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

4th International Conference and Exhibition on Food Processing & Technology

London 10-12 August 2015

Chemical structure of phenolic acids

Acid X Y

Protocatechuic H OH

Vanillic OCH 3 H

Syringic OCH 3 OCH 3

Gallic OH OH

p -Hydroxybenzoic H H

Acid X Y

p -Coumaric H H

Caffeic H OH

Ferulic H OCH 3

Sinapic OCH 3 OCH 3

X

OH COOH

Y

X

OH CH

Y

CH COOH

C OHOH

O

OCH3

CH3O

O

OHC OOH

O

OCH3

CH3

OCH2

OHOH

OH

C OOH

O

OCH3

CH3O

CH2 CH2 N+

CH3

CH3

CH3

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 F1

Characteristic of the cultivation conditions

Fertilization Control Intensive Spare

Phosphorus(Autumn)

Potassium(Autumn)

Nitrogen-Autumn- Spring I-Spring II-Total

Sulphur(Spring)

60 kg

120 kg

30 kg120 kg60 kg

210 kg

45 kg

80 kg

150 kg

30 kg120 kg80 kg

230 kg

60 kg

40 kg

60 kg

30 kg120 kg40 kg

190 kg

-

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 plant plantation was inoculated with spores of fungal Alternaria brassica.The seeds of stage of full maturity were analysed.

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

Extraction:

Phenolic compounds were extracted from the defatted seeds with 80% (v/v) aqueous methanol at 80o 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 oC in a rotary evaporator. The remaining water solution was lyophilised.

HPLC analysis of phenolic compounds

A Shimadzu HPLC system was employed:

LC-10ADVP pumpControler SCL-10AVPPhotodiode array detector UV-VIS SPD-M10AVP,Controler SCL-10AVP

Conditiones of separations:

Prepacked LUNA C8 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)

Control Intensive Spare0

10

20

30

40

50

60

70

53.949.8 53.2

Nelson F1

To

tal p

he

no

lics

(mg

/g)

Control Intensive Spare0

10

20

30

40

50

60

70

48.9 48.3 49.4

Castilla

To

tal p

he

no

lics

(mg

/g)

Control Intensive Spare55

56

57

58

59

60

61

62

60.9

59.4

57.1

California

To

tal p

he

no

lics

(mg

/g)

Control Intensive Spare0.3

0.4

0.5

0.468

0.417

0.469

California

TE

AC

(m

mo

l Tro

lox/

g)

Control Intensive Spare0.0

0.1

0.2

0.3

0.4

0.5

0.366 0.360 0.365

Castilla

TE

AC

(m

mo

l Tro

lox/

g)

Control Intensive Spare0.4

0.5

0.4110.418

0.432

Nelson F1

TE

AC

(m

mo

l Tro

lox/

g)

TEAC of the extracts (mmol Trolox/g)

Control Intensive Spare1.3

1.4

1.5

1.6

1.7

1.611.66

1.43

California

FR

AP

(m

mo

l Fe

2+

/g)

Control Intensive Spare0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

1.34 1.30 1.30

Castello

FR

AP

(m

mo

l Fe

2+

/g)

Control Intensive Spare0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

1.351.15

1.31

Nelson F1

FR

AP

(m

mo

l Fe

2+

/g)

FRAP of the extracts (mmol Fe2+/g)

Antiradical activity of the extracts against DPPH radical

0.0 0.2 0.4 0.6 0.8 1.00.0

0.2

0.4

0.6

0.8

1.0

1.2California

Control

Intensive

Spare

Content (mg/assay)

Ab

sorb

an

ce a

t 51

7 n

m

0.0 0.2 0.4 0.6 0.8 1.00.0

0.2

0.4

0.6

0.8

1.0

1.2Castilla

Control

Intensive

Spare

Content (mg/assay)

Ab

sorb

an

ce a

t 51

7 n

m

0.0 0.2 0.4 0.6 0.8 1.00.0

0.2

0.4

0.6

0.8

1.0

1.2Nelson F1

Control

Intensive

Spare

Content (mg/assay)

