loss of myrosi-

nase activity. These data show that the blanching process com-

pletely destroyed the myrosinase. Both soups contained small

quantities of sulforaphane nitrile (0.08 mg and 0.196 mg

for lightly cooked fresh and frozen broccoli, respectively).

3.3 Kinetics and bioavailability of sulforaphane

conjugates in urine and plasma

Before consumption of broccoli soups, sulforaphane and sul-

foraphane conjugates were not detected in samples of plasma

or urine from any of the volunteers. Following consumption

of the soups, sulforaphane and a range of thioconjugates

representing the entire mercapturic acid metabolic/excretory

pathway (Fig. 1) were detected and quantified. The order of

prevalence of the conjugates in plasma was sulforaphane

> sulforaphane-cysteine-glycine > sulforaphane-cysteine >

sulforaphane-glutathione ∼ sulforaphane-N-acetyl-cysteine.

The mean concentrations were as follows (lightly cooked from

fresh, lightly cooked from frozen broccoli soups, respectively)

sulforaphane (0.6194, 0.0336 �M), sulforaphane-cysteine-

glycine (0.4037, 0.0084 �M), sulforaphane-cysteine (0.1616,

0.0013 �M), sulforaphane-N-acetyl-cysteine (0.1229, 0.0031

�M) sulforaphane-glutathione (0.1151, 0.0004 �M). The or-

der of prevalence of the conjugates in urine was sulforaphane-

N-acetyl-cysteine > sulforaphane-cysteine > sulforaphane

> sulforaphane-cysteine-glycine > sulforaphane-glutathione.

The mean concentrations were as follows (lightly cooked

from fresh, lightly cooked from frozen broccoli soups,

respectively) sulforaphane-N-acetyl-cysteine (10.103, 1.680

�M), sulforaphane-cysteine (2.676, 0.445 �M), sulforaphane

(1.105, 0.136 �M), sulforaphane-cysteine-glycine (0.020,

0.001 �M) sulforaphane-glutathione (0.005, 0.000 �M).

When volunteers consumed a soup made from a stan-

dard portion of fresh broccoli that was lightly cooked, sul-

foraphane conjugates appeared in plasma within 15 min and

total conjugate concentrations peaked at 0.21 �M after 2 h

(Fig. 2A). After peaking, plasma levels declined to very low

levels (mean 0.5 nM) at 48 h. In contrast, when volunteers

consumed a soup made from frozen broccoli that had been

cooked in the same way, sulforaphane conjugates were not

detected in plasma until 1 h after consumption of the soup,

peaked much later at 6 h, and the maximal concentration

achieved in plasma was substantially lower (0.020 versus

0.21 �M, p for difference <0.0001; Fig. 2A). The rate of uri-

nary excretion (�mol total sulforaphane h−1) from the fresh

soup was highest in the 0–2 h collection and declined there-

after, whereas from the frozen broccoli soup the rate of excre-

tion peaked in the 4–6 h sample (Fig. 2B). Total urinary excre-

tion of sulforaphane from the lightly cooked fresh soup was

substantially and significantly higher than that for the lightly

cooked frozen soup (58.5% versus 9.6%; p for difference

< 0.0001). The pharmacokinetic parameters are shown in

Table 1.

Recently, we reported a significant difference between

GSTM1 positive and null genotypes in the urinary yield of

sulforaphane from an oral dose [16]. Accordingly, we strat-

ified our subjects according to GSTM1 genotype. However,


Mol. Nutr. Food Res. 2012, 56, 1906–1916 1911

Figure 1. Structure of the broccoli

glucosinolate glucoraphanin, its break

down products sulforaphane (isothio-

cyanate) and sulforaphane-nitrile, and

the sulforaphane human metabolites

produced via the mercapturic acid

metabolic pathway.

there were no significant differences between the GSTM1

positive and null groups in either plasma pharmacokinetic

parameters (Cmax, Tmax, area under the curve) or total urinary

excretion (Table 1). Also, there were no significant differences

between the GSTM1 positive and null genotypes in the rela-

tive amounts of individual sulforaphane metabolites present

in plasma or urine (data not presented).

