Experimental Food Chemistry - 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 : 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

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, pollutants, cigarette smokes, etc.

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 - 1O2 Nitric oxide - NO* Peroxyl nitrite - ONOO-

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.

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

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

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

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

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.

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.

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 Resveratrol & Flavonoids !!

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 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 !!

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

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

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.

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

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

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

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

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

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)

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

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 Rg3 0 kGy 10 kGy 30 kGy Gamma irradiation B) Radiation-induced Ginsenoside conversion 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 (1-1-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

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

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 1-1-diphenyl-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 Transfer to 96 well plate Abs at 515nm Micro plate reader Sample + DPPH

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

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

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!