1. Experimental Food Chemistry
RCA/UNDP training course workshop
Experimental Food Chemistry
- Antioxidative properties of Foods -
Advanced Radiation Technology Institute,
Korea Atomic Energy Research Institute
Jong-Heum Park

2. Contents : Free Radicals, Oxidative Stress and Diseases
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
Chapter IV
: Experimental Food Chemistry
- Measurement of Antioxidant Potentials of Foods

3. Chapter I: Free Radicals, Oxidative Stress
and Diseases

4. 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.

5. Types of Free Radicals
Reactive Oxygen Species (ROS)
Reactive Nitrogen Species (RNS)
Reactive Metabolites or Intermediates
- metabolic activation of drugs, toxins, pollutants, cigarette smokes, etc.

6. Partial reduction of oxygen are highly reactive
Types of Free Radicals
Partial reduction of oxygen are highly reactive
Members:
Superoxide anion radical - O2-*
Hydroperoxyl radical HOO*
Hydrogen peroxide H2O2
Hydroxyl radical OH-
Lipid peroxide radicals - ROO*
Singlet oxygen O2
Nitric oxide NO*
Peroxyl nitrite ONOO-

7. Generation of Free Radicals
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.
due to leaks in the electron transport chain in mitochondria.
Some enzymes, xanthine oxidase and aldehyde oxidase form superoxide anion radical or hydrogen peroxide.
Macrophage also produces NO from arginine by the enzyme nitric oxide synthase.

8. Generation of Free Radicals
2. Environmental effects:
High fat foods
due to xenobiotic metabolism.
due to damages caused by UV ray
cigarette or alcohol
Industrial and environmental pollution
Stress
High fat diets
Pesticides and herbicides
UV and X-ray
Excess alcohol and smoking
Air pollution

9. Harmful Effects of Free Radicals
H2O2
OH•
1O2
Molecular Reaction with Biological Substances
Enzymes
(-SH)
Carbohydrates
(R-OO•)
Nucleic Acids
(=O)
Cellular Disturbances
Tissue Injuries
Diseases

10. Harmful Effects of Free Radicals
Extent of Oxidative Stress and Biological Consequences
Extent of Oxidative Stress
Biological Consequences
A. Low level & gradual ->
Aging
B. Medium level & rapid ->
Carcinogenesis,
Mutagenesis
C. Large level & rapid >
Death, Stroke

11. Harmful Effects of Free Radicals
Cardiovascular diseases
Cancers
Inflammatory diseases
Respiratory Diseases
Diabetes mellitus
Infertility
Aging process
Parkinson’s disease Alzheimer disease
Others

12. Chapter II: Antioxidants and Foods

13. 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.

14. 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.

15. 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.

16. Antioxidants and foods The Power of Antioxidants
Rich in fruits and green leafy vegetables.

17. 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
Resveratrol &
Flavonoids !!

18. 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)

19. Polyphenols

20. Commercial polyphenol products
Beneficial activity of polyphenols
Anti-oxidative activity
Anti-inflammatory activity
Anti-mutagenic activity
Immune enhancing
: macrophage activation
Linked to a reduced risk
of coronary heart disease
Prevents occurrence
of a higher disease rate
(cardiovascular diseases, diabetes)
Hair-growing activity
Alleviation of pigmentation problem
Skin protection
Commercial products of polyphenols
Shampoo
- Hair growing
Favorite food
Functional food
Therapy
- Skin care
Polyphenols !!

21. Chapter III: Applications of Ionizing Technology for Screening New Compounds from Polyphenols

22. Applications of Ionizing technology
Application to Food Industries
Application to sterilization industries
Hygienic production and safe distribution
Stable supply of agricultural commodities
Efficient and scientific quarantine measures
Quality assurance of public health-related products without the use of the chemical fumigants

23. Basic concept of structural modification using
radiation technology
Ionizing radiation
(UV, -, -Ray)
Target
materials
Changes of
Structure
Denature
maintain
Irradiating a target material,
the properties of material can be changed
because the structure changes to diverse forms.
Development of new biological materials is designed in the basic concept of structural modification technology
with radiation technology.
When a target material is irradiated, the irradiated material can be structurally changed and the properties can be also changed.
Using this hypothesis, the various products can be obtained.
My team has been being performed this field of research from 2000.

