purification, analysis, and structures of avenanthramides from oats: unique components for targeted...

Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted

Nutraceutical Applications

Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted

Nutraceutical Applications

F. William CollinsF. William Collins

Eastern Cereals and Oilseeds Research CentreOttawa, ON.

Eastern Cereals and Oilseeds Research CentreOttawa, ON.

8th IOC Minneapolis, MN, June 20088th IOC Minneapolis, MN, June 2008

Agri-Food CanadaAgri-Food CanadaAgri-Food CanadaAgri-Food CanadaAgriculture andAgriculture andAgriculture andAgriculture and Agriculture etAgriculture etAgriculture etAgriculture et

Agroalimentaire CanadaAgroalimentaire CanadaAgroalimentaire CanadaAgroalimentaire Canada

OVERVIEWOVERVIEW

Group separation for reproducibility Group separation for reproducibility

2-D HPLC-MSMS techniques to map total avenanthramide diversity

2-D HPLC-MSMS techniques to map total avenanthramide diversity

Co-extracted compounds and their effects on quantitation

Co-extracted compounds and their effects on quantitation

UV Spectral properties of avenanthramides UV Spectral properties of avenanthramides

Need for improved methods of analysis Need for improved methods of analysis

22 Purification, Analysis, and Structures of Avenanthramides from Oats

Purification, Analysis, and Structures of Avenanthramides from Oats



NEED FOR IMPROVED METHODSNEED FOR IMPROVED METHODS

Avenanthramide components are complex mixture of over 30 individual members

Avenanthramide components are complex mixture of over 30 individual members

Several other groups of compounds in oat extracts can interfere with UV-based HPLC estimation of avenanthramides

Several other groups of compounds in oat extracts can interfere with UV-based HPLC estimation of avenanthramides

Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more

accurate/robust analytical techniques

Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more

accurate/robust analytical techniques

33





1. Stabilization and extraction with aqueous EtOH1. Stabilization and extraction with aqueous EtOH

SUMMARY OF PROCEDURESUMMARY OF PROCEDURE

2. Removal of lipophilic phenolics and prolamines using hydrophobic binding to Octyl

Sepharose

2. Removal of lipophilic phenolics and prolamines using hydrophobic binding to Octyl

Sepharose 3. Removal of flavonoids glycosides and avenacins

using aromatic absorption on Sephadex LH-203. Removal of flavonoids glycosides and avenacins

using aromatic absorption on Sephadex LH-20

4. Concentration of avenanthramides 4. Concentration of avenanthramides

5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A

5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A

44



Anthranilic acid (2-aminobenzoic)

= AVENANTHRAMIDE: TYPE I

p-Coumaric acid (4-hydroxycinnamic)+

2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported

1. Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported

OH

NH

O

OHO

3

4

5

6

2'

3'

5'

6'

7'

8'

THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS…THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS…

55



= AVENANTHRAMIDE: TYPE 2

Anthranilic acid (2-aminobenzoic)

Avenalumic acid 5-(4-hydroxyphenyl)-2,4-pentadienoic acid

+

1. Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported

…..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID

…..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID

2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported

9'

10' 8'

7'

OH

NH

O

OHO

66



10 g ground oats

1. 75 mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH

Stir for 20 min with ambient cooling and decant into graduated glass column

Allow to settle and pack by gravity to form a percolation extraction bed, Vb

Procedure is scaleable

Denatures enzymes (e.g. peroxidases, esterases, etc)

Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...


HPLC evaluation at this point, detection at 330nm

Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent

Drain extract and percolate a further 3xVb fresh solvent to give a “percolate”

Percolation is an extremely efficient extraction method

77



Avenacosylate peaks (in part)

“Prolamine” peaks (in part)“Glycolipid” peaks (in part)“Phosphatide” peaks (in part)

Avenanthramide zone

HPLC Profiles During Group Separation Of Avenanthramides...


VAO 48(hulless)

Crude EtOH extract

5 10 15 20 25 30 35 40 45 50 55 60 65

20

40

60

0

80

100

120 OD 330nm(mAU)

Time (min)

88



There are 3 major classes of avenacosylates in oats

O

HO

ORO

O

HO

OR1O O

OH

OOR2

R = CH3; 1-O-feruloyl-n-hexacosan-1-ol

R = H; 1-O-caffeoyl-n-hexacosan-1-ol

(C26 alcohol)

(C26 alcohol)

R1 = R2 = CH3; 1,26-di-O-feruloyl-n- hexacosan-1,26-diol

(C26 α,ω -diol)

R1 = H; 1-O caffeoyl-26-O-feruloyl-n- R2 = CH3 hexacosan-1,26-diol

(C26 α,ω -diol)

R1 = R2 = CH3; 1,26-di-O-caffeoyl-n- hexacosan-1,26-diol

(C26 α,ω -diol)