3.4 Effects of cooking broccoli

The complete lack of sulforaphane in the soups made from

commercial frozen broccoli that had been cooked only briefly

(75 s) is likely to be due to the blanching step used during

commercial freezing of broccoli. We investigated the effects

of the cooking process and the cooking time on the poten-

tial for sulforaphane production through the action of resid-

ual endogenous broccoli thioglucosidase activity. Samples of

fresh broccoli yielded 50–60% of sulforaphane and 30–50% of

sulforaphane-nitrile (molar yields) upon hydrolysis of freeze-

dried tissue in buffer. When cooked by boiling, sulforaphane

molar yields remained largely unaltered for the first 1.5 min

and then declined, while sulforaphane nitrile molar yields

were less than 5% for all of the cooked samples. Using a

microwave, sulforaphane molar yields increased to 80% at

75 s and then declined to 30% at 3 min and less than 6%

thereafter, while nitrile yields remained near 50% for up to

45 s before declining rapidly to less than 10% after 75 s.

Steamed broccoli retained high molar yields (>60%) up to

2 min before they declined rapidly; nitrile yields remained

above 30% up to 75 s before declining rapidly. These data

indicate that the best cooking methods for time-dependent

retention of sulforaphane production in broccoli are steam-

ing and microwave cooking. Broccoli boiled for just 2 min

generated less than 15% mole yield of sulforaphane from

glucoraphanin.

3.5 Erucin N-acetyl-cysteine in urine

To assess the possibility of sulforaphane and erucin inter-

conversion, six urine samples were analysed for the presence

of erucin N-acetyl-cysteine conjugate (the major metabolite

in urine) from three volunteers following consumption of

either the lightly cooked or frozen broccoli. In each case,

this compound could be detected. The pharmacokinetics of

erucin was not similar to that of sulforaphane. The rate

of urinary excretion (�mols) from the lightly cooked fresh

soup was highest in the 4–6 h collection period and declined

thereafter, whereas from the lightly cooked frozen broccoli

soup, the rate of excretion peaked in the 8–24 h (see Fig. 3).

The ratio of erucin N-acetyl-cysteine and sulforaphane N-

acetyl-cysteine was similar in between subjects (three sub-

jects) in lightly cooked fresh broccoli consumption group



Figure 2. Plasma pharmacokinetic profiles (A) and urinary excre-

tion rates (B) following consumption of broccoli florets that were

lightly cooked from fresh (filled circles and bars) or from frozen

(open circles and bars). The lightly cooked fresh broccoli soup

contained 23.5 �moles of sulforaphane and no glucoraphanin;

the lightly cooked frozen broccoli soup contained 42.5 �moles of

glucoraphanin and no sulforaphane.

but variable in frozen broccoli consumption group (Table 2).

The sulforaphane N-acetyl-cysteine was approximately three

times higher than erucin N-acetyl-cysteine in both groups

(Table 2).

3.6 Bioconversion of glucoraphanin to glucoerucin

in the human gut

Glucoerucin is the major glucosinolate in Rocket seeds [14]

providing a convenient source of material in good yields.

In order to obtain glucoraphanin, the reduced form of glu-

coerucin was oxidised with hydrogen peroxide followed by

purification on DEAE-A25 Sephadex and Sephadex G10. The

bioconversion of glucoraphanin in a simple batch fermenta-

tion model of human gut microbiota was followed by HPLC

and GC-MS analysis (Fig. 4). The time course curve (Fig. 4C)

shows that the glucoraphanin concentration remained near

the initial concentration at the 4-h sample point and then de-

creased dramatically in the 8 and 24 h samples. Glucoerucin

was not detected at the 0- and 4-h sample points but appeared

in appreciable concentrations in the 8 and 24 h samples.

The hydrolysis products at each time point were analysed by

GC-MS, and product formation was observed only in the 8

and 24 h samples. Erucin, erucin-nitrile, sulforaphane and

sulforaphane-nitrile were all detected in the 24 h sample

(Table 3). However, no hydrolysis products were detected

in the 0 and 4 h samples while only erucin nitrile was de-

tected in the 8 h sample. The peak abundances for erucin

(28.06%) and erucin-nitrile (68.53%) were much higher than

for sulforaphane and sulforaphane-nitrile where only trace

amounts were detected (Table 3). No glucoerucin or gluco-

raphanin breakdown products (sulforaphane, sulforaphane

nitrile, erucin and erucin nitrile) were detected in incubations

conducted with autoclave-sterilised faecal samples indicating

that the origins of the reduction of glucoerucin and hydrolysis

products are entirely enzymatic.