24. Basic concept of structural modification using
radiation technology
Effect of Irradiation
Function Improvement
Organic Substances
Polymerization
Fragmentation
Modification
Physicochemical Properties
Increase of solubility
Decrease of viscosity
Increase of extracting yield
Physiological Properties
- Increase of immune, anti-tumor & anti-oxidative activities
Toxic or unfavorable compounds
Decolorization of pigments
Degradation of carcinogens
Formation of structurally modified compounds
Carbohydrates
Proteins / Peptides
Polyphenols
Development of functionally improved materials or products

25. 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
Industrial application of natural extracts is too difficult
Antioxidant activity
Whitening activity
0 kGy
5 kGy
10 kGy
20 kGy
Natural extracts can be decolorized by ionizing radiation without change of functional activity

26. Screening of biological activities from -irradiated
polyphenols
C) HPLC Analysis
Control
12.5
Non-irradiated 25 50
100 (mM)
Irradiated
Necrosis 
Apoptosis 
A) In vitro cytotoxicity
B) Apoptosis
New Compounds
Polyphenol 1
Decrease
of cytotoxicity

27. Screening of biological activities from -irradiated
polyphenols
A) Vascular relaxation
B) Tyrosinase inhibition
C) Antioxidant activity
E) HPLC analysis
0 kGy
150 kGy
100 kGy
70 kGy
200 kGy
New Compound O H
Polyphenol 2

28. Isolation of new compounds from -irradiated Antioxidant activities
polyphenols
New compounds 2
Naturally occurring
compounds = 5
Polyphenol 3 ⑤ ⑥ ⑦ ② ③ ④
30kGy
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

29. Isolation of new compounds from -irradiated
polyphenols 1 2 3 4 5 6
B) Purity check (new compounds 1, 2, 3, 4, 5, 6)
Polyphenol 4
A) Isolation of new compounds
Irradiated Polyphenol
Irradiation (50 kGy)

30. Structural analysis of new compounds isolated
from -irradiated polyphenols
B) Functional Analysis
A) Structural Analysis of new compounds 1 and 2
1H NMR (compound 1)
MALDI-TOF
(compound 1)
New compound 1
1H NMR (compound 2)
(compound 2)
New compound 2

31. Proposed mechanism on the formation of new
polyphenol compounds by radiation
Polyphenol 4
New compounds 1, 2 (Steroisomer)

32. Radiation-induced molecular conversion
of Ginsenoside
A) HPLC analysis
mAU
Rb1
Rg3
0 kGy
10 kGy
30 kGy
Gamma irradiation
B) Radiation-induced Ginsenoside conversion
Ginsenoside Rb1
Ginsenoside Rg3

33. 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.

34. 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.

35. Chapter IV: Experimental Food Chemistry
- Measurement of Antioxidant Potentials of Foods -

36. 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 (1-1-diphenyl-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

37. Methods Used to Determine Antioxidant Potential
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

38. 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

39. 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 1-1-diphenyl-2-picryl-hydrazine
This transformation results in color change from purple to yellow, which is measured spectrophotometrically

40. 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.

41. Measurement of Antioxidative Activity of Foods
Procedure of DPPH assay
Procedure:
Reagent: 0.3 mM DPPH (0.012 g DPPH 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)

42. 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
Transfer to
96 well plate
Abs at 515nm
Micro plate reader
Sample + DPPH

43. 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

44. 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)

45. 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.

46. 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.

47. 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
Transfer to
96 well plate
Abs at 593nm
Micro plate reader
Sample +
FRAP reagent

48. 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
Transfer to
96 well plate
Abs at 593nm
Micro plate reader
Sample +
FRAP reagent

49. 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

50. Measurement of Antioxidative Activity of Foods
Thank You
for Your Attention!