Transcript Measurement of Antioxidative Activity of Foods

- 2014 RCA/UNDP training course workshop
Experimental Food Chemistry
- Antioxidative properties of Foods -
Advanced Radiation Technology Institute,
Korea Atomic Energy Research Institute
Jong-Heum Park
Contents
Chapter I
: Free Radicals, Oxidative Stress and Diseases
Chapter II
: Antioxidants and Foods
Chapter III : Applications of Ionizing Technology for Screening
New Compounds from Polyphenols
: Experimental Food Chemistry
Chapter IV
- Measurement of Antioxidant Potentials of Foods
Chapter I: Free Radicals, Oxidative Stress
and Diseases
Free Radicals
• Groups of atom with a one or more unpaired
electrons in its outer orbital (R*)
• Leads to formation of bi-products that are toxic
such as super oxide (02-) anion.
Types of Free Radicals
• Reactive Oxygen Species (ROS)
• Reactive Nitrogen Species (RNS)
• Reactive Metabolites or Intermediates
- metabolic activation of drugs, toxins, pollutant
s, cigarette smokes, etc.
Types of Free Radicals
Partial reduction of oxygen are highly reactive
Members:
• Superoxide anion radical
• Hydroperoxyl radical
• Hydrogen peroxide
• Hydroxyl radical
• Lipid peroxide radicals
• Singlet oxygen
• Nitric oxide
• Peroxyl nitrite
-
O2-*
HOO*
H2O2
OHROO*
1O
2
NO*
ONOO-
Generation of Free Radicals
1. Cellular metabolism
About 1-4% of oxygen taken up in the
body is converted to free radicals.
They are constantly produced during
the normal oxidation of foodstuffs.
a) due to leaks in the electron transport chain in mitochondria.
b) Some enzymes, xanthine oxidase and aldehyde oxidase form
superoxide anion radical or hydrogen peroxide.
c) Macrophage also produces NO from
arginine by the enzyme nitric oxide
synthase.
Generation of Free Radicals
2. Environmental effects:
a) High fat foods
b) due to xenobiotic metabolism.
c) due to damages caused by UV ray
d) cigarette or alcohol
e) Industrial and environmental pollution
f) Stress
UV and X-ray Excess alcohol and smoking
Air pollution
High fat diets
Pesticides and herbicides
Harmful Effects of Free Radicals
Free Radicals
1O
2
O2•-
OH•
H2O2
Molecular Reaction with Biological Substances
Enzymes
(-SH)
Carbohydrates
(R-OO•)
Cellular Disturbances
Tissue Injuries
Diseases
Nucleic Acids
(=O)
Harmful Effects of Free Radicals
Extent of Oxidative Stress and
Biological Consequences
Extent of Oxidative Stress
A. Low level & gradual
Biological Consequences
->
B. Medium level & rapid ->
C. Large level & rapid
->
Aging
Carcinogenesis,
Mutagenesis
Death, Stroke
Harmful Effects of Free Radicals
• Cardiovascular
diseases
• Cancers
• Inflammatory diseases
• Respiratory Diseases
• Diabetes mellitus
• Infertility
• Aging process
• Parkinson’s disease
Alzheimer disease
• Others
Chapter II: Antioxidants and Foods
Antioxidants
The Healing Power of Foods (Antioxidants)
• Greek physician Hippocrates,
"Let food be your medicine and
medicine be your food."
• Four of the 10 leading causes of death
in the U.S. (heart disease, cancer, stroke and
diabetes) are directly related to way we eat.
• Significant relationship between foods and diseases
prevention.
Antioxidants
The Power of Antioxidants
• Substances from foods may protect cells from damage
caused by unstable molecules known as free radicals.
• Most common form of free radicals is oxygen.
When an oxygen molecule (O2) becomes electronically
charged or “radicalized” it tries to steal electrons from
other molecules, causing damage to DNA and other
cellular molecules.
• In addition, such damage may become irreversible and
lead to disease including cancer.
Antioxidants
The Power of Antioxidants
• Antioxidants interact with and stabilize free radical
molecules and prevent some of the damage from free
radicals otherwise might cause.
 Examples of antioxidants include beta-carotene, lycopene,
vitamines C, E, and A, and other substances.
