Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted Nutraceutical Applications F. William Collins Eastern Cereals and Oilseeds Research Centre Ottawa, ON. 8th IOC Minneapolis, MN, June 2008

Purification, Analysis, and Structures of Avenanthramides from Oats 2 OVERVIEW  Need for improved methods of analysis  Group separation for reproducibility  Co-extracted compounds and their effects on quantitation  UV Spectral properties of avenanthramides  2-D HPLC-MSMS techniques to map total avenanthramide diversity

NEED FOR IMPROVED METHODS 3 NEED FOR IMPROVED METHODS  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  Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more accurate/robust analytical techniques

Purification, Analysis, and Structures of Avenanthramides from Oats 4 SUMMARY OF PROCEDURE Stabilization and extraction with aqueous EtOH 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-20 4. Concentration of avenanthramides 5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A

THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS… 5 Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported 2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported Anthranilic acid (2-aminobenzoic) p-Coumaric acid (4-hydroxycinnamic) + = AVENANTHRAMIDE: TYPE I

…..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID 6 Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported Avenalumic acid 5-(4-hydroxyphenyl)-2,4-pentadienoic acid + 9' 10' 8' 7' 2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported Anthranilic acid (2-aminobenzoic) = AVENANTHRAMIDE: TYPE 2

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 7 10 g ground oats Procedure is scaleable 1. 75 mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH Denatures enzymes (e.g. peroxidases, esterases, etc) Stir for 20 min with ambient cooling and decant into graduated glass column Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties 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” Percolation is an extremely efficient extraction method HPLC evaluation at this point, detection at 330nm Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent

HPLC Profiles During Group Separation Of Avenanthramides... 8 5 10 15 20 25 30 35 40 45 50 55 60 65 80 100 120 OD 330nm (mAU) Time (min) VAO 48 (hulless) Avenacosylate peaks (in part) Crude EtOH extract “Prolamine” peaks (in part) “Glycolipid” peaks (in part) “Phosphatide” peaks (in part) Avenanthramide zone

AVENACOSYLATES: UV Interfering Components from Oats 9 There are 3 major classes of avenacosylates in 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

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 10 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 Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent Collins et al, US Patent #6,495,140 (2002)

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 11 Re-suspend in acidified 50% EtOH and transfer with washings to top of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% 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 Elute with 3xVb acidified 50% EtOH Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH HPLC evaluation at this point, detection at 330nm 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 Profiles During Group Separation Of Avenanthramides... 12 5 10 15 20 25 30 35 40 45 50 55 60 65 80 100 120 OD 330nm (mAU) Time (min) VAO 48 (hulless) Crude EtOH extract after removal of lipophilics “flavone glycosides” “Prolamine” peaks (in part) “Avenacins” Avenanthramide zone luteolin aglycone apigenin aglycone tricin aglycone

Other UV Absorbing Components from Oats: The Avenacins 13 Triterpene saponins with a chromophoric group with UV absorption max at ~ 325 nm (A-1, B-1)

Other UV Absorbing Components from Oats: Flavones 14 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%?)

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 15 Re-suspend in acidified 50% EtOH and transfer with washings to top of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% 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 Elute with 3xVb acidified 50% EtOH Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH HPLC analysis to look at profile 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

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 16 Re-suspend in acidified 50% EtOH and transfer with washings to top of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% 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 Elute with 3xVb acidified 50% EtOH Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH HPLC analysis to look at profile 3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound Elute with 2xVb acidified 40% EtOH to remove polar components

EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES... 17 All avenanthramides are not bound to Sephadex LH-20 in 95% EtOH 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 Procedure results in base-line resolution of all the avenanthramides and can be easily scaled up to gram quantities HPLC-rapid scan (“diode array”) HPLC-diode array-MSMS Molecular Weight Range scanned between 100-700 daltons Can also use different HPLC column:solvent configurations

HPLC Profiles During Group Separation Of Avenanthramides... 18 5 10 15 20 25 30 35 40 45 50 55 60 65 80 100 120 OD 330nm (mAU) Time (min) VAO 48 (hulless) Final purified avenanthramide extract Avenanthramide zone Flat base line for ease of integration

Comparison of HPLC Profiles During Separation Of Avenanthramides... 19 Avenanthramide zone 5 10 15 20 25 30 35 40 45 50 55 60 65 80 100 120 Time (min) Crude EtOH extract After Octyl Sepharose After LH-20

Spectral analyses of Avenanthramides 20 Spectral analyses of Avenanthramides 5 10 15 20 25 30 35 40 45 50 55 60 65 80 100 120 OD 330nm (mAU) VAO 48 (hulless) Time (min) High degree of peak resolution facilitates spectral analysis and identification

Spectral analyses of avenanthramides 21 Spectral analyses of avenanthramides OD 330nm (mAU) (mAU) B m/z = 329 Retention Time (min) 250 300 350 275 325 375 λ (nm) 400 200 100 (mAU)