Ab

sorb

an

ce a

t 51

7 n

m

HPLC chromatograms of the extracts

Content of individual phenolic compounds in the extracts of California (mg/g)

Compound Control Intensive Spare

123456

80.1 ± 1.48.4 ± 0.35.6 ± 0.36.3 ± 0.4

-8.5 ± 0.5

81.9 ± 5.17.7 ± 0.65.1 ± 0.26.2 ± 0.3

-8.3 ± 0.2

76.2 ± 4.07.6 ± 0.65.1 ± 0.36.1 ± 0.3

-7.6 ± 0.5

Content of individual phenolic compounds in the extracts of Castilla (mg/g)

Compound Control Intensive Spare

123456

76.3 ± 3.47.1 ± 0.95.9 ± 0.55.9 ± 0.7

-6.0 ± 0.7

73.0 ± 5.67.1 ± 1.25.9 ± 1.05.2 ± 0.6

-5.7 ± 0.2

70.1 ± 1.46.4 ± 0.25.4 ± 0.55.5 ± 0.1

-6.4 ± 1.5

Content of individual phenolic compounds in the extracts of Nelson F1 (mg/g)

Compound Control Intensive Spare

123456

65.8 ± 3.04.0 ± 0.15.2 ± 0.13.8 ± 0.2

18.3 ± 0.53.8 ± 0.4

64.5 ± 3.32.5 ± 0.24.9 ± 0.23.9 ± 0.5

16.9 ± 1.03.2 ± 0.4

70.8 ± 5.33.1 ± 0.85.0 ± 0.64.4 ± 0.8

17.3 ± 0.93.5 ± 0.8

Content of total phenolics in the extracts and seeds

Control Incubated0

10

20

30

40

50

60

70

57.6

45.9

To

tal p

he

no

lics

(mg

/g o

f ext

ract

)

Inoculated Control Incubated6

8

10

8.38

9.68

Tot

al p

heno

lics

(mg/

g of

def

atte

d se

eds)

Inoculated

TEAC of the extracts and seeds

Control Incubated0.0

0.1

0.2

0.3

0.4

0.338000000000001

0.246

TE

AC

(m

mo

l Tro

lox/

g o

f ext

ract

)

Control Incubated0.05

0.05

0.05

0.0490000000000001

0.051

TE

AC

(m

mol

Tro

lox/

g of

def

atte

d se

eds)

InoculatedInoculated

FRAP of the extracts and seeds

Control Incubated0

100

200

300

400

500

600

700

800

747

507

FR

AP

(u

mo

l Fe

2+

/g o

f ext

ract

)

Inoculated Control Incubated106.4

106.6

106.8

107

107.2

107.4

107.6

107.8

108

108.2

108

107F

RA

P (

um

ol F

e2

+/g

of d

efa

tted

se

ed

s)

Inoculated

Antiradical activity of the extracts aginst DPPH radical

0.0 0.4 0.8 1.2 1.6 2.00.0

0.2

0.4

0.6

0.8

1.0

1.2

Control Incubated

Content (mg/assay)

Abs

orba

nce

at 5

17 n

m

Inoculated

HPLC chromatograms of the extracts

Content of individual phenolic compounds in rapeseed extracts (mg/g of extract)

Compound Control Inoculated

1 (sinapine)2345678

58.2 ± 2.23.3 ± 0.21.7 ± 0.21.6 ± 0.23.1 ± 0.2

11.2 ± 0.82.0 ± 0.12.1 ± 0.1

37.9 ± 1.82.3 ± 0.21.4 ± 0.10.7 ± 0.12.2 ± 0.17.0 ± 0.51.0 ± 0.11.1 ± 0.1

Content of individual phenolic compounds in rapeseeds (mg/g of defatted seeds)

Compound Control Inoculated

1 (sinapine)2345678

8.49 ± 0.410.48 ± 0.030.25 ± 0.010.24 ± 0.010.45 ± 0.021.63 ± 0.050.29 ± 0.010.30 ± 0.01

8.00 ± 0.390.48 ± 0.030.29 ± 0.010.16 ± 0.010.46 ± 0.021.47 ± 0.050.21 ± 0.010.23 ± 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 F1 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