• Considerable laboratory evidence using in vitro and
in vivo experimental models
 Antioxidants may slow or possibly prevent the development
of various diseases.
Antioxidants and foods
The Power of Antioxidants
• Rich in fruits and green leafy vegetables.
Antioxidants and foods
The “French Paradox”
• In certain regions of France, the
incidence of cardiovascular
disease is relatively low, despite
a diet high in saturated fats
Antioxidants and foods
Sources of Antioxidants in the Diet:
 Polyphenols, carotenoids, vitamins
•
•
•
•
•
•
•
•
Red wine (tannins, resveratrol, flavonoids)
Cranberries & blue berries (flavonoids, tannins)
Strawberries (ellagic acid, ellagitannins)
Tea (EGCG, other cathechin, tannins)
Chocolate (cathechin)
Onions (quercetin)
Spinach & leafy greens (lutein, zeaxanthin)
Citrus fruits (vitamin C)
Polyphenols
Commercial polyphenol products
• Beneficial activity of polyphenols
• Commercial products of polyphenols
 Anti-oxidative activity
 Anti-inflammatory activity
 Anti-mutagenic activity
 Immune enhancing
: macrophage activation
 Linked to a reduced risk
of coronary heart disease
Shampoo
- Hair growing
Favorite food
Polyphenols !!
 Prevents occurrence
of a higher disease rate
(cardiovascular diseases, diabetes)
Therapy
- Skin care
 Hair-growing activity
 Alleviation of pigmentation problem
 Skin protection
Functional food
Chapter III: Applications of Ionizing Technology
for Screening New Compounds from
Polyphenols
Applications of Ionizing technology
 Application to Food Industries
• Hygienic production and safe distribution
• Stable supply of agricultural commodities
• Efficient and scientific quarantine measures
 Application to sterilization industries
• Quality assurance of public health-related
products without the use of the chemical
fumigants
Basic concept of structural modification using
radiation technology
Ionizing radiation
(UV, -, -Ray)
Denature
maintain
Target
materials
Changes of
Structure
Basic concept of structural modification using
radiation technology
Organic Substances
Effect of Irradiation
Function Improvement
 Physicochemical Properties
Carbohydrates
Polymerization
- Increase of solubility
- Decrease of viscosity
- Increase of extracting yield
Fragmentation
Proteins / Peptides
Modification
 Physiological Properties
- Increase of immune, anti-tumor &
anti-oxidative activities
 Toxic or unfavorable compounds
- Decolorization of pigments
- Degradation of carcinogens
Polyphenols
- Formation of structurally modified
compounds
Development of functionally improved materials or products
Structural modification using radiation technology
Development of valuable materials from natural products
Green tea extract
•
•
•
Deep dark color
Amount of addition is limited
Functional activity is not certified
0 kGy
5 kGy
10 kGy
Industrial application of natural
extracts is too difficult
Antioxidant activity
Whitening activity
20 kGy
Natural extracts can be decolorized by ionizing radiation
without change of functional activity
Screening of biological activities from -irradiated
polyphenols
A) In vitro cytotoxicity
Polyphenol 1
Decrease
of cytotoxicity
Necrosis 
B) Apoptosis
C) HPLC Analysis
Control
Apoptosis 
Non-irradiated
Irradiated
New Compounds
12.5
25
50
100 (mM)
Screening of biological activities from -irradiated
polyphenols
A) Vascular relaxation
Polyphenol 2
OH
OH
D AD 1 B, Sig=280,4 R ef=off (TR S_2012\2012-11-14TR S_KJK_1000_20121114 2012-11-14 15-50-08\001-0101.D )
mAU
HO
O
100
0
OH
OH
0
10
30
O20H
D AD 1 B, Sig=280,4 R ef=off (TR S_2012\2012-11-14TR S_KJK_1000_20121114