4 Discussion

4.1 Bioavailability

Epidemiological evidence consistently supports a link be-

tween increased cruciferous vegetable consumption and re-

duced risk of cancer at several sites [17]. Sulforaphane,

the isothiocyanate released from glucoraphanin, has at-

tracted particular interest for its anticarcinogenic effect. Sul-

foraphane was present predominantly as free sulforaphane

in plasma (see results section 3.3, Fig. 2A) but also as a num-

ber of conjugates (glutathione, cysteine-glycine, cysteine, N-

acetyl-cysteine) [6]. Here, we report that consumption of a

standard portion of lightly cooked broccoli provides a reason-

able dose of sulforaphane (100 �M in soup; 23.5 �mol), and

leads to the rapid appearance in plasma of a number of thiol-

conjugates that are subsequently excreted via the urine. These

data are mainly consistent with a report describing the plasma

pharmacokinetics and urinary excretion of sulforaphane fol-

lowing consumption of a soup made from standard broccoli,

and a soup made from broccoli with enhanced levels of sul-

foraphane [16]. Further, none of our subjects excreted all the

ingested sulforaphane; mean urinary sulforaphane excretion

was 58.5% of dose and the highest individual urinary yield

was 79.6%. The reasons for the differences in urinary yield

between this study and that reported previously [16] are not

clear, but it is plausible that the different dosages are an im-

portant factor.

Broccoli is consumed largely as a cooked vegetable, al-

though a small proportion is eaten raw, for example when

used in salads. It has been established that extended cook-

ing of broccoli and other cruciferous vegetables inactivates

endogenous myrosinase enzyme activity and that as a con-

sequence, the glucosinolates enter the gastrointestinal tract

intact [2]. Shorter cooking times can lead to enhanced


Mol. Nutr. Food Res. 2012, 56, 1906–1916 1913

Table 1. Comparison of plasma pharmacokinetic and urinary excretion data for the study population and stratified according to GSTM1

genotype

GSTM1-positive (n = 8) GSTM null (n = 10) p-valuea)

Broccoli type Genotype

Plasma sulforaphanec)

Cmax (�mol/L)

Fresh broccoli 0.271 ± 0.127b) 0.224 ± 0.140 <0.0001 0.45

Frozen broccoli 0.031 ± 0.030 0.021 ± 0.020

Tmax (h)

Fresh broccoli 1.47 ± 0.74 2.13 ± 1.01 <0.0001 0.39

Frozen broccoli 9.14 ± 6.72 7.00 ± 1.07

AUC (�mol h/L)

Fresh broccoli 1.26 ± 0.80 1.27 ± 0.96 <0.0006 0.61

Frozen broccoli 0.414 ± 0.576 0.144 ± 0.131

Urinary sulforaphanec)

Total urinary excretion (0–48 h) (�mol)

Fresh broccoli 13.39 ± 1.52 14.03 ± 2.93 <0.0001 0.35

Frozen broccoli 1.90 ± 0.62 2.55 ± 0.99

a) Derived using ANCOVA model.b) Data presented as mean ± standard deviation.c) “Sulforaphane” values represent the combined total of free sulforaphane plus the glutathione, cysteine-glycine, cysteine and N-acetyl-cysteine conjugates.Volunteers consumed the broccoli as a soup in a single seating. The lightly cooked fresh broccoli soup contained 23.5 �moles of sul-foraphane and no glucoraphanin; the lightly cooked frozen broccoli soup contained 42.5 �moles of glucoraphanin and no sulforaphane.

Figure 3. Urinary excretion of erucin-N-acetyl-cysteine after con-

sumption of fresh (black bar) and frozen (grey bar) broccoli soup.

