Ronald Madl, Ph.D Research Professor

Director Emeritus, Bioprocessing and Industrial Value Added Program

Department of Grain Science & Industry

Reaching New Heights

Antioxidants in Wheat

Food and Agriculture

Lecture Series

July 18, 2013

Perth, Australia

Trends

Fiber Content

Metabolic Changes

Antioxidant Activity

Prebiotic properties

Oxidation processes are responsible for: - Chronic inflammatory disease - Coronary artery disease - Rheumatoid arthritis - Alzheimer’s disease - Cataracts Free radicals of oxygen are primary mechanism

Beta Carotene Vitamin E Vitamin C Phytochemicals - Chelating agents (Phytate) - Estrogens - Isoflavones - Lignans - Phenolic Acids

COOHHO

HO

Caffeic acid

Chlorogenic acid

Ferulic acid

O

O

OH

HO

OHHOOC

OH

OH

OCH3

COOH

HO

trans - Ferulic Acid

Decker, et al, 2002, CFW, 47, 8, 370

History: In 1990s medical literature reported that wheat bran suppressed cancer cell growth in vitro. Later results conflicted.

1994 mouse study showed red wheat bran had protective effect against chemically induced colon cancer vs white wheat bran with equal fiber content.

KSU scientists undertook a survey of over 90 wheat cultivars, representing 5 classes to determine variation in antioxidant activity.

Ortho-phenolic (PheOH ortho) concentration. How much of these antioxidants do they contain? Cell culture assay (IC50). How good are they at killing colon cancer cells? Anti-tumor activity: In vivo min Mouse study

Screen wheat- Develop in vitro assays - representing 5 classes and 34 cultivars by:

PheOHortho Concentration Values are given in apparent

µg (caffeic acid equivalents) per gram of dry grain meal.

High values are better.

Do grains contain different proportions of ortho phenolics?

YES!

µg (caffeic acid equivalents) /g of dry grain

ortho - Phenolic Acid Content of Wheats

Madison

Red WIne

Ernie Betty

Karl 92Arapahoe

0

500

1000

1500

2000

2500

SRW SRW HWW HRW HRW

Are Blood Levels of Polyphenolic Acids

Elevated?

Yes!

Arapahoe

201* (caffeic acid) 249 (ferulic acid)

Betty 456 (caffeic acid) 295 (ferulic acid)

Ernie 468 (caffeic acid) 266 (ferulic acid)

Madison 716* (caffeic acid) 329 (ferulic acid)

Phenolic Acid levels in Mouse Blood Wheat Variety Polyphenolic acid level ng/ml serum

HPLC/MS

IC50 is the lethal dose of grain meal slurry needed to kill 50% of the colon cancer cells.

Low values are better.

Are some grains more potent cancer killers than

others?

YES!

µg protein /

ml of medium

In Vitro anticancer activity

Low is better

WardMadisonRed Wine

Ernie

Betty

Araphahoe

0

20

40

60

80

100

120

HRW HWW SRW SRW Durum

Cancer Suppression and Antioxidant Content

Cultivar LD501 PheOHortho

2

Arapahoe* >100 400

Karl 92 >100 500

Tam 107 >100 900

Betty* 80 1100

Ernie* 50 1100

Coker 9474 25 1800

Elkhart 20 1700

Madison* 5 2400 1 Concentration needed to kill 50% of colon cancer cell population; reported as ug protein/ml of total assay; where >100 means no cell killing; results are means of three determinations. 2 Modified methods of Gutfinger (1981) and Nergiz (1993); reported as ug (caffeic acid equivalents)/g of dry grain. * Wheat selected for in vivo diet studies using Min mice.

Will Wheat Diets High

in Phenolic Acids

Prevent Tumor Formation?

1.) Choose wheats with high, low, and mid protective range Arapaho- low Betty, Ernie- mid Madison- high 2.) Diet fed to Min mice for 10 weeks.

