ANTIOXIDANT PROPERTIES OF RAPESEED CAN BE MODIFIED BY

ANTIOXIDANT PROPERTIES OF RAPESEED CAN BE MODIFIED BY CULTIVATION AND BIOLOGICAL STRESS R. Amarowicz Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland 4 th International Conference and Exhibition on Food Processing & Technology London 10 -12 August 2015

Chemical structure of phenolic acids

Sinapic acid Sinapine Glucopyranosyl sinapate

The major objective of the present study was to investigate the effect of cultivation (different level of fertilization) and action of pathogen fangus on the rapessed phenolic compounds present in the extract and their antioxidant properties.

Cultivars: California, Castilla, Nelson F 1 Characteristic of the cultivation conditions Fertilization Control Intensive Spare Phosphorus (Autumn) 60 kg 80 kg 40 kg Potassium (Autumn) 120 kg 150 kg 60 kg 30 kg 120 kg 60 kg 210 kg 30 kg 120 kg 80 kg 230 kg 120 kg 40 kg 190 kg 45 kg 60 kg - Nitrogen -Autumn - Spring I -Spring II -Total Sulphur (Spring)

Effect of pathogen: Cultivar: Hybryda 1 Green house of the University of Warmia and Mazury Cultivation in the vason (volum of 9 l). During the phase of budding plantation was inoculated with spores of fungal Alternaria brassica. The seeds of stage of full maturity were analysed.

Extraction: Phenolic compounds were extracted from the defatted seeds with 80% (v/v) aqueous methanol at 80 o C for 15 min at a solid to solvent ratio of 1: 10 (w/v). Extraction was carried out in dark-colored flakes using a shaking water bath. The extraction was repeated twice more, supernatants combined and acetone evaporated under vacuum at 40 o. C in a rotary evaporator. The remaining water solution was lyophilised. Chemical analysis: Total phenolics (Folina – Ciocalteu’a phenol reagent) Antiradical activity against DPPH radical (Yen i Chen, 1995) Antiradical actiovity against ABTS cation radical (TEAC) (Re et al. , 1999) FRAP (Prior et al. , 2005) RP-HPLC

HPLC analysis of phenolic compounds A Shimadzu HPLC system was employed: LC-10 ADVP pump Controler SCL-10 AVP Photodiode array detector UV-VIS SPD-M 10 AVP, Controler SCL-10 AVP Conditiones of separations: Prepacked LUNA C 8 column (5μm, 4. 6 x 250 mm; Phenomenex) A gradient: A - water-methanol (90: 10; v/v) with 1. 25% o-phosphoric acid, B - methanol with 0. 1% o-phosphoric acid; linear gradient from 0 to 60% B for 50 min. Flow rate – 1 ml/min; injection volume 20 μl; the detector was set at 330 nm.

Content of total phenolics in the extracts (mg/g) 66 California 70 70 Castilla Nelson F 1 62 60 58 56 54 52 Control Intensive Spare 60. 9 59. 4 57. 1 65 65 Total phenolics (mg/g) 64 60 55 50 45 Control Spare 48. 9 Intensive 48. 3 49. 4 60 55 50 45 Control Intensive 53. 9 49. 8 Spare 53. 2

TEAC of the extracts (mmol Trolox/g) 0. 5 California 0. 5 Castilla Nelson F 1 0. 3 0. 2 0. 366 0. 360 0. 365 TEAC (mmol Trolox/g) 0. 4 0. 1 0. 0 0. 4 Control Spare 0. 468 Intensive 0. 417 0. 469 Control Intensive Spare 0. 3 Control Intensive 0. 411 0. 418 Spare 0. 432

FRAP of the extracts (mmol Fe 2+/g) 1. 7 California 1. 8 Castello 1. 8 1. 7 1. 6 1. 5 1. 4 FRAP (mmol Fe 2+/g) 1. 7 1. 6 1. 5 1. 4 1. 2 1. 0 0. 8 0. 6 1. 35 1. 15 1. 31 0. 4 1. 3 1. 4 Nelson F 1 0. 2 1. 3 Control Spare 1. 61 Intensive 1. 66 1. 43 1. 2 Control Spare 1. 34 Intensive 1. 30 0. 0 Control Intensive Spare

Antiradical activity of the extracts against DPPH radical 1. 2 California Absorbance at 517 nm 1. 0 0. 8 0. 6 0. 4 Control 0. 2 Intensive Spare 0. 0 0. 2 0. 4 0. 6 0. 8 Content (mg/assay) 1. 0 1. 2 Absorbance at 517 nm 1. 2 Castilla 1. 0 0. 8 0. 6 0. 4 Control Intensive 0. 2 Spare 0. 0 0. 2 0. 4 0. 6 0. 8 Content (mg/assay) Nelson F 1 1. 0 0. 8 0. 6 0. 4 Control Intensive 0. 2 Spare 0. 0 0. 2 0. 4 0. 6 0. 8 Content (mg/assay) 1. 0