Urine was collected for the time periods indicated and the con-

tent of erucin-N-acetyl-cysteine quantified using LC-MS. Data are

average for three subjects with error bars indicating the SD.

isothiocyanate production due to destruction of ESP that

appears to be responsible for the conversion of the un-

stable thiohydroxamate-O-sulfonate to isothiocyanate-nitriles

[2]. We were interested in establishing the isothiocyanate ex-

posure derived from standard portions of typical, commer-

cially available broccoli. Broccoli is almost exclusively avail-

able to the consumer in two forms–fresh broccoli heads and

frozen broccoli florets. In this report, we show that while

light cooking of fresh broccoli facilitated the production of

significant quantities of sulforaphane upon maceration of

the broccoli, frozen broccoli, when cooked in the same man-

ner, generated no sulforaphane. Further, when the subjects

consumed soups made from lightly cooked fresh broccoli, up

to 80% of the sulforaphane was excreted in urine and the

concentrations in plasma reached 0.21 �M (mean for 18

subjects). In contrast, when the volunteers consumed soups

made with lightly cooked frozen broccoli, only a small fraction

Table 2. Sulforaphane N-acetyl cysteine and erucin N-acetyl-cysteine urinary excretion after consumption of soups made with lightly

cooked fresh and frozen broccoli

Subject Total Total Ratio (total erucin-NAC/total

sulforaphane-NACa) erucin-NAC sulforaphane-NAC)

Fresh broccoli

(�moles)

Frozen broccoli

(�moles)

Fresh broccoli

(�moles)

Frozen broccoli

(�moles)

Fresh broccoli Frozen broccoli

1 8.3 1.1 2.4 0.39 0.29 0.35

2 10.5 1.3 3.6 0.68 0.34 0.52

3 6.6 1.5 2.3 0.38 0.35 0.25

Mean 8.5 1.3 2.8 0.48 0.33 0.38

a) NAC, N-acetyl cysteine conjugate.



Figure 4. Conversion of glucoraphanin to glucoerucin in a simple

batch fermentation model. Glucoraphanin (starting concentration

6.3 nmoles/mL) was added to a basal growth medium that had

been inoculated with human faecal slurry and incubated at 37�C.

Samples were removed at the indicated time points and analysed

for glucoraphanin and glucoerucin by HPLC. The chromatogram

(A) is shown for glucosinolate analysis at 8-h sample, the chro-

matogram (B) is shown for glucosinolate analysis at 24-h sample

and the time course curve is shown in (C).

of the glucoraphanin was excreted in urine as sulforaphane

(≈5% on average) and mean plasma concentrations peaked

at 0.025 �M (Fig. 2). A number of reports concerned with

the effects of cooking broccoli on sulforaphane production

have been published. Shapiro et al. reported that the to-

tal urinary excretion of isothiocyanates from fresh broccoli

sprouts was about sixfold higher than for an equivalent dose

of cooked broccoli sprouts [18]. Conaway et al. reported that

steaming reduced the mean plasma concentrations and uri-

nary excretion of sulforaphane from broccoli significantly [7].

Vermeulen et al. reported that consumption of raw broccoli

results in faster absorption, higher bioavailability and higher

concentration of sulforaphane compared to cooked broccoli in

urine and plasma samples [19]. Hence, domestic processing

is a major factor determining the exposure to isothiocyanates

from cruciferous vegetable consumption. Our data show that

commercial frozen broccoli lacks the thio-glucosidase activ-

ity required for sulforaphane generation and, as a result, only

glucosinolates are delivered to the gastrointestinal tract. We

observed that the appearance and peak of sulforaphane in

plasma occurred later than for lightly cooked fresh broc-

coli, presumably because intestinal microflora provide the

thio-glucosidase activity required for sulforaphane produc-

tion, and absorption cannot occur in the stomach/small in-

testine. These data suggest that the efficiency of sulforaphane

absorption from glucosinolates delivered to the colon is low

compared with sulforaphane delivered to the upper gastroin-

testinal tract. Since the majority of broccoli is cooked prior

to consumption, and in most cases for periods sufficiently

long to destroy myrosinase activity, the sulforaphane expo-

sure level for the vast majority of the population will be sim-

ilar to that reported here for frozen (cooked) broccoli (see

Fig. 2).

