AVEQAVEQ

Analysis of Tocopherols/Tocotrienols Analysis of Tocopherols/Tocotrienols

and Avenanthramides in Oats and Avenanthramides in Oats

WP 8

Rita Redaelli and Lena Dimberg

Tocopherols/ TocotrienolsTocopherols/ Tocotrienols

Tocopherols

Tocotrienols

Isomers R' R''

α CH3 CH3

β CH3 H

γ H CH3

δ H H

Pisacane et al., 2004Solvent: MethanolFlour/solvent ratio: 1 g/14 ml Time: 2*1 minSolubilized: Methanol(or hexane/1,4-dioxane, 95/5)

SLU-labSolvent - MethanolFlour/solvent ratio:(1 g/21 ml)Time: 3*20 min Solubilized - Hexane

Peterson et al., 2007Solvent: Methanol (or saponified KOH in ethanol + antioxidant)Flour/solvent ratio: 1 g/28 mlTime: 2* 20 minSolubilized: Hexane(or hexane/2-propanol, 99.5/0.5)

Extraction of Tocopherols/Tocotrienols

Nielsen and Hansen, 2008Solvent: HexaneFlour/solvent ratio: 1 g/30 mlTime: 1* 10 minSolubilized: Hexane

SLU-labNP- column: 250 mmIsocratic solvent:Hexane/1,4-dioxane (96:4 v/v)Flow: 1.5 ml/minTime: 21 minFluorescence detector: ex 294 nm; em 326 nmReference: 8 isomers

Nielsen and Hansen, 2008NP- column: 125 mmIsocratic solvent:Hexane/ethyl acetate/acetic acid (94.6/3.6/1.8)Flow: 1 ml/minTime: 16 minFluorescence detector: ex 290 nm; em 330 nmReferences: 8 isomers

Pisacane et al., 2004NP- column: 250 mmIsocratic solvent:Hexane/1,4-dioxane (95:5 v/v)Flow: 1,5 ml/min Time: 11 minFluorescence detector: ex 294 nm; em 326 nmReference: 8 isomers

Peterson et al., 2007NP- column: 250 mmIsocratic solvent:Hexane/2-propanol (99.5/0.5 v/v)Flow: 1 ml/min Time:?Fluorescence detector: ex 295 nm; em 330 nmReferences: α-tocopherol

HPLC analysis of Tocopherols/ Tocotrienols

0.0475

Volts

0.0225

0.0250

0.0275

0.0300

0.0325

0.0350

0.0375

0.0400

0.0425

0.0450

0.0475

Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12

volt

s

0.0225

0.0250

0.0275

0.0300

0.0325

0.0350

0.0375

0.0400

0.0425

0.0450

αT

αT3

βT

γT

βT3

γT3

δT

δT3

Pisacane et al. 2004

NP-HPLC OF TOCOLSNP-HPLC OF TOCOLS

AvenanthramidesAvenanthramides

R

R

NH

O

OH

R

R

COOH

n

R= H, OH or OCHR= H, OH or OCH33

n= 1 or 2n= 1 or 2

AvenanthramidesAvenanthramides(ca 30 different compounds in oats)(ca 30 different compounds in oats)

1 2 3

p N

O

OHCOOHH

N

O

OHCOOHH

HO

N

O

OHCOOHH

HO

OMe

f N

O

OHCOOHH

OMe

N

O

OHCOOHH

OMe

HO

N

O

OHCOOHH

OMe

HO

OMe

c N

O

COOHH

OH

OH

N

O

COOHH

HO

OH

OH N

O

COOHH

HO

OH

OH

OMe

HPLC-chromatogram (UV 340 nm) HPLC-chromatogram (UV 340 nm) oat extract and reference compoundsoat extract and reference compounds

0

10

20

30

40

50

15 20 25 30 35 40

Time (min)

Ab

so

rba

nc

e (

mA

U)

a

2c

2p2f

2fd

3f

2pd

0

40

80

120

160

15 20 25 30 35 40

Time (min)

Ab

so

rba

nc

e (

mA

U)

b

2c3c

2p

p

2f

3p

3f1c 2fd

1p1f

Tran

AvenanthramidesAvenanthramidesUV-spectraUV-spectra

0

20

40

60

80

100

120

140

160

200 250 300 350 400

Wavelength (nm)

Ab

sorb

ance

(mA

U)

2c

0

20

40

60

80

100

120

140

200 250 300 350 400

Wavelength (nm)

Ab

sorb

ance

(mA

U)

3c

0

50

100

150

200

250

300

200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm)

Ab

sorb

ance

(mA

U)

2p

0

50

100

150

200

250

200 250 300 350 400

Wavelength (nm)

Ab

so

rba

nc

e (

mA

U)

2f

0

50

100

150

200

250

200 250 300 350 400

Wavelength (nm)

Ab

sorb

ance

(mA

U)

3p

0

20

40

60

80

100

120

140

200 250 300 350 400

Wavelength (nm)

Ab

so

rba

nc

e (

mA

U)

3f

0

20

40

60

80

100

120

140

160

200 250 300 350 400

Vawelength (nm)

Ab

so

rba

nc

e (

mA

U)

1c

020406080

100120140160180200

200 250 300 350 400

Wavelength (nm)

Ab

so

rba

nc

e (

mA

U)

2fd

0

50

100

150

200

250

200 250 300 350 400

Vawelength (nm)

Ab

sorb

ance

(mA

U)

1p

0

50

100

150

200

250

200 250 300 350 400

Wavelength (nm)

Ab

so

rba

nc

e (

mA

U)

1f

020406080

100120140160180200

200 250 300 350 400

Wavelength (nm)

Ab

sorb

ance

(nm

)