O

HO

OR2O

O

O

O

OH

OOH

O

HO

OR1O

R1 = CH3; 1-O caffeoyl-2-O-feruloyl-3-O-(26'-O- R2 = OH caffeoyl)-n-hexacosanoyl-D-glycerol

AVENACOSYLATES: UV Interfering Components from Oats

AVENACOSYLATES: UV Interfering Components from Oats

Hydroxy-cinnamoyl esters of n-

alkanols

Hydroxy-cinnamoyl esters of n-alkan-

α,ω-diols

Mixed hydroxy-cinnamoyl esters of

ω-hydroxy-n-alkanoyl glycerides

99



10 g ground oats

1. 75 mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH

Stir for 20 min with ambient cooling and decant into graduated glass column

Allow to settle and pack by gravity to form a percolation extraction bed, Vb

Drain extract and percolate a further 3xVb fresh solvent to give a “percolate”

Procedure is scaleable

Denatures enzymes (e.g. peroxidases, esterases, etc)

Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties

Percolation is an extremely efficient extraction method


EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...1010

Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent

Collins et al, US Patent #6,495,140 (2002)



2. Re-suspend in acidified 50% EtOH and transfer with washings to top

of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% EtOH)

Elute with 3xVb acidified 50% EtOH

Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH

Prolamines and lipophilic phenolics are bound to the beads in 50% EtOH

All avenanthramides and hydrophilic components are not bound to Octyl Sepharose in 50% EtOH

3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH

Elute with 2xVb acidified 40% EtOH to remove polar components

All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound



HPLC evaluation at this point, detection at 330nm

1111



Avenanthramide zone

VAO 48(hulless)

Crude EtOH extract after removal of lipophilics

“Prolamine” peaks (in part)“flavone glycosides”

luteolin aglycone apigenin aglycone

tricin aglycone

5 10 15 20 25 30 35 40 45 50 55 60 65

20

40

60

0

80

100

120 OD 330nm(mAU)

Time (min)

1212 HPLC Profiles During Group Separation Of Avenanthramides...


“Avenacins”



Other UV Absorbing Components from Oats: The Avenacins

Other UV Absorbing Components from Oats: The Avenacins

O

O

OH

OHH

O

H

O

O

HO

O

O

OHHO

HO

HO

O

OHHO

HOHO

R

O HNCH3

OR =

Avenacin A-1

O

OR =

Avenacin A-2

R

O

O

OH

O

H3C H

O

H

O

O

HO

O

O

OHHO

HO

HO

O

OHHO

HOHO O HN

CH3

O

Avenacin B-1

R =

O

O

Avenacin B-2

R =

1313

Triterpene saponins with a chromophoric group with UV

absorption max at ~ 325 nm (A-1, B-1)



O

OH

OH

HO

O

4',5,7-(OH)4-flavone(Apigenin)

O

OH

OH

HO

O

OH

3',4',5,7-(OH)4-flavone(Luteolin)

O

OH

OH

HO

O

4',5,7'-(OH)3-3',5'-(OMe)2-flavone

(Tricin)

OCH3

OCH3

O

OH

OH

H3CO

O

O

HOHOHO

H3C

OOH

HO

OHO

Isoswertisin-2"-O-rhamnoside(Apigenin-8-C-(2"-O-α-L-rhamno-pyranosyl)-β-D-glucoside-7-O-

methyl ether)

O

OHHO

HOHO O

OH

OH

HO

O

IsovitexinApigenin-6-C-β-D-glucoside

1414 Other UV Absorbing Components from Oats: Flavones

Other UV Absorbing Components from Oats: Flavones

• UV absorption spectral maxima similar to those of avenanthramides

• Chromatographic properties overlap with avenanthramides

• However, flavone derivatives appear to be minor components in oat groat tissues (~5%?)













HPLC analysis to look at profile

1515









All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound




HPLC analysis to look at profile

1616




Recover avenanthramides by eluting column with 3xVb 95% EtOH

4. Evaporate eluate to dryness, take up in known volume of 50% EtOH (~ 2-5 mL),

pass sub-sample through 0.45μ filter

HPLC-rapid scan (“diode array”)

HPLC-diode array-MSMS

Can also use different HPLC column:solvent configurations

All avenanthramides are not bound to Sephadex LH-20 in 95% EtOH

Procedure results in base-line resolution of all the avenanthramides and can be easily scaled up to gram quantities

Molecular Weight Range scanned between 100-700daltons


EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...1717



Avenanthramide zone

VAO 48(hulless)

5 10 15 20 25 30 35 40 45 50 55 60 65

20

40

60

0

80

100

120 OD 330nm(mAU)

Time (min)

1818 HPLC Profiles During Group Separation Of Avenanthramides...


Final purified avenanthramide

extract

Flat base line for ease of

integration



1919

Avenanthramide zone

After LH-20

After Octyl Sepharose

Crude EtOH extract

Comparison of HPLC Profiles During Separation Of Avenanthramides...

