antioxidant properties of rapeseed can be modified by cultivation and biological stress r. amarowicz...
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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.
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