Development of Vegetables with Improved Health-Promoting Qualities

Elizabeth JefferyDepartment of Food Science and Human

Nutrition, University of Illinois

Fruits, Vegetables & Cancer Prevention: Epidemiology is undecided

Variety or Category % Positive

Vegetables 80% (59/74)

Fruits 64% (36/56)Raw vegetables 87% (40/46)

Cruciferous Vegetables 69% (38/55)Allium Vegetables 77% (27/35)

Green vegetables 77% (68/88)Carrots 81% (59/73)

Tomatoes 71% (36/51)

Citrus Fruit 66% (27/41)

The 1997 World Cancer Research Fund and the American Institute for Cancer Research (WCRF/AICR) report:Food, Nutrition and the Prevention of Cancer: a global perspective, p442. by John D Potter and other panel members

Case-Control Studies Cohort Studies

Riboli and Norat, 2003

Epidemiology of Dietary Cancer Prevention : Fruit and Vegetables

SAFE EFFECTIVE

DOSE

Food Variability: environment, genotype

Human Variability: environment, genotype

BFC i.d. Formulation/ food preparation

Bioavailability Biomarkers

DNA RNA Protein Metabolites

Nutrigenomics

Nutrigenetics TranscriptomicsEpigenetics

proteomics metabolomics

Post-genomics

PHENOTYPE

Human Variability

Crucifers/non-smokers 0.70 (ns)

Crucifers/smokers 0.31 (p<0.05)

Crucifers Lower Risk for Lung Cancer Crucifers Lower Risk for Lung Cancer More Effectively in those at high riskMore Effectively in those at high risk

Human Variability

Zhao et al, 2001, Cancer Epi Bio Prev 10:1063-7

Relative riskRelative risk

0

0.2

0.4

0.6

0.8

1

1.2

Q1 Q2 Q3 Q4

Wildtype, p trend=NS

Null, P trend<0.03

OR

, B

reas

t C

ance

r

Quartile of Crucifer Intake

Fowke et al., Cancer Res. 63: 3980-3986, 2003

Breast Cancer Risk, Dietary Crucifers and the GSTT1 Null Phenotype

Human Variability

2005 and 2003 USDA Nutritional Data for RAW BROCCOLI (abridged): Mean value per 100.00 grams edible partName Unit Amount Amount #data S.E.

2003 2005 points

Food energy kcal: 28.00 28.00 1

Protein g : 2.98 2.98 22 .11

Total lipid (fat) g : 0.35 0.35 22 .03

Carbohydrate g : 5.24 5.24 1

Total saturated fat g : 0.05 0.05 1

Cholesterol mg : 0 0 1

Total dietary fiber g : 3.0 -

Vitamin A IU : 1542 3000 1

Ascorbic acid mg : 93.2 93.2 15 2

Carotenoid content of Broccoli

0 10 20 30 40 50 60

Peto-13

Zeus

Packman

Su003

Marathon

Cavolo

Peto-6

Eu8-1

GEM

Brigadar

Majestic

MA-191

Peto-7

Baccus

EV6-1

VI158

Pirate

Green Comet

Shogan

BNC

High Sierra

Atlantic

mic

ro m

ol /

100

g DW

Genotypes

lutienbeta- carotene

Means, 22 different broccoli genotypes (mol/100g DW)

10-fold difference

Lutein

Beta-carotene

Glucosinolates: precursors to active components in cruciferous

vegetables

0

2

4

6

8

10

12

Brocc

oli

Bruss

els

Cabbag

e

Caulif

lower

Kale

progoitringlucoraphaninglucobrassicinsinigrin

S

C

O

N O SO3-

S C6H11O5

glucoraphanin

ITC

Isothiocyanate Anticarcinogen

Myrosinase

S

C

O

N O SO3-

S C6H11O5

S

N

O

C S

Glucoraphanin

Sulforaphane

Glucosinolates break down to ITC when plants are crushed or chewed

VARIATION IN CONTENT:

Determination of scientific basis for variation

CONTROL

Examples (taken from broccoli):