2012-11-14O
15-50-08\002-0201.D )
HO
OH
40
mAU
50
min
50
min
OH
100
OH
OH
OH
HO
0
0
O
10
20
30
D AD 1 B, Sig=280,4 R ef=off (TR S_2012\2012-11-14TR S_KJK_1000_20121114 2012-11-14 15-50-08\003-0301.D )
40
mAU
100
B) Tyrosinase inhibition
OH
E) HPLC analysis
OH
0
0
10
20
30
DDAD
AD11 B,B, Sig=280,4
Sig=280,4 RRef=off
ef=off (TR
(TRS_2012\2012-11-14TR
S_2012\2012-11-14TRS_KJK_1000_20121114
S_KJK_1000_20121114 2012-11-14
2012-11-14 15-50-08\001-0101.D
15-50-08\004-0401.D))
40
50
min
00
1010
2020
3030
DDAD
AD11 B,B, Sig=280,4
Sig=280,4 RRef=off
ef=off (TR
(TRS_2012\2012-11-14TR
S_2012\2012-11-14TRS_KJK_1000_20121114
S_KJK_1000_20121114 2012-11-14
2012-11-14 15-50-08\002-0201.D
15-50-08\005-0501.D))
4040
5050
min
min
00
1010
2020
3030
DDAD
AD11 B,B, Sig=280,4
Sig=280,4 RRef=off
ef=off (TR
(TRS_2012\2012-11-14TR
S_2012\2012-11-14TRS_KJK_1000_20121114
S_KJK_1000_20121114 2012-11-14
2012-11-14 15-50-08\003-0301.D
15-50-08\006-0601.D))
4040
5050
min
min
00
1010
2020
3030
AD11 B,B, Sig=280,4
Sig=280,4 RRef=off
ef=off (TR
(TRS_2012\2012-11-14TR
S_2012\2012-11-14TRS_KJK_1000_20121114
S_KJK_1000_20121114 2012-11-14
2012-11-14 15-50-08\004-0401.D
15-50-08\007-0701.D))
DDAD
4040
5050
min
min
00
1010
2020
3030
AD11 B,B, Sig=280,4
Sig=280,4 RRef=off
ef=off (TR
(TRS_2012\2012-11-14TR
S_2012\2012-11-14TRS_KJK_1000_20121114
S_KJK_1000_20121114 2012-11-14
2012-11-14 15-50-08\005-0501.D
15-50-08\008-0801.D))
DDAD
4040
5050
min
min
00
1010
2020
3030
D AD 1 B, Sig=280,4 R ef=off (TR S_2012\2012-11-14TR S_KJK_1000_20121114 2012-11-14 15-50-08\006-0601.D )
4040
5050
min
min
0
40
50
min
mAU
mAU
0 kGy
100
100
00
mAU
mAU
70 kGy
100
100
00
mAU
mAU
C) Antioxidant activity
100 kGy
100
100
00
mAU
mAU
150 kGy
100
100
00
New Compound
mAU
mAU
200 kGy
100
100
00
mAU
100
0
10
20
30
Isolation of new compounds from -irradiated
polyphenols
20.526
16.264
6.914
5.397
5.800
4.937
3.683
0.25
2.594
0.50
⑤
④ ②
4.544
2.896
3.425
⑥⑦
Polyphenol 3
8.704
③
mV
0.75 Detector A Ch2:280nm
30kGy
0.00
0.0
New compounds 2
2.5
Naturally occurring
compounds = 5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
Antioxidant
activities
0 kGy
15 kGy
30 kGy
SEM
DPPH
57.15b
74.63ab
83.72a
0.37
Antioxidant Index
69.49b
76.68a
80.39a
1.50
Phenolic contents
26.76b
29.70ab
35.09a
0.37
min
Isolation of new compounds from -irradiated
polyphenols
Polyphenol 4
A) Isolation of new compounds
B) Purity check (new compounds 1, 2, 3, 4, 5, 6)
1
3
2
Irradiation (50 kGy)
2
1
Irradiated Polyphenol
4
3
4
5
6
5
6
ST_GE1 228 (4.483) Cm (227:229)
1: TOF MS ES2.91e4
Structural analysis of new compounds isolated
from -irradiated polyphenols
317.1062
100
%
316.0991
318.1298
635.2240
137.0190
633.2058 636.2407
315.0956 319.0946
183.0194
632.1968 637.2169
A) Structural Analysis of new compounds 1 and 2
1H NMR (compound 1)
319.9574363.1174 409.1158
229.0267 245.0742
0
100 150 200 250
ST_GE1 219 (4.315) Cm (217:220)
300
350
ST_GE2 225 (4.427) Cm (224:227)
100
450
657.1981
500
550
676.2220
m/z
650 700
1: TOF MS ES8.96e4
600
MALDI-TOF
1: TOF MS ES(compound 1)
2.98e4
316.0987
100
400
443.2415
317.1274
316.1209
%
317.1057
%
633.2043
315.0948
318.1422
632.1991
137.0258
318.1429
634.2443
319.0552
137.0146315.1129
166.0276
362.1117 408.1222
255.0529
229.0312
443.1091
0
636.2645
319.0804
632.2358
100 183.0313150 200 250 300 350 400 450 500
360.1478
229.0385
631.2311 679.2900 792.3104 951.4415
1H NMR (compound 2)
0
100 200 300 400
ST_GE2 233 (4.589) Cm (231:234)
500
600
800
636.2153
631.1920
638.0284 678.2347
550
1110.6692
600
650
1269.7590
m/z
700
New compound 1
1428.8049
m/z
900 1000 1100 1200 1300 1400