HPLC chromatograms of the extracts

Content of individual phenolic compounds in the extracts of California (mg/g) Compound Control Intensive Spare 1 2 3 4 5 6 80. 1 ± 1. 4 8. 4 ± 0. 3 5. 6 ± 0. 3 6. 3 ± 0. 4 8. 5 ± 0. 5 81. 9 ± 5. 1 7. 7 ± 0. 6 5. 1 ± 0. 2 6. 2 ± 0. 3 8. 3 ± 0. 2 76. 2 ± 4. 0 7. 6 ± 0. 6 5. 1 ± 0. 3 6. 1 ± 0. 3 7. 6 ± 0. 5

Content of individual phenolic compounds in the extracts of Castilla (mg/g) Compound Control Intensive Spare 1 2 3 4 5 6 76. 3 ± 3. 4 7. 1 ± 0. 9 5. 9 ± 0. 5 5. 9 ± 0. 7 6. 0 ± 0. 7 73. 0 ± 5. 6 7. 1 ± 1. 2 5. 9 ± 1. 0 5. 2 ± 0. 6 5. 7 ± 0. 2 70. 1 ± 1. 4 6. 4 ± 0. 2 5. 4 ± 0. 5 5. 5 ± 0. 1 6. 4 ± 1. 5

Content of individual phenolic compounds in the extracts of Nelson F 1 (mg/g) Compound Control Intensive Spare 1 2 3 4 5 6 65. 8 ± 3. 0 4. 0 ± 0. 1 5. 2 ± 0. 1 3. 8 ± 0. 2 18. 3 ± 0. 5 3. 8 ± 0. 4 64. 5 ± 3. 3 2. 5 ± 0. 2 4. 9 ± 0. 2 3. 9 ± 0. 5 16. 9 ± 1. 0 3. 2 ± 0. 4 70. 8 ± 5. 3 3. 1 ± 0. 8 5. 0 ± 0. 6 4. 4 ± 0. 8 17. 3 ± 0. 9 3. 5 ± 0. 8

Content of total phenolics in the extracts and seeds 60 50 40 30 57. 6 45. 9 20 10 0 Control Incubated Inoculated Total phenolics (mg/g of defatted seeds) Total phenolics (mg/g of extract) 70 10 8 6 9. 68 4 8. 38 2 0 Control Incubated Inoculated

TEAC of the extracts and seeds 0. 05 0. 3 0. 2 0. 338000 0000 1 0. 246 0. 1 0. 0 Control Incubated Inoculated TEAC (mmol Trolox/g of defatted seeds) TEAC (mmol Trolox/g of extract) 0. 4 0. 05 0. 04900000 Control 00000001 Incubated Inoculated 0. 051

800 109. 5 700 109 600 500 400 747 300 507 200 100 0 Control Incubated Inoculated FRAP (umol Fe 2+/g of defatted seeds) FRAP (umol Fe 2+/g of extract) FRAP of the extracts and seeds 108. 5 108 107. 5 107 106. 5 106 105. 5 Control 108 Incubated Inoculated 107

Antiradical activity of the extracts aginst DPPH radical Absorbance at 517 nm 1. 2 Control 1. 0 Inoculated Incubated 0. 8 0. 6 0. 4 0. 2 0. 0 0. 4 0. 8 1. 2 Content (mg/assay) 1. 6 2. 0

HPLC chromatograms of the extracts

Content of individual phenolic compounds in rapeseed extracts (mg/g of extract) Compound Control Inoculated 1 (sinapine) 2 3 4 5 6 7 8 58. 2 ± 2. 2 3. 3 ± 0. 2 1. 7 ± 0. 2 1. 6 ± 0. 2 3. 1 ± 0. 2 11. 2 ± 0. 8 2. 0 ± 0. 1 2. 1 ± 0. 1 37. 9 ± 1. 8 2. 3 ± 0. 2 1. 4 ± 0. 1 0. 7 ± 0. 1 2. 2 ± 0. 1 7. 0 ± 0. 5 1. 0 ± 0. 1 1. 1 ± 0. 1

Content of individual phenolic compounds in rapeseeds (mg/g of defatted seeds) Compound Control Inoculated 1 (sinapine) 2 3 4 5 6 7 8 8. 49 ± 0. 41 0. 48 ± 0. 03 0. 25 ± 0. 01 0. 24 ± 0. 01 0. 45 ± 0. 02 1. 63 ± 0. 05 0. 29 ± 0. 01 0. 30 ± 0. 01 8. 00 ± 0. 39 0. 48 ± 0. 03 0. 29 ± 0. 01 0. 16 ± 0. 01 0. 46 ± 0. 02 1. 47 ± 0. 05 0. 21 ± 0. 01 0. 23 ± 0. 01

Conclusions: • All the rapeseed extracts were characterized by the high content of phenolic compound (phenolic acids). • Strong antioxidant activities of the rapeseed extracts were observed and assayed using different chemical methods. • In the seeds of Nelson F 1 we found sinapic acid derivative which was absent in the seeds of California and Castilla. • The weak effect of fertilization on the antioxidant properties was observed. However, it was different for the individual rapeseed cultivars and the chemical methods used for the measure the antioxidant activity. • In the extract of the seeds treated by Alternaria brassica the content of phenolic compounds as well as antioxidant activity were lower than in the extracts of the untreated seeds.

Thank you