The lack of myrosinase activity in frozen broccoli not only

caused a substantial reduction in the amount of sulforaphane

absorbed and the resulting circulating concentrations (∼10-

fold lower in this study; see Fig. 2A), but also changed the site

of absorption, and this could have important effects on the

potential for bioactivity at these absorption sites. For fresh

and lightly cooked fresh broccoli, the oesophagus, stomach

and small intestine are exposed to the relatively high con-

centrations of sulforaphane released from the crushed broc-

coli through the action of the plant myrosinase (100 �M in

soup), whereas for frozen broccoli the direct exposure to sul-

foraphane would be extremely low. The cells of these upper

gastrointestinal tract tissues would be expected to take up

sulforaphane from fresh broccoli rapidly and accumulate sul-

foraphane and its glutathione conjugate at very high concen-

trations (several hundred micromolar), as has been reported

elsewhere for several cell types [20, 21]. For frozen broccoli,

the upper gastrointestinal tract would only be exposed to sul-

foraphane via the peripheral circulation, and in this case,

the exposure concentrations would be orders of magnitude

lower (20 nM in this study; Fig. 2A). Zhang and Talalay re-

ported that the ability of an isothiocyanate (ITC) to induce a

phase-2 enzyme (quinone reductase, QR) correlated with the

extent to which the ITC was taken up, accumulated and conju-

gated with glutathione when treating murine hepatoma cells

with micromolar concentrations of ITCs [20]. But, detectable

changes in QR induction were only observed concentration at


Mol. Nutr. Food Res. 2012, 56, 1906–1916 1915

Table 3. Total ion currenta) of hydrolysis products from the incubation of glucoraphanin with a faecal slurry over 24 h

Hydrolysis product T = 0 h T = 4 h T = 8 h T = 24 h

Sulforaphane- ITC 0 0 0 6.24 (1.6)b)

Sulforaphane-nitrile 0 0 0 7.36 (1.9)b)

Erucin-ITC 0 0 0 111.76 (28.1)b)

Erucin-nitrile 0 0 11.9 (100)b) 272.94 (68.5)b)

a) Total ion current obtained by GC-MS analysis of dichloromethane extracts of the fermentation.b) Figures in parentheses show the proportion of hydrolysis products calculated from the total ion current obtained by GC-MS analysis ofdichloromethane extracts of the fermentation.

>0.2 �M, which is tenfold higher than the observed peak in

peripheral blood reported here following ingestion of frozen

broccoli (i.e. QR is not likely to be induced by frozen broccoli).

It was subsequently demonstrated that significant increases

in GSTA1 transcripts could be induced in vivo by perfusion

of the proximal jejunum with a solution containing 11 �M

sulforaphane [21]. Thus, the tissues of the upper gastroin-

testinal tract may benefit from the direct luminal exposure

to micromolar concentrations of sulforaphane delivered by

fresh broccoli, whereas sulforaphane from frozen broccoli is

only likely to influence the upper gastrointestinal tract tissues

via the peripheral circulation, with substantial differences in

the exposure concentrations. Nevertheless, peripheral expo-

sure to low concentrations of sulforaphane conjugates derived

from frozen broccoli can still induce significant changes in

gene expression of tissues, as recently demonstrated in the

prostate tissue of men with prostatic intraepithelial neoplasia

who had consumed frozen broccoli for 12 months [22].

In our study, we also looked at bioconversion of sul-

foraphane to its reduced analogue erucin, in human urine

samples. Urinary erucin N-acetyl-cysteine accounted for 10–

15% of ingested sulforaphane after consumption of lightly

cooked fresh broccoli soup and 1–2% after consumption of

lightly cooked frozen broccoli soup. Thus, the differences in

the yields of sulforaphane and erucin for frozen compared

to fresh broccoli were similar (∼10-fold lower). Interestingly,

our glucosinolate analyses did not confirm the presence of

glucoerucin in either the lightly cooked fresh or the frozen

broccoli soup. Erucin mercapturic acid in urine had been pre-

viously reported after consumption of cruciferous vegetables,

such as broccoli, red and white cabbage that do not contain

glucoerucin [23]. These results show that the reduction of the

sulfinyl group, changing sulforaphane into erucin, can take

place. This was also confirmed in rats by Kassahun et al. [3]

and Bheemreddy et al. [9] and in humans by Clarke et al. [10].

It is not clear what drives the conversion of the sulfoxide sul-

foraphane to the thioether erucin but this is an important

area for future study.