Tumor Data Summary Tumor Incidence

Wide range of antioxidant potential in wheat. Naturally occurring.

Wheat antioxidants affect blood chemistry of animal models.

Wheat with high antioxidant potential kill cancer cells in vitro.

Wheat antioxidants suppress tumor development in animal models.

Plant lignans

Lignans are produced in the outer fiber-containing layers

The main lignans in wheat bran

Enterodiol Enterolactone

Minor constituents to form the building blocks of lignin in the plant cells

The regulation of plant growth

Plant host defense system antifungal insecticidal

HPLC chromatograms of Lignans

-200

20406080

100120140

0 10 20 30 40 50 60

Retention Time (min)

mAU

(Are

a)SDG

Flavone

-10

0

10

20

30

40

50

0 10 20 30 40 50 60

Retention Time (min)

mA

U (A

rea)

SDG

Flavone

Madison

A

B

0

20

40

60

80

100

120

Madison Ernie Betty Arapahoe

µg /g

Und

etec

tabl

e

Mean±SE, n=2-3

0.00%

40.00%

0 20 40 60 80 100

Lignan Contents (µg/g)

Ant

i-tum

or A

ctiv

ities

(%)

r=0.73 , p<0.02

The cells were cultured in the 6-well plates

Treated by enterolactone and enterodiol respectively or in combination at 0μM-40μM for 24-72hours

The number of cells were counted with a hemacytometer

0

1

2

3

24h 48h 72h

% o

f con

trol

Enterolactone

****

**

0

1

2

3

24h 48h 72h

Enterodiol

*

**

* **

0

1

2

3

24h 48h 72h

*

****

**

**

Combined

0 µM 10 µM 20 µM 40 µM **p<0.01, *p<0.05

300

200

100

0

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

Treated cells were fixed in 70% ethanol

Cell cycle was

analyzed by Flow cytometry

0

0.5

1

1.5

24h 48h 72h

S- p

hase

Cel

ls (%

of C

ontro

l)

Enterolacone *

* **

*

0

0.5

1

1.5

24h 48h 72h

Enterodiol

** **

0

0.5

1

1.5

24h 48h 72h

** **

*

**

Combined

40 µM 20 µM 10 µM 0 μM **p<0.01, *p<0.05

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

150

100

50

0

Treated cells (floating cells and adherent cells) were fixed by 1% paraformaldehyde and 70% ice cold ethanol

APO-BrdU TUNEY

assay—detect DNA fragment

Apoptosis cell%

% Apoptosis cells

7.62 18.7 41.7367.02

Control Enterolactone Enterodiol Combined

7 2 h o u r s , 4 0 µ M

0

2 0

4 0

6 0

8 0

1 0 0

C o n tro l E n te ro la c to ne E nte ro d io l C o m b ine d

T re a tm e n t

Ap

op

tos

is c

el

**

* *

Effects of lignan metabolites on apoptosis

(Means ± SD; n=4, *p<0.05 vs. vehicle control)

Lignan content of selected wheat bran from four cultivars were significantly correlated with their anti-tumor activities (r=0.73, p<0.02), suggesting that lignan may contribute cancer prevention

Treatment of human colon carcinoma SW480 cells with either enterolactone or enterodiol or combined, resulted in a dose-and time-dependent decrease of cell numbers

The inhibition of cell growth by lignan metabolites appears to be mediated by cytostatic and apoptotic mechanisms

Segregate existing varieties with high AOX Confirm analytical content of AOX

for grain selected from general market

Develop new varieties with enhanced AOX content

Learn cause of high AOX

Research on wheat shows it has: Wide range of antioxidant levels:

Among genotypes Single variety, different locations/years (1.5 fold)

35

0

200

400

600

800

1000

1200

1400

1600

1800

2000

ug o

f FA

E/g

bran

1.5 fold difference

Karl 92 Greenhouse & growth chambers Stress factors:

Insect feeding Rust infestation Heat

Control plants Harvested grains Tempered and milled Extraction & Analysis: Total

phenolic content and DPPH radical scavenging

Insect feeding: Bird cherry oat aphids (Rhopalosiphum padi) Control: Insect-free plants

Rust infestation: avirulent strain of Leaf rust (Puccinia triticinia) Control: Rust-free plants grown under optimum environmental conditions (T & %RH)

39

Stress factor 3: Heat

Mature heads Harvested Dried Hand-threshed

Kernels Cleaned & sorted Tempered Milled

Acid Extract

0.002.004.006.008.00

10.0012.0014.00

Heat Rust/HeatCont.