Comparison of HPLC Profiles During Separation Of Avenanthramides...

5 10 15 20 25 30 35 40 45 50 55 60 65

20

40

60

0

80

100

120

Time (min)



5 10 15 20 25 30 35 40 45 50 55 60 65

20

40

60

0

80

100

120 OD 330nm(mAU)

VAO 48(hulless)

Time (min)

2020 Spectral analyses of AvenanthramidesSpectral analyses of Avenanthramides

High degree of peak

resolution facilitates spectral

analysis and identification



OD 330nm(mAU)

Retention Time (min)

250

300

350

275

325

375

λ(nm)

400

300

200

100

0

(mAU)

BOH

OCH3NH

O

OHO

HO

m/z = 329

2121 Spectral analyses of avenanthramidesSpectral analyses of avenanthramides

(mAU)



OD 330nm(mAU)

(mAU)

2222 Spectral analyses of avenanthramidesSpectral analyses of avenanthramides

OH

NH

O

OHO

HO

OCH3

m/z = 355

P

Retention Time (min)

250

300

350

275

325

375

λ(nm)

400

300

200

100

0

(mAU)



BOH

OCH3NH

O

OHO

HO

m/z = 329

OH

NH

O

OHO

HO

OCH3

m/z = 355

P

2323

300s

335 350s

Spectral analyses of avenanthramidesSpectral analyses of avenanthramides

All Type I avenanthramides show a shoulder between 290

and 310nm

All Type II avenanthramides lack this shoulder

360340s



Time A(%) B(%) C(%) Flow rate

0 40 55 5 0.8 mL/min 80 60 35 5 0.8 mL/min 85 80 15 5 0.8

mL/min 88 100 0 0 0.8 mL/min 91 100 0 0 0.8 mL/min 94 40 55 5 0.8 mL/min105 40 55 5 0.8

mL/min

Hypersil® C18 5μ column:

SolventsA: MethanolB: H2OC: 5% Acetic acid

Gradient

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES


2424



Time A(%) B(%) C(%) Flow rate

0 55 40 5 0.8 mL/min

96 95 0 5 0.8 mL/min 100 95 0 5 0.8 mL/min 105 55 40 5 0.8

mL/min 115 55 40 5 0.8

mL/min

Polaris® Amide C18 5μ column:

SolventsA: MethanolB: H2OC: 5% Formic acid

Gradient



2525





Thermo Finnigan LCQ Advantage® with DA detectorMode: electrospray ionization neg. modeSource voltage: 4.5 KvoltsCapillary voltage: -10vCapillary temp: 300C Sheath gas flow = 80% fullAux gas flow: 20% max. (no stream splitting)

MASS SPEC-MASS SPEC:(MS-MS)

INSTRUMENTATIONINSTRUMENTATION HPLC:

Thermo Separation Products® (Thermo Finnigan)

P4000 pumpSN 4000 ControllerSpectraSystem UV3000 detector UV 250-400nm; Chromquest software

2626



OD 330nm(mAU)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

0 30 4020 5010 60 70 80Retention Time (min)



More than 30 peaks

detectected

AC Baton(hulless)

2727



OD 330nm(mAU)

0

100

200

300

400

500

600

700

800

900

1000

0 30 4020 5010 60 70 80Retention Time (min)

90 100



More than 20 peaks

detectected

AC Baton(hulless)