Carotenoids and Tocopherols

Glucosinolates

Sulforaphane production from glucoraphanin

Genotype

Metabolome

Content of metabolite of interest: Phenotype

Genome

Genotype

Environment

Metabolome

Content of metabolite of interest

Genome

Genotype

Environment

Metabolome

Content of metabolite of interest in food product

Genome

Processing

Glucosinolate variation among 50 broccoli varieties; one season

0

5

10

15

20

25

Brigadier Packman Peto 7 50 varieties

progoitringlucoraphaninglucobrassicin

Glucosinolate variation due to genotype, environment and genotype x environment interaction _______________________________________________________________________ Gluco- Total Gluco- Total Sinigrin Progoitrin rapahnin aliphatics brassicin indolyl-GS ________________________________________________________________________ Genotype 68.2%* 71.0%* 54.2%* 61.1%* 18.7% 12.0%

Environment 0.1% 5.6% 4.7% 4.5% 19.2%* 33.0%*

GXE 15.5%* 10.4%* 9.3%* 10.3%* 20.0%* 21.0%*

Residual 16.0% 12.9% 31.2% 24.1% 42.1% 32.1%

significant at p<0.05

10 genotypes of broccoli grown over four environments (Fall 1996, Fall 1997, Spring 1998, Fall 1998)

Glucosinolate variation due to genotype, environment and genotype x environment interaction _______________________________________________________________________ Gluco- Total Gluco- Total Sinigrin Progoitrin rapahnin aliphatics brassicin indolyl-GS ________________________________________________________________________ Genotype 68.2%* 71.0%* 54.2%* 61.1%* 18.7% 12.0%

Environment 0.1% 5.6% 4.7% 4.5% 19.2%* 33.0%*

GXE 15.5%* 10.4%* 9.3%* 10.3%* 20.0%* 21.0%*

Residual 16.0% 12.9% 31.2% 24.1% 42.1% 32.1%

significant at p<0.05

10 genotypes of broccoli grown over four environments (Fall 1996, Fall 1997, Spring 1998, Fall 1998)

G

E

GxE

Slice 4

G

E

GxE

Slice 4

Aliphatic Indolyl

Variation in glucosinolates due to genotype, environment and G x E

Genetics x Environment Interaction

E1 E2

G1

G2

Meta

bolite

Con

ten

t

E1 E2

G3

G4

Meta

bolite

Con

ten

t

Selective increase of the potential anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli. Carcinogenesis. 1998 ;19(4):605-9

Faulkner K, Mithen R, Williamson G.John Innes Centre, Norwich Research Park, UK.

The putative anticarcinogenic activity of Brassica vegetables has been associated with the presence of certain glucosinolates. 4-Methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding glucosinolate found in broccoli, has previously been identified as a potent inducer of the anticarcinogenic marker enzyme quinone reductase [NADP(H):quinone-acceptor oxidoreductase] in murine hepatoma Hepa 1c1c7 cells. We have therefore produced a broccoli hybrid with increased levels of this anticarcinogenic glucosinolate and tested the ability of extracts to induce quinone reductase. A 10-fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n = 9) complex. Tissue from these hybrids exhibited a >100-fold increase in the ability to induce quinone reductase in Hepa 1c1c7 cells over broccoli cultivars, due to both an increase in 4-methylsulphinylbutyl glucosinolate content and increased percentage conversion to sulphoraphane.

Sulforaphane formation: glucoraphanin hydrolysis Glucoraphanin

Sulforaphane

Myrosinase(Crushing)

Unstable Intermediate

S

C

O

N O SO3-

S C6H11O5

S

N

O

C S

S

C

O

N O SO3-

SH

+ C6H11O5

Glucoraphanin Hydrolysis

Glucoraphanin

Sulforaphane Sulforaphane Nitrile

Myrosinase(Crushing)

Unstable Intermediate

S

C

O

N O SO3-

S C6H11O5

S

C

O

N

S

N

O

C S

S

C

O

N O SO3-

SH

+ C6H11O5

10-15% 85-90%

Defatted Broccoli Seed(1 kg)

Water Extract

Methylene Chloride Extract

Preparative HPLC

Sulforaphane(4.8 g)

SulforaphaneNitrile (3.8 g)

Refractive IndexUV 254 nm

Matusheski et al, 2001

GC FID Detection GC FID Detection

Induction of QR in Cell Culture1

1Matusheski and Jeffery, 2001

0

50

100

150

200

250

300

0.01 0.1 1 10 100 1000 10000

Log Concentration (µM)