4.2 Bioconversion of glucoraphanin by human gut

microbiota

We investigated the ability of the human gut microbiota

to transform specific glucosinolates in a simple batch fer-

mentation model. Our time course study showed that gluco-

raphanin started to decrease beyond 4 h of incubation and

was substantially reduced after 24 h of incubation, and there

was a corresponding appearance of glucoerucin that was first

detected in 8 h samples and further increased after 24-h in-

cubation. The conversion of glucoraphanin to glucoerucin

did not occur with heat-sterilised samples, and this is the

first report of bioconversion of glucoraphanin to glucoerucin

by human gut microbiota. The formation of sulforaphane

by hydrolysis of glucoraphanin in the cecum of rats and

its ability to cross the cecal enterocyte for systemic absorp-

tion was reported by Lai et al. [24]. They also found very low

levels of the hydrolytic metabolite erucin nitrile by anaero-

bic incubation in rat cecal microbiota. It is known that the

unstable intermediate formed during glucosinolate hydroly-

sis can undergo non-enzymatic rearrangement to a nitrile.

In our study, erucin-nitrile was also detected in the faecal

flora incubations by MS and the abundance was higher than

erucin.

In conclusion, we have demonstrated that the peripheral

exposure to sulforaphane from a single serving of lightly

cooked fresh broccoli is around 0.2 �M, but that the expo-

sure derived from broccoli as it is typically consumed is about

10-fold lower. These physiological concentrations are below

those reported to cause a range of responses in cellular mod-

els. In this study, we reported that erucin-N-acetyl-cysteine

was detected in the urine sample after consumption of either

the lightly cooked form of fresh or frozen broccoli and the

bioavailability of erucin in urine dramatically lowered when

subjects consumed lightly cooked frozen broccoli soup com-

pared to lightly cooked fresh broccoli soup. Further evidence

in this study reported that glucoraphanin and glucoerucin

interconvert in the human microbiota.

The authors thank all the staff at the IFR Human Nutrition

Unit for their assistance with the study, and all our dedicated

volunteers who made the study possible. We also thank Jim Bacon

(IFR) for assistance with the genotyping and Jack Dainty for

assistance with the statistical analysis. We thank Tozer Seeds Ltd.

for their kind gift of Rocket seed. This work was funded by the

Food Standards Agency (UK) with additional support from the

Biotechnology and Biological Sciences Research Council (BBSRC

grant no. 42/D 20475 and 42/D1800) UK.

The authors have declared no conflict of interest.



5 References

[1] Kristal, A. R., Lampe, J. W., Brassica vegetables and prostate

cancer risk: a review of the epidemiological evidence. Nutr.

Cancer 2002, 42, 1–9.

[2] Matusheski, N. V., Juvik, J. A., Jeffery, E. H., Heating de-

creases epithiospecifier protein activity and increases sul-

foraphane formation in broccoli. Phytochemistry 2004, 65,

1273–1281.

[3] Kassahun, K., Davis, M., Hu, P., Martin, B. et al., Biotrans-

formation of the naturally occurring isothiocyanate sul-

foraphane in the rat: identification of phase I metabolites

and glutathione conjugates. Chem. Res. Toxicol. 1997, 10,

1228–1233.

[4] Ye, L., Dinkova-Kostova, A. T., Wade, K. L., Zhang, Y. et al.,

Quantitative determination of dithiocarbamates in human

plasma, serum, erythrocytes and urine: pharmacokinetics of

broccoli sprout isothiocyanates in humans. Clin. Chim. Acta.

2002, 316, 43–53.

[5] Kristensen, M., Krogholm, K. S., Frederiksen, H., Duus, F.

et al., Improved synthesis methods of standards used for

quantitative determination of total isothiocyanates from

broccoli in human urine. J. Chromatogr. B Analyt. Technol.

Biomed. Life Sci. 2007, 852, 229–234.

[6] Al Janobi, A. A., Mithen, R. F., Gasper, A. V., Shaw, P. N. et al.,

Quantitative measurement of sulforaphane, iberin and their

mercapturic acid pathway metabolites in human plasma and

urine using liquid chromatography-tandem electrospray ion-

isation mass spectrometry. J. Chromatogr. B Analyt. Technol.

Biomed Life Sci. 2006, 223–234.

[7] Conaway, C. C., Getahun, S. M., Liebes, L. L., Pusateri, D.

J. et al., Disposition of glucosinolates and sulforaphane in

humans after ingestion of steamed and fresh broccoli. Nutr.

Cancer 2000, 38, 168–178.