Rust Insect Insect Cont.

*Red columns indicate percentage of total Phenolics relative to control **Error bars indicate ± 1SD

43

Wheat plant growth stages

P G S T A K R

0 2 12 14 15 21 Weeks 11

B H WINTER

Insect feeding at specific stages

Research results suggest that the longer the period of stress, the higher AND more consistent the amount of phenolics=> Not specific physiological stage

0

2

4

6

8

10

12

14

16

Tillering Early GF Tillering through Grain filling

mg

FAE/

g b

ran

(% o

f co

ntro

l)

All the original cancer suppression research was conducted on wheat with natural levels of AOX

Are the induced AOX compounds the same? Do they have the same cancer suppression

capacity?

• Cancer cell cultures treated with AOX extracts to determine the AOX extracts’ abilities to inhibit cancer cell propagation.

• Caco-2 , HT-29, and SW480 cell lines are human colorectal adenocarcinoma.

• Cancer cells are cultured in 96 well plates and treated with

AOX extracts

• Differences in cell proliferation between control and experimental samples, measured by MTS assay, elucidate the anti-proliferation properties of specific AOX extracts.

47

48

y = 19.559x - 65.1

y = 0.0911x - 3.8751

y = 8.6192x - 1.105

y = -0.7874x - 11.608

-40

-20

0

20

40

60

80

100

0 2 4 6 8 10 12 14

% G

row

th I

nh

ibit

ion

ug FAE/ well

Extracts Applied to SW480 Culture

Spring Triticale Free-Conj

Spring Triticale Bound

Ike 2010 Free-Conj

Ike 2010 Bound

Linear (Spring Triticale Free-Conj)

Linear (Spring Triticale Bound)

Linear (Ike 2010 Free-Conj)

Linear (Ike 2010 Bound)

49

Wheat can be good source of dietary antioxidants, but levels are variable,

The AOX affect blood chemistry, arrest growth of cancer cells, and suppress tumor growth in model animal systems,

Insect stress of wheat plants induces AOX levels in grain,

These AOX compounds are physiologically important.

Carol Klopfenstein, Grain Science & Ind., KSU Delores Takemoto, Dept of Biochemistry, KSU Frank Padula, Hudson Valley CC, Troy, NY John Carter, Dept of Physical Therapy, WSU George Wang, Dept of Human Nutrition, KSU Ruth MacDonald, University of Missouri, (ISU) Scott Haley, Soil & Crop Sciences, CSU Oscar Ramos, PhD Student Jerry Sullivan, MS Student Kansas Wheat Commission Elizabeth Arndt, ConAgra Midwest Advanced Food Manufacturing Alliance

(MAFMA)

Lignin Synthesis Pathway

An extraction method has been developed by our lab to isolate free, soluble-conjugated, and bound phenolics from wheat bran.

Free AOX extraction:

Acidified (pH 2) methanol, acetone, and water based solvent to extract free phenolics

Soluble-Conjugated AOX extraction: Alkali (2M NaOH) hydrolysis of soluble-conjugated phenolics from crude free phenolic extract.

Basic AOX extraction:

Alkali (4M NaOH) hydrolysis of bound, structural phenolics from left over bran residue

Aqueous extracts are then subjected to a liquid/liquid extraction by 1:1 ethyl ether, ethyl acetate solvent to reduce compounds that interfere with analytical assays

Extracts are then analyzed using various AOX assays.

54