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AVEQ

AVEQ. Analysis of Tocopherols/Tocotrienols and Avenanthramides in Oats WP 8 Rita Redaelli and Lena Dimberg. Tocopherols/ Tocotrienols. Tocopherols. Tocotrienols. Extraction of Tocopherols/Tocotrienols. Pisacane et al., 2004 Solvent: Methanol Flour/solvent ratio: 1 g/14 ml

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AVEQ

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  1. AVEQ Analysis of Tocopherols/Tocotrienols and Avenanthramides in Oats WP 8 Rita Redaelli and Lena Dimberg

  2. Tocopherols/ Tocotrienols Tocopherols Tocotrienols

  3. Extraction of Tocopherols/Tocotrienols Pisacane et al., 2004 Solvent: Methanol Flour/solvent ratio: 1 g/14 ml Time: 2*1 min Solubilized: Methanol (or hexane/1,4-dioxane, 95/5) Peterson et al., 2007 Solvent: Methanol (or saponified KOH in ethanol + antioxidant) Flour/solvent ratio: 1 g/28 ml Time:2* 20 min Solubilized: Hexane (or hexane/2-propanol, 99.5/0.5) SLU-lab Solvent - Methanol Flour/solvent ratio:(1 g/21 ml) Time: 3*20 min Solubilized - Hexane Nielsen and Hansen, 2008 Solvent: Hexane Flour/solvent ratio: 1 g/30 ml Time: 1* 10 min Solubilized: Hexane

  4. HPLC analysis of Tocopherols/ Tocotrienols • Peterson et al., 2007 • NP- column: 250 mm • Isocratic solvent: • Hexane/2-propanol(99.5/0.5 v/v) • Flow: 1 ml/min • Time:? • Fluorescence detector: • ex 295 nm; em 330 nm • References: α-tocopherol • Pisacane et al., 2004 • NP- column: 250 mm • Isocratic solvent: • Hexane/1,4-dioxane(95:5 v/v) • Flow: 1,5 ml/min • Time: 11 min • Fluorescence detector: • ex 294 nm; em 326 nm • Reference: 8 isomers • Nielsen and Hansen, 2008 • NP- column: 125 mm • Isocratic solvent: • Hexane/ethylacetate/acetic acid(94.6/3.6/1.8) • Flow: 1 ml/min • Time: 16 min • Fluorescence detector: • ex 290 nm; em 330 nm • References: 8 isomers • SLU-lab • NP- column: 250 mm • Isocratic solvent: • Hexane/1,4-dioxane(96:4 v/v) • Flow: 1.5 ml/min • Time: 21 min • Fluorescence detector: • ex 294 nm; em 326 nm • Reference: 8 isomers

  5. NP-HPLC OF TOCOLS Pisacane et al. 2004

  6. Avenanthramides R= H, OH or OCH3 n= 1 or 2

  7. Avenanthramides(ca 30 different compounds in oats)

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

  9. AvenanthramidesUV-spectra

  10. Extraction of Avenanthramides Collins Solvent - 80% Ethanol (acidified) Flour/solvent ratio:(1 g/25 ml) Temperature: 100 C Time: 3*20 min Purification: Octyl-sepharose, Sephadex-LH20 Solubilized – 50% Ethanol Matsukawa et al., 2000 Solvent - Methanol Flour/solvent ratio:(1 g/30 ml) Temperature: ambient? Time: 3*? min Solubilized -Methanol SLU-lab Solvent - 80% Ethanol (pH 2) Flour/solvent ratio:(1 g/21 ml) Temperature: 50 C Time: 3*20 min Solubilized - Methanol Wise et al., 2009 Solvent: 80%Ethanol Flour/solvent ratio: 1 g/30 ml Temperature: 50 C Time: 3*20 min Solubilized: Methanol

  11. HPLC analysis of Avenanthramides • Collins et al., 2004 • RP C-18 column: ? mm • Solvent: A. 5% acetic acid • B. methanol • Gradient: 40 to 100% B in A for 88 min • Flow: 0.8 ml/min • Time: 50 min • Diod array: 330 nm • Reference: ? compounds • Matsukawa et al., 2000 • RP C-18 column: 150 mm • Solvent: A. 0.5% TFA, • B. acetonitrile • Gradient: 15 to 30% B in A for 60 min • Flow: ? ml/min • Time: ? min • Diod array: 340 nm • Reference ? compounds • SLU-lab • RP C-18 column: 125 mm • Solvent: A. = 0.01 M formic acid • (pH 2.9), 5% acetonitrile • B. acetonitrile • Gradient: 5 to 38% B in A in 35 min • Flow: 1 ml/min • Time: 30 min • Diod array: 340 nm • Reference: 17 compounds • Wise et al., 2009 • RP C-18 : 50 mm • Solvent: A. 0.1% formic acid, 5% acetonitrile • B. acetonitrile, 0.1% formaric acid • Gradient: 13-30% B in A for 20 min • Flow: 1 ml/min • Time: 20 min • Diode array detector: 280 and 330 nm • References: 3 compounds

  12. Analysis of Avenanthramides LC-MS a UV detection, 340 nm b-g Selective ion monitoring (SIM) detection [M+H]+ for 2c, 2p, 2f, 3f, 2pd and 2fd, respectively

  13. Molecular weights of avenanthramides

  14. Effect of germination

  15. Thank you

  16. Antioxidant activity of different avenanthramides

  17. Avenanthramides in crown rust susceptible and resistant cultivars

  18. Radical scavenging activity of different avenanthramides 1 min 5 min

  19. Radical scavenging activity of different avenanthramides 1 min 5 min

  20. HPLC-chromatogram (UV-detection 340 nm) -oat extract and reference compounds-

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