Tran

SLU-labSolvent - 80% Ethanol (pH 2)Flour/solvent ratio:(1 g/21 ml)Temperature: 50 CTime: 3*20 min Solubilized - Methanol

Extraction of Avenanthramides

Wise et al., 2009Solvent: 80% EthanolFlour/solvent ratio: 1 g/30 ml Temperature: 50 CTime: 3*20 minSolubilized: Methanol

Matsukawa et al., 2000Solvent - MethanolFlour/solvent ratio:(1 g/30 ml)Temperature: ambient?Time: 3*? min Solubilized -Methanol

CollinsSolvent - 80% Ethanol (acidified)Flour/solvent ratio:(1 g/25 ml)Temperature: 100 CTime: 3*20 minPurification: Octyl-sepharose,

Sephadex-LH20 Solubilized – 50% Ethanol

SLU-labRP C-18 column: 125 mmSolvent: A. = 0.01 M formic acid

(pH 2.9), 5% acetonitrileB. acetonitrile

Gradient: 5 to 38% B in A in 35 minFlow: 1 ml/minTime: 30 minDiod array: 340 nmReference: 17 compounds

Wise et al., 2009RP C-18 : 50 mmSolvent: A. 0.1% formic acid, 5% acetonitrile

B. acetonitrile, 0.1% formaric acidGradient: 13-30% B in A for 20 minFlow: 1 ml/minTime: 20 minDiode array detector: 280 and 330 nm References: 3 compounds

Collins et al., 2004RP C-18 column: ? mmSolvent: A. 5% acetic acid

B. methanolGradient: 40 to 100% B in A for 88 minFlow: 0.8 ml/minTime: 50 minDiod array: 330 nmReference: ? compounds

Matsukawa et al., 2000RP C-18 column: 150 mmSolvent: A. 0.5% TFA,

B. acetonitrileGradient: 15 to 30% B in A for 60 minFlow: ? ml/minTime: ? minDiod array: 340 nmReference ? compounds

HPLC analysis of Avenanthramides

a

b c d e f g

2c

2p

2f

3f

2pd

2fd

2c

2p

2f

3f

2pd

15 17 19 21 23 25 27 29

Time (min)

2fd

Analysis of AvenanthramidesAnalysis of AvenanthramidesLC-MSLC-MS

b-g Selective ion monitoring (SIM) detection[M+H]+

for 2c, 2p, 2f, 3f, 2pd and 2fd, respectively

aUV detection, 340 nm

Molecular weights of avenanthramidesMolecular weights of avenanthramides

   anthranilic

acid 5-OH 5-OH, 4-MeOH 4-OH 4-OH, 5-OH

  1 2 3 4 5

p-coumaric p 283 299 329 299 315

ferulic f 313 329 359 329 345

sinapic s 343 359 389 359 375

caffeic c 299 315 345 315 331

             

p-coumaric p diene 309 325 355 325 341

ferulic f diene 339 355 385 355 371

sinapic s diene 369 385 415 385 401

caffeic c diene 325 341 371 341 357

0

2

4

6

8

10

2 4 6 8 10 12 14 16 18 20

Time (min)

Pea

k ar

ea (

AU

)

120 h germination Raw grains

1

2

32c

567 8

2p

2f

1112

13

14=3f

15

16

171819 20

21

Effect of germinationEffect of germination

Thank youThank you

Antioxidant activity of different Antioxidant activity of different avenanthramidesavenanthramides

0

400

800

1200

1600

0 30 60 90 120

Time (min)C

on

jug

ated

die

ne

hyd

rop

ero

xid

es

(uM

)

1a

2a

3a

Control

a-tocopherol

0

400

800

1200

1600

0 30 60 90 120

Time (min)

Co

nju

gat

ed d

ien

e h

ydro

per

oxi

des

(u

M)

3a

3p

3f

3s

3c

Control

a-tocopherol

0

100

200

300

400

500

600

2c 2p 2f

Co

nce

ntr

atio

n (

pea

k ar

ea/g

)

s

r

Avenanthramides in crown rust Avenanthramides in crown rust susceptible and resistant cultivarssusceptible and resistant cultivars

Radical scavenging activity of Radical scavenging activity of different avenanthramidesdifferent avenanthramides

a p f s c

12

30

10

20

30

40

50

60

70

Ra

dic

al

sc

av

en

gin

g

ac

tiv

ity

(d

elt

a A

(1 m

in) A

U)

a p f s c

12

30

10

20

30

40

50

60

70

Ra

dic

al

sc

av

en

gin

g

ac

tiv

ity

(d

elt

a A

(5 m

in) A

U)

1 min

5 min

Radical scavenging activity of Radical scavenging activity of different avenanthramidesdifferent avenanthramides

a p f s c

12

30

10

20

30

40

50

60

70

Ra

dic

al

sc

av

en

gin

g

ac

tiv

ity

(d

elt

a A

(1 m

in) A

U)

a p f s c

12

30

10

20

30

40

50

60

70

Ra

dic

al

sc

av

en

gin

g

ac

tiv

ity

(d

elt

a A

(5 m

in) A

U)

1 min

5 min

HPLC-chromatogram HPLC-chromatogram (UV-detection 340 nm) (UV-detection 340 nm)

--oat extract and reference compounds-oat extract and reference compounds-

0

10

20

30

40

50

15 20 25 30 35 40

Time (min)

Ab

so

rba

nc

e (

mA

U)

a

2c

2p2f

2fd

3f

2pd

0

40

80

120

160

15 20 25 30 35 40

Time (min)

Ab

so

rba

nc

e (

mA

U)

b

2c3c

2p

p

2f

3p

3f1c 2fd

1p1f

Tran