Spectral analyses of avenanthramides 22 Spectral analyses of avenanthramides OD 330nm (mAU) m/z = 355 P Retention Time (min) 250 300 350 275 325 375 λ (nm) 400 200 100 (mAU)

Spectral analyses of avenanthramides 23 Spectral analyses of avenanthramides 300s B m/z = 329 335 350s All Type I avenanthramides show a shoulder between 290 and 310nm m/z = 355 P 360 340s All Type II avenanthramides lack this shoulder

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES 24 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/min 105 40 55 5 0.8 mL/min Hypersil® C18 5μ column: Solvents A: Methanol B: H2O C: 5% Acetic acid Gradient

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES 25 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: Solvents A: Methanol B: H2O C: 5% Formic acid Gradient

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES 26 INSTRUMENTATION HPLC: Thermo Separation Products® (Thermo Finnigan) P4000 pump SN 4000 Controller SpectraSystem UV3000 detector UV 250-400nm; Chromquest software Thermo Finnigan LCQ Advantage® with DA detector Mode: electrospray ionization neg. mode Source voltage: 4.5 Kvolts Capillary voltage: -10v Capillary temp: 300C Sheath gas flow = 80% full Aux gas flow: 20% max. (no stream splitting) MASS SPEC-MASS SPEC: (MS-MS)

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES 27 OD 330nm (mAU) 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 30 40 20 50 10 60 70 80 Retention Time (min) AC Baton (hulless) More than 30 peaks detectected

HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES 28 100 200 300 400 500 600 700 800 900 1000 OD 330nm (mAU) AC Baton (hulless) More than 20 peaks detectected 10 20 30 40 50 60 70 80 90 100 Retention Time (min)

KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m2 29 MS parameters optimized for generation of [M-1] - Parent ion [M-1]- n - R Parent ion [M-1]- - R n Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide R Daughter ion m1 - - Daughter ion m2 - n R - Daughter m1 - gives molecular weight of the anthranilic moiety Daughter m2 - gives molecular weight of both ring moieties

STRUCTURES OF TYPE I AVENANTHRAMIDES 30 STRUCTURES OF TYPE I AVENANTHRAMIDES R = H R = OCH3 R = OH D E F A B C G H K AA BB CC X Y Z

STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES 31 TYPE 1 AVENANTHRAMIDES 20 40 60 80 100 SUMMARY 5 Different anthranilic acid amides with either p-coumaric, ferulic, or caffeic acids anthranilic Rt (min) Amide Rt (min) C18 5-OH anthranilic 4-OH anthranilic 4-OH,5-OMe anthranilic 4,5-di(OH) anthranilic

STRUCTURES OF TYPE II AVENANTHRAMIDES 32 STRUCTURES OF TYPE II AVENANTHRAMIDES R = H R = OCH3 R = OH L M N O P Q R S T OO PP QQ U V W

STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES 33 TYPE 2 AVENANTHRAMIDES 20 40 60 80 100 SUMMARY 5 Different anthranilic acid amides with either avenalumic, 3’-OMe-avenalumic, or 3’-OH-avenalumic acids Rt (min) Amide Rt (min) C18 5-OH anthranilic 4-OH anthranilic 4,5-di(OH) anthranilic 4-OH,5-OMe anthranilic

2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES 34 Based on: 1) Rt in 2 solvents 2) UV spectra 3) MS pattern 20 40 60 80 100 Rt (min) Amide Rt (min) C18 K G H A B C D E F L M N CC AA BB X Y Z O P Q QQ OO PP V U W R S T There are about 30 different avenanthramides not including dimeric forms Each one identified by an alphabetic designation Agri-Food Canada Agriculture and Agriculture et Agroalimentaire Canada

RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE….. 35 Rt (min) Amide OD (AU) 0.5 1.0 1.5 2.0 2.5 AC Baton (hulless) Relative quantitative estimations: 20 40 60 80 100 Major Avenanthramides A, B, C O, P, Q OO, PP, QQ AA, BB, CC Rt (min) C18

GENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELS 36 CONCENTRATION IN PPM 50 100 200 300 400 500 VARIETIES / LINES AV A AV B AV C TOTAL Strong genotype x environment interaction as well

TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS…. 37 USDA TUFTS UNIVERSITY Mohsen Medani Liping Liu Ligia Zubik Melissa Marko Weimin Guo Vascular Biology Lab: In vitro studies AAFC F. William Collins Vern Burrows Winson Orr Dwight Colley Nicole Fillion Synthesis , GMP Purification, Analytical, Germplasm Jeffrey Blumberg Paul Milbury Chung-Yen Chen Ting Li Jennifer O’Leary Antioxidant Research Lab: In vivo studies