Prooxidant Mechanism of bCarotene in Lipid Oxidation

1
Prooxidant Mechanism of b-Carotene in Lipid
Oxidation

• D. B. Min
• Department of Food Science and Technology
• The Ohio State University
• Columbus, Ohio 43210

2
Effects of Lipid Oxidation
? Flavor Quality Consumer
Acceptance
? Nutritional Quality Essential
Fatty Acids
Vitamins
? Health Growth Retardation
Heart Diseases
Carcinogens
3
Effects of Reactive Oxygen Species on Health

• Dermatitis
• Psoriasis
• Skin

• Cataractogenesis
• Retinal damage
• Eye

• Hepatitis
• Liver injury
• GI

• Heart attack
• Heart

• Arthritis
• Joints

• Periodontis
• Teeth

Reactive Oxygen Species

• Lung
• Asthma
• Hyperoxia

• Multiorgan failure
• Aging
• Cancer

• Vessels
• Atherosclerosis
• Vasospasms

• Brain
• Trauma
• Stroke

4
Molecular Orbital of Triplet Oxygen
Molecular Orbital
s

Atomic Orbital
Atomic Orbital
p

p

p
p
2Pz
2Py
2Px
2Px
2Py
2Pz
s
Energy
s

2S
2S
s
s

1S
1S
s
5
Mechanisms of Autoxidation
14 13 12 11 10 9
C
H
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
2
2
3
3
- ? H
13 12 11 10 9
(
C
H
)
C
H
C
H
R
C
H
C
H
C
H
C
H
C
H
2
4
3
2
?
3O2
13 12 11 10 9
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
2
4
3
2
O
O
? H
?
6
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
- ? OH
H
13 12 11 10 9
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
O
?
12 11 10 9
O
?
C
C
H
C
H
C
H
C
H
C
H
R

C
C
H
(
C
H
)
H
2
3
2
3
2
H
? H
C
H
(
C
H
)
C
H
3
2
3
3
7
Molecular Orbital of Singlet Oxygen
Molecular Orbital
Atomic Orbital
s
Atomic Orbital

p

p

p
p
2Pz
2Py
2Px
2Px
2Py
2Pz
s
Energy
s

2S
2S
s
s

1S
1S
s
8
Singlet Oxygen Formation by Sensitizer
Excited State
1Sensitizer
ISC, K1- 20?108/sec
hv
3Sensitizer
Excited
K1- 3?109/sec
K 2?108/sec
3O2
K10- 104/sec
Singlet Oxygen
Ground State
1Sensitizer
Chlorophyll, Myoglobin, Riboflavin, and Red No 3
9
2-Pentenylfuran Formation from Linolenic Acid in
Soybean Oil
1O2
C
H
C
H
(
C
H
)
C
O
O
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
6
2
3
2
2
O
- O
H
O
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
O
1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
2
2

O
O
O
H
- H2O
C
H
C
H
C
H
C
H
C
H
3
2
2
O
10
Physical Quenching Mechanism of ?- Carotene on
Singlet Oxygen
1O2 1?-Carotene 3O2 3?-Carotene
3 ?-Carotene
1?-Carotene
Energy Transfer
Radiationless Energy Transfer
11
R, RO, O2- HO, HOO, H2O2 ROO ,
1O2
(b-Carotene)
Nutraceuticals
R, RO, O2- HO HOO H2O2, ROO, 1O2,
12
?-Carotene
13
Introduction

• b-Carotene has been known as an antioxidant in
  lipid oxidation by quenching singlet oxygen.
• b-Carotene scavenges free radicals of oil and
  acts as an antioxidant in the oxidation of oil.
• 3. The free radical scavenging mechanism of
  b-carotene has been reported due to the electron
  transfer and / or hydrogen transfer mechanism.
• 4. The free radical scavenging mechanism of
  b-carotene has been controversial due to the lack
  of scientific information.