2828



Parent ion [M-1]-

nn

-

RRRR

RR

OH

NH

O

OO

KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m2

KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m2

Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide

Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide

MS parameters optimized for

generation of [M-1] -

MS parameters optimized for

generation of [M-1] -

Daughter m2 - gives

molecular weight of both ring moieties

Daughter m2 - gives

molecular weight of both ring moieties

RR

Daughter ion m1 -

N

O

OO

-

RRDaughter m1

- gives molecular weight of the anthranilic moiety

Daughter m1 - gives

molecular weight of the anthranilic moiety

Parent ion [M-1]-

-

RRRR

nnRR

OH

NH

O

OO

Daughter ion m2 -

nn

RR

-

RR

RR

OH

NH

O

2929



OH

NH

O

OHO

R

DD EE FF

OH

NH

O

OHO

HO

RAA BB CC

OH

NH

O

OHO

R

OH

GG HH KK

OH

NH

O

OHO

HO

R

OH

AAAA BBBB CCCC

OH

NH

O

OHO

HO

R

OCH3

XX YY ZZ

R = HR = H R = OCH3R = OCH3 R = OHR = OH

STRUCTURES OF TYPE I AVENANTHRAMIDES

STRUCTURES OF TYPE I AVENANTHRAMIDES

3030



Rt (min) Amide

Rt (min) C18

0

0

20

20

40

40

60

60 80

80

100

STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES


DDEEFF

BBCCAA

KKGG

HHCCCC

AAAA BBBB

TYPE 1 AVENANTHRAMIDESSUMMARY

5 Different anthranilic acid amides with either p-coumaric, ferulic, or caffeic acids

SUMMARY

5 Different anthranilic acid amides with either p-coumaric, ferulic, or caffeic acids

5-OH anthranilic

4-OH anthranilic

anthranilic

4,5-di(OH) anthranilic

ZZXX YY

4-OH,5-OMe anthranilic

3131



OH

NH

O

OHO

R

R = HR = H R = OCH3R = OCH3 R = OHR = OH

LL MM NN

OH

NH

O

OHO

R

HO

OO PP QQ

OH

NH

O

OHO

R

OH

RR SS TT

OH

NH

O

OHO

R

OH

HO

OOOO PPPP QQQQ

OH

NH

O

OHO

R

OCH3

HO

UU VV WW

STRUCTURES OF TYPE II AVENANTHRAMIDES

STRUCTURES OF TYPE II AVENANTHRAMIDES

3232



Rt (min) Amide

Rt (min) C18

0

0

20

20

40

40

60

60 80

80

100



QQ

OOPP

MMNN

LL

QQQQOOOO

PPPP

4,5-di(OH) anthranilic

WWUU

VV

SSRRTT

SUMMARY

5 Different anthranilic acid amides with either avenalumic, 3’-OMe-avenalumic, or 3’-OH-avenalumic acids

TYPE 2 AVENANTHRAMIDESanthranilic

5-OH anthranilic

4-OH anthranilic

4-OH,5-OMe anthranilic

3333


Agroalimentaire CanadaAgroalimentaire CanadaAgroalimentaire CanadaAgroalimentaire CanadaAgri-Food CanadaAgri-Food CanadaAgri-Food CanadaAgri-Food CanadaAgriculture andAgriculture andAgriculture andAgriculture and Agriculture etAgriculture etAgriculture etAgriculture et


Rt (min) Amide

Rt (min) C18

0

0

20

20

40

40

60

60 80

80

100

KK

GG

HH

AA

BBCC

DD

EEFF

LL

MMNN

CCCCAAAA

BBBB

XX

YY

ZZ

OO

PPQQ

QQQQ

OOOOPPPP

VV

UU

WW

RR

SSTT

2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES

2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES

Based on:1) Rt in 2 solvents2) UV spectra3) MS pattern

Based on:1) Rt in 2 solvents2) UV spectra3) MS pattern

There are about 30 different avenanthramides not including dimeric forms

There are about 30 different avenanthramides not including dimeric forms

Each one identified by an alphabetic designation

Each one identified by an alphabetic designation

3434



Rt (min) Amide

Rt (min) C18

0

0

20

20

40

40

60

60 80

80

100

OD (AU)

0

0.5

1.0

1.5

2.0

2.5 Relative quantitative estimations:

Relative quantitative estimations:

Major Avenanthramides

Major Avenanthramides

A, B, CA, B, C

O, P, QO, P, Q

OO, PP, QQOO, PP, QQ

AA, BB, CCAA, BB, CC

AC Baton(hulless)

RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE…..

RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE…..

3535



CO

NC

EN

TR

AT

ION

IN

PP

M

0

50

100

200

300

400

500

VARIETIES / LINES AV AAV BAV C TOTAL

AV AAV BAV C TOTAL

3636 GENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELSGENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELS

Strong genotype x environment interaction as wellStrong genotype x environment interaction as well



TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS….

TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS….

AAFCAAFC

F. William CollinsVern BurrowsWinson OrrDwight ColleyNicole Fillion

F. William CollinsVern BurrowsWinson OrrDwight ColleyNicole Fillion

Synthesis ,GMP Purification, Analytical,Germplasm

USDA TUFTS UNIVERSITYUSDA TUFTS UNIVERSITY

Mohsen MedaniLiping LiuLigia Zubik

Melissa MarkoWeimin Guo

Mohsen MedaniLiping LiuLigia Zubik

Melissa MarkoWeimin Guo

Vascular Biology Lab:In vitro studies

Jeffrey BlumbergPaul MilburyChung-Yen ChenTing LiJennifer O’Leary

Jeffrey BlumbergPaul MilburyChung-Yen ChenTing LiJennifer O’Leary

Antioxidant Research Lab:In vivo studies

3737

purification, analysis, and structures of avenanthramides from oats: unique components for targeted...

Documents

structures of avenanthramides

types of avenanthramides

overview group separation

chromophoric group

uv absorbing components

uv interfering components

uv absorption max

hplc evaluation