1: TOF MS ES1.14e5
MALDI-TOF
(compound 2)
316.1173
100
700
635.2169
%
317.1283
633.2433
634.2498
315.1133
632.2382
318.1183
137.0246
0
100
B) Functional Analysis
319.0665
229.0435
200
408.1450
300
400
631.2332
453.1794
500
635.2388
636.2126
950.4166
638.2437
600
700
800
1109.6584
1267.7272
1427.2856
900 1000 1100 1200 1300 1400
m/z
New compound 2
Proposed mechanism on the formation of new
polyphenol compounds by radiation
Polyphenol 4
New compounds 1, 2 (Steroisomer)
Radiation-induced molecular conversion
of Ginsenoside
A) HPLC analysis
mAU
Rb1
0 kGy
mAU
10 kGy
mAU
Rg3
30 kGy
B) Radiation-induced Ginsenoside conversion
Gamma irradiation
Ginsenoside Rb1
Ginsenoside Rg3
Conclusions (Chapter I, II and III)
• Free radicals are any molecular species capable of independent
existence that contains an unpaired eletron in an atom orbital.
• These radicals attack important cellular macromolecules
leading to cell damage and homeostatic disruption.
• Oxidative stress, arising as a result of an imbalance between
free radicals formation and antioxidant defense, makes a
significant contribution to human diseases.
• Antioxidants can decrease oxidative stress by scavenging free
radicals and slow or possibly prevent the development of
various diseases.
Conclusions (Chapter I, II and III)
• Antioxidants are found in many foods including fruits and
green leafy vegetables, and various forms of polyphenols or
non-phenolic substances such as tannins, resveratrol,
flavonoids, ellagic acid, ellagitannins, EGCG, other cathechin,
cathechin, vitamins, etc.
• Ionizing irradiation is effective food sanitary technology that
can inactivate food-borne pathogens in foods and extend their
shelf stability.
• Recently, the technology has been effectively applied to
decolor the pigment of green tea extract for the development
of cosmetic component without changing its antioxidant and
whitening activities, or develop new compounds having
improved biological activity from natural substances.
Chapter IV: Experimental Food Chemistry
- Measurement of Antioxidant Potentials of Foods -
Measurement of Antioxidative Activity of Foods
Objectives
• Determine antioxidative activities of cherry tomato and effect
of ionizing radiation on its antioxidant potential
• Determine antioxidative activity of cherry tomato using DPPH (1-1diphenyl-2-picryl-hydrazyl) and FRAP (ferric reducing antioxidant
power) assays
• Investigate the effects of ionizing radiation on antioxidant activity of
cherry tomato
For testing cherry tomato, its metanol and water extracts should be
prepared before test
Measurement of Antioxidative Activity of Foods
Methods Used to Determine
Antioxidant Potential
• DPPH (1-1-diphenyl-2-picryl-hydrazyl) assay
• FRAP (ferric reducing antioxidant power) assay
Measurement of Antioxidative Activity of Foods
DPPH assay
• DPPH (1-1-diphenyl-2-picryl-hydrazyl); a molecule containing
a stable free radical
• Based on the reduction of DPPH in methanol solution in the
presence of a hydrogen-donating antioxidant due to the
formation of the non radical from DPPH-H
Measurement of Antioxidative Activity of Foods
• When a solution of DPPH is mixed with antioxidants which can
donate a hydrogen atom, the DPPH is converted to 1-1diphenyl-2-picryl-hydrazine
• This transformation results in color change from purple to
yellow, which is measured spectrophotometrically
Measurement of Antioxidative Activity of Foods
• Discoloration of DPPH from purple to yellow indicates the
scavenging potential of antioxidant. The change in the
absorbance can be detected at 515 nm.