14
Objectives

• To study the free radical mechanism of b-carotene
  in 2, 2-diphenylpicryl hydrazyl (DPPH) by visible
  and NMR spectroscopy
• To study the effects of b-carotene on the
  oxidation of soybean and palm oils

15
Volatile Compound Formation from Linoleic Acid
O
13 12 11 10 9
CH3 (CH2)3 -CH2 -CH CH-CH2 -CH CH-(CH2)7 -C-OH
_
H
O
CH3 (CH2)3 -CH2 -CH CH-CH CH-CH-(CH2)7 -C-OH

3O2
O
CH3 (CH2)3 -CH2 -CH CH-CHCH-CH-(CH2)7 -C-OH
O-O
H
CH3 (CH2)3 -CH2-CHCH-CHCH-CH-(CH2)7 -C-OH
O-O-H
_
O H
O
CH3 (CH2)3 -CH2 - CHCH-CHCH-C-H
16
Hydrogen Donation from Antioxidant
.
.
.
- H
R..., ROO.
RH, ROOH
O
C
(
C
H
)
3
3
.
O
C
H
3
17
2, 2-Diphenyl-1-picryl-hydrazyl radical (DPPH)
N
.
.H
.H
.
N
N
O
O
N
2
2
N
O
2
No Absorption at 517 nm
Absorption at 517 nm
18
Reaction between 2, 2-Diphenyl-1-picryl-hydrazyl
radical and b-Carotene
.

No Absorption at 517 nm
b-Carotene
(Absorption at 517 nm)
DPPH
19
2, 2-Diphenyl-1-Picryl-Hydrazyl Radical
N
.
.
.
. or .H
.H
or
.
N
.
N
O
O
N
2
2
N
O
2
No Absorption at 517 nm
Absorption at 517 nm
20
Hydrogen or Electron Donation of b-Carotene
H
H
- ? H
?
H
H
H
- ?
H

?
- ?
H
21
Effects of b-Carotene on the Absorbance of DPPH
0.7

nm


0.6


0.5



0.00 mM
0.4



0.06 mM
0.3


Absorbance of DPPH at 517

0.12 mM
0.2







0.1

0.20 mM

0


0

100

200

300



Storage Time in Minutes
22
NMR Spectra of BHA and DPPH for 0, 30, and 60
Minutes

Peak from BHA


0 min

N
30 min

.H
.
N
New peak


N
O
O
N
2
2
N
O
60 min

2


ppm

8.0

7.0

6.0

5.0

4.0

23
NMR Spectra of b-Carotene and DPPH for 0, 30, and
60 Minutes

0 min

N
.H
.
N
30 min

N
O
O
N
2
2
N
O
2
60 min

TMS


ppm

0

1

2

3

4

5

6

7

8

9

10

24
Effects of Dark and Light on Absorbance of DPPH

100

Dark

80

Light
Absorbance of DPPH at 517 nm ()

60



40


20







0
30
60
90
120
150
Time in Minutes

25
Effects of Solvent Polarity on the Absorbance of
DPPH

100


Methanol
80

Acetone
Absorbance of DPPH at 517 nm ()

60



40


20







0
30
60
90
120
150
Storage Time in Minutes

26
Effects of Oxygen Content on the Absorbance of
DPPH

1.0


0.8

1 Oxygen
0.6

Absorbance of DPPH at 517 nm
0.4

21 Oxygen

0.2


0

0
30
60
90




Storage Time in Minutes

27
Effects of pH on the Absorbance of DPPH

1.0


0.9

0.8

0.7

0.6

Absorbance of DPPH at 517 nm
0.5

pH 4

0.4

pH 5

pH 6

0.3

pH 7

pH 8

0.2

pH 9

0.1

pH 10

0
0

30

60

90

120

Storage Time in Minutes
28
Factors Affecting the Absorbance of DPPH

• The absorbance of DPPH at 517 nm is changed by
  light exposure, polarity of solvent, oxygen
  content, and pH
• The absorbance of DPPH at 517 nm does not give a
  reliable information for the evaluation of
  antioxidant
• To confirm the antioxidant activity evaluation of
  a compound by DPPH Absorbance method, another
  antioxidant analytical method should be used.