[8] Melchini, A., Traka, M. H., Biological profile of erucin: a

new promising anticancer agent from cruciferous vegeta-

bles. Toxins 2010, 2, 593–612.

[9] Bheemreddy, R. M., Jeffery, E. H., The metabolic fate of pu-

rified glucoraphanin in F344 rats. J. Agric. Food Chem. 2007,

55, 2861–2866.

[10] Clarke, J. D., Hsu, A., Riedl, K., Bella, D. et al., Bioavailability

and inter-conversion of sulforaphane and erucin in human

subjects consuming broccoli sprouts or broccoli supplement

in a cross-over study design. Pharmacol. Res. 2011, 64, 456–

463.

[11] Zabala, M. d. T., Grant, M., Bones, A. M., Bennett, R. et al.,

Characterisation of recombinant epithiospecifier protein and

its over-expression in Arabidopsis thaliana. Phytochemistry

2005, 66, 859–867.

[12] Magrath, R., Bano, F., Morgner, M., Parkin, I. et al., Genet-

ics of aliphatic glucosinolates. I. Side chain elongation in

Brassica napus and Arabidopsis thaliana. Heredity 1994, 72,

290–299.

[13] Thies, W., Isolation of sinigrin and glucotropaeolin from cru-

ciferous seeds. Fett Wissenschaft Technologie-Fat Sci. Tech-

nol. 1988, 90, 311–314.

[14] Iori, R., Bernardi, R., Gueyrard, D., Rollin, P. et al., Formation

of glucoraphanin by chemoselective oxidation of natural glu-

coerucin: a chemoenzymatic route to sulforaphane. Bioorg.

Medic. Chem. Lett. 1999, 9, 1047–1048.

[15] Cotton, S. C., Sharp, L., Little, J., Brockton, N., Glutathione

S-transferase polymorphisms and colorectal cancer: a HuGE

review. Am. J. Epidemiol. 2000, 151, 7–32.

[16] Gasper, A. V., Al-Janobi, A., Smith, J. A., Bacon, J. R.

et al., Glutathione S-transferase M1 polymorphism and

metabolism of sulforaphane from standard and high-

glucosinolate broccoli. Am. J. Clin. Nutr. 2005, 82, 1283–

1291.

[17] Bianchini, F., Vainio, H., Isothiocyanates in cancer prevention.

Drug Metab. Rev. 2004, 36, 655–667.

[18] Shapiro, T. A., Fahey, J. W., Wade, K. L., Stephenson, K. K.

et al., Chemoprotective glucosinolates and isothiocyanates

of broccoli sprouts: metabolism and excretion in humans.

Cancer Epidemiol. Biomarkers Prev. 2001, 10, 501–508.

[19] Vermeulen, M., Klopping-Ketelaars, I. W., van den Berg, R.,

Vaes, W. H., Bioavailability and kinetics of sulforaphane in

humans after consumption of cooked versus raw broccoli.

J. Agric. Food Chem. 2008, 56, 10505–10509.

[20] Zhang, Y. S., Talalay, P., Mechanism of differential potencies

of isothiocyanates as inducers of anticarcinogenic phase 2

enzymes. Cancer Res. 1998, 58, 4632–4639.

[21] Petri, N., Tannergren, C., Holst, B., Mellon, F. A. et al., Absorp-

tion/metabolism of sulforaphane and quercetin, and regula-

tion of phase II enzymes, in human jejunum in vivo. Drug.

Metab. Dispos. 2003, 31, 805–813.

[22] Traka, M., Gasper, A. V., Melchini, A., Bacon, J. R. et al.,

Broccoli consumption interacts with GSTM1 to perturb onco-

genic signalling pathways in the prostate. PLoS One 2008, 3,

e2568.

[23] Vermeulen, M., van den Berg, R., Freidig, A. P., van Bladeren,

P. J. et al., Association between consumption of cruciferous

vegetables and condiments and excretion in urine of isoth-

iocyanate mercapturic acids. J. Agric. Food Chem. 2006, 54,

5350–5358.

[24] Lai, R. H., Miller, M. J., Jeffery, E., Glucoraphanin hydroly-

sis by microbiota in the rat cecum results in sulforaphane

absorption. Food Funct. 2010, 1, 161–166.


saha et al (2012) sfn and erucin from fresh frozen broccoli (mnfr) (published)

Documents