Measurement of Antioxidative Activity of Foods
Procedure of DPPH assay
Procedure:
• Reagent: 0.3 mM DPPH (0.012 g DPPH + 100 mL MeOH)
Method:
• Extract the antioxidants from cherry tomato using MeOH
• Prepare Sample (tomato extract), Control
- Sample: Tomato extract (300 L) + DPPH solution (300 L) +
MeOH (300 L)
- Control: DPPH (300 L) + MeOH (600 L)
Measurement of Antioxidative Activity of Foods
Procedure of DPPH assay
• Incubate for 10 min in dark and transfer 150 L of Samples,
Blank and Control to 96 well plate
• Then, the absorbance is measured at 515 nm
Abs at 515nm
Sample + DPPH
Transfer to
96 well plate
Micro plate reader
Measurement of Antioxidative Activity of Foods
Procedure of DPPH assay
• The percentage inhibition can be calculated using below
formula
Inhibition (%) = ((A0-A1)/A0) X 100
• Where: A0: - Control
A1: - Samples
Measurement of Antioxidative Activity of Foods
FRAP assay
• FRAP; ferric reducing antioxidant power assay
• Based on the reduction of ferric tripyridyl triazine (Fe III-TPTZ)
complex to ferrous (Fe II) ion formation by antioxidants at low
pH.
• This coloration gives rise to the intense blues color of Fe2 +TPTZ from colorless Fe3 +-TPTZ
Fe3+-TPTZ (colorless) + antioxidant
Fe2 +-TPTZ (blue color)
Measurement of Antioxidative Activity of Foods
FRAP assay
• This transformation can be measured spectrophotometrically
• Coloration of colorless Fe3+-TPTZ to blue color of Fe2 +-TPTZ
indicates the scavenging potential of antioxidants. The change
in the absorbance can be detected at 593 nm.
Measurement of Antioxidative Activity of Foods
Procedure of FRAP assay
Procedure:
• Reagent: 1) 300 mM sodium acetate buffer (pH3.6)
 3.1g sodium acetate + 16 mL acetic acid /1L
2) 10 mM TPTZ solution in 40 mM HCl,
3) 20 mM FeCl36H2O
Method:
• Prepare FRAP reagent by combing above reagent 1) 25 mL +
reagent 2) 2.5 mL + reagent 3) 2.5 mL
• Extract antioxidants from cherry tomato using D.W.
Measurement of Antioxidative Activity of Foods
Procedure of FRAP assay
• Then, prepare Sample (tomato extract), Control
- Sample: Tomato extract (90 L) + FRAP reagent (900 L) +
D.W (90 L)
- Control: FRAP reagent (900 L) + D.W. (120 L)
• Incubate for 10 min in dark, then transfer 150 L of Samples
and Control to 96 well plate
Abs at 593nm
Sample +
FRAP reagent
Transfer to
96 well plate
Micro plate reader
Measurement of Antioxidative Activity of Foods
Procedure of FRAP assay
• Finally, the absorbance is measured at 593 nm
• For preparation of std calibration curve using Ascorbic Acid
std (100 M- 1,000 M), diluted gallic acid is added to the
reaction mixture instead of tomato extract
Abs at 593nm
Sample +
FRAP reagent
Transfer to
96 well plate
Micro plate reader
Measurement of Antioxidative Activity of Foods
Procedure of FRAP assay
• FRAP value can be calculated using below formula,
FRAP value (M) = (Changes in abs of samples for 10 min)/
(An X-intercept of the equation from
calibration curve of Ascorbic acid)
X intercept
Example
Measurement of Antioxidative Activity of Foods
Thank You
for Your Attention!