29
Headspace Oxygen Analyses by Gas- Chromatography
for Oil Oxidation
The higher the headspace oxygen depletion is, the
greater the oil oxidation is.
30
Effects of BHA and b-Carotene on Headspace Oxygen
of Soybean Oil.

22




20


18

Headspace Oxygen Content ()
Soybean oil BHA
16

14

Soybean oil
12

Soybean oil
b-Carotene
10

0

20

40

60

80

100

Storage Time in Hours

31
Effects of ?-Carotene on the Peroxide Values of
Soybean Oil without Chlorophyll under Light
15.0
b-Carotene 25 PPM
b-Carotene 35 PPM
12.5
b-Carotene 50 PPM
10.0
7.5
Peroxide Value
5.0
2.5
0
0
4
8
12
16
20
24
Storage Time in Hours
32
Effects of ?-Carotene on the Headspace O2
Depletion of Soybean Oil without Chlorophyll
under Light
5
b-Carotene 25 PPM
(u mole)
b-Carotene 35 PPM
4
b-Carotene 50 PPM
3
2
Headspace O2 depletion
1
0
24
16
0
4
8
12
20
Storage Time in Hours
33
Effects of ?-Carotene on Peroxide Values of
Soybean Oil with Chlorophyll under Light
15.0
b-Carotene 0 ppm
b-Carotene 5 ppm
b-Carotene 25 ppm
12.5
b-Carotene 50 ppm
10.0
7.5
Peroxide Value
5.0
2.5
0.0
0
4
8
12
16
20
24
Storage Time in Hours
34
Effects of ?-Carotene on the Headspace O2
Depletion of Soybean Oil with Chlorophyll under
Light
b-Carotene 0 ppm
5

(umoles)
b-Carotene 5 ppm
B-Carotene 25 ppm
4
B-Carotene 50 ppm
3
2
Headspace O2 depletion
1
0
24
16
20
8
12
4
0
Storage Time in Hours
35
Standard One-Electron Reduction Potential
Compounds E (mV)
HO H / H2O 2310 RO H /
ROH 1600 ROO H / ROOH 1000 R H /
RH 600 Catechol H / Catechol 530 ?-
Tocopheroxyl H / ?- Tocopherol 500 Ascorbate-
H / Ascorbate 282

36
One-Electron Reduction Potential of b-Carotene
- e
(1060 mV)
?

37
Standard One-Electron Reduction Potential
Compounds E (mV)
HO H / H2O 2310 RO H /
ROH 1600 ROO H / ROOH 1000 R H /
RH 600 Catechol H / Catechol 530 ?-
Tocopheroxyl H / ?- Tocopherol 500 Ascorbate-
H / Ascorbate 282

38
Conclusions

• b-Carotene is an antioxidant in singlet oxygen
  oxidation of oil by quenching singlet oxygen
• 2. b-Carotene scavenges free radicals by electron
  donation only. It did not donate hydrogen to
  lipid free radicals. Therefore, b-carotene is
  not an antioxidant
• Headspace oxygen depletion and peroxide value
  results indicated that b-carotene acted as a
  prooxidant in soybean oil oxidation
• b-Carotene has higher one electron reduction
  potential (1060 mV) than fatty acid (600mV).
  Therefore, b-carotene act as a prooxidant.

39
(No Transcript)
40
Degradation of b-Carotene at 25, 37, and 55 C
m
g
700
600
500
400
300
25 C
200
37 C
100
55 C
0
0
3
6
9
12
Storage Time in Weeks
41
Headspace Oxygen Depletion of Soybean Oil after
8 Days at 55?C in the Dark
6.0
5.9
5.8
Headspace Oxygen Depletion (umole)
5.7
5.6
5.5
Soybean Oil
Soybean Oil 50 ppm Oxidized b-Carotene
42
Comparison of 3O2 and 1O2

3O2
1O2 ENERGY LEVEL 0
22.5 Kcal NATURE
DIRADICAL NONRADICAL REACTION
RADICAL NONRADICAL C 18
REACTION 1 1500
43
Triplet Oxygen and Singlet Oxygen Reaction

- H
R
R
'
3O
2
O
O
H
R
R
'
R
R
'
O
1O
O
H
2