QR

Sp

ecif

ic A

cti

vit

y (

nm

ol M

TT

re

du

ced

/mg

pro

tein

/min

)

Sulforaphane Sulforaphane Nitrile

Hepatic QR1

1Matusheski and Jeffery, 2001

*Significantly different from pair fed group (Student’s T-test, p<0.05). Mean ± Standard Error

0

20

40

60

80

100

120

140

160

200 500 1000

Treatment

QR

Act

ivity

(nm

ol D

PIP

re

du

ced

/mg

pro

tein

/min

) Pair Fed

Sulforaphane

200 500 1000 SF 500

Treatment

Pair Fed

Sulforaphane Nitrile

Experiment 1 Experiment 2

*

*

*

Effect of Heating on Broccoli Florets

-0.01

0.19

0.39

0.59

0.79

0.99

1.19

0 20 40 60 80 100 1205 Minute Pre-Heating Temperature

µmol

/g F

resh

Wei

ght

Sulforaphane Nitrile

Sulforaphane

ESP may remove S in glucosinolates that have no receiving alkenyl group

Alkenyl glucosinolates

Non Alkenyl glucosinolates

Nitrile

EpithioNitrile

ESP

IsothiocyanateNitrile

Isothiocyanate

Measurements of ESP activity:

Epithionitrile:nitrile ratio

Sulforaphane: sulforaphane nitrile ratioESPESP

Recombinant Epithiospecifier protein; Effect of Heating

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 20 40 60 80 100 120

5 Minute Pre-Heating Temperature (°C)

ES

P A

ctiv

ity

(mo

lar

rati

o o

f E

TN

/CH

B)

200-

116-97.4-

66-

45-

31-

MW 100 10 1StdDilution

Broccoli ESP Activity and Bioactivity

** *

*

0

20

40

60

80

100

120

140

160

0 20 40 60 80 100 120

Temperature (°C)*Significantly greater than control (p<0.05).

QR

Act

ivity

(nm

ol M

TT re

duce

d/m

in/m

g pr

otei

n)

ESP Activity

Bioactivity

N

S S

O

FeS

S

O

S

O

N C S C N

N

SS

O

b-D-Glucose

Glucoraphanin

Myrosinase

Lossen-TypeRearrangement

Sulforaphane Sulforaphane Nitrile

b-D-Glucose

SO3-

SO3-

SO42-

SO42-

ESP

y = 1.4278x + 0.4182

R2 = 0.2466

0

0.5

1

1.5

2

2.5

3

3.5

0 0.2 0.4 0.6 0.8 1 1.2ESP Activity (ETN/CHB Ratio)

SF

N/S

F R

atio

ESP activity (epithionitrile: nitrile) correlated with the sulforaphane nitrile: sulforaphane ratio

P < 0.05

How can we harness this in processing to optimize sulforaphane ?

-0.01

0.19

0.39

0.59

0.79

0.99

1.19

0 20 40 60 80 100 1205 Minute Pre-Heating Temperature

µmol

/g F

resh

Wei

ght

Sulforaphane Nitrile

Sulforaphane

Microwave heating for different periodsPinnacle - Microwave

Heating Time (min)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Hyd

roly

sis

Pro

duc

ts (

umol

/g w

w)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

SFN SF SF/(SF+SFN)

Processing to optimize sulforaphane formation

Pinnacle

Time (min)

0.00 0.25 0.50 0.75 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Su

lfora

ph

ane

( m

ol/g

ww

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

MicrowavingBoilingSteaming

Time since meal (h)

Baseline 0-5.9 h 6-11.9 h 12-23.9 h 24-35.9 h 36-49 h

SF

C/c

reat

inin

e (

mol

/mg)

0

10

20

30

40

50

60

Raw 1.5 min steamed5.0 min steamed

Sulforaphane excretion

Genotype

Environment

Phenotype

Stable content of metabolite of interest in food product

processing

Acknowledgements

• Jack Juvik• Barbara Klein• Mosbah Kushad• Matthew Wallig• Richard Mithen• Malcomb Bennett• Anna Keck

• Allan Brown• Anne Kurilich• In-Gyu Park• Grace Wang• Kanta Kobira• Nathan Matusheski• Ranjan Swarup