RNS) Scavenging Assays, Oxidative Stress - ACS Publications

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Antioxidant activity/capacity measurement: III. Reactive oxygen and nitrogen species (ROS/RNS) scavenging assays, oxidative stress biomarkers, and chromatographic/chemometric assays Re#at Apak, Mustafa Özyürek, Kubilay Guclu, and Esra Capanoglu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04744 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 26, 2015

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Journal of Agricultural and Food Chemistry

Antioxidant activity/capacity measurement: III. Reactive oxygen and nitrogen species (ROS/RNS) scavenging assays, oxidative stress biomarkers, and chromatographic/chemometric assays

Reşat Apak1, Mustafa Özyürek1, Kubilay Güçlü1, Esra Çapanoğlu2

1

Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320,

Istanbul-Turkey

2

Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering,

Istanbul Technical University, Maslak 34469, Istanbul-Turkey

* Corresponding Author. Tel.: +90 212 4737070 Fax: +90 212 473 7180 E-mail: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

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There are many studies in which the antioxidant potential of different foods have been analyzed.

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However, there are still conflicting results and lack of information as a result of unstandardized

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assay techniques, and differences between the principles of the methods applied. The

6

measurement of antioxidant activity, especially in case of mixtures, multifunctional or complex

7

multiphase systems, cannot be evaluated satisfactorily using a simple antioxidant test due to the

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many variables influencing the results. In the literature, there are many antioxidant assays which

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are used to measure the total antioxidant activity/capacity of food materials. In this review,

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reactive oxygen and nitrogen species (ROS/RNS) scavenging assays are evaluated with respect

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to their mechanism, advantages, disadvantages, and their potential use in food systems. On the

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other hand, in vivo antioxidant activity (AOA) assays including oxidative stress biomarkers and

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cellular based assays are covered within the scope of this review. Finally, chromatographic and

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chemometric assays are reviewed focusing on their benefits especially with respect to their time

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saving, cost-effective and sensitive nature.

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Keywords: Antioxidant activity, Total antioxidant capacity; ROS/RNS scavenging assays;

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Oxidative stress biomarkers; Chemometric methods, Chromatographic methods.

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1. INTRODUCTION

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ROS is a collective term often used to include oxygen radicals [superoxide (O2−), hydroxyl

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(OH), peroxyl (ROO) and alkoxyl (RO)] and certain nonradicals that are either oxidizing

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agents and/or easily radical-convertible species, such as hypochlorous acid (HOCl), ozone (O3),

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singlet oxygen (1O2) and hydrogen peroxide (H2O2). RNS is a similar collective term that

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includes nitric oxide radical (NO), peroxynitrite anion (ONOO−), nitrogen dioxide radical

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(NO2), other oxides of nitrogen and products arising from the reaction of NO with O2−, RO

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and ROO.1 ROS and RNS are essential for humans to maintain homeostasis and health, but

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uncontrolled and excess ROS/RNS have been implicated in the pathogenesis of various diseases

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including cancer, cardiovascular and neurodegenerative diseases as well as aging.2,3 The

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mechanisms by which these pathologies develop generally involve oxidative alteration of

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physiologically critical molecules, including proteins, lipids, carbohydrates and nucleic acids,

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along with modulation of gene expression and the inflammatory response.4

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The human organism has developed defense systems to neutralize the excessive levels

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of ROS and RNS and compensate for oxidative stress.4,5 These include enzymatic systems,

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especially superoxide dismutases (SOD), catalases, gluthatione peroxidases and thioredoxin

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systems, which are recognized as being highly efficient in ROS detoxification. Other than

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albumin and antioxidative proteins (rich in thiols and certain amino acids), the main small-

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molecule nonenzymatic antioxidants present in the human organism are gluthatione, bilirubin,

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estrogenic sex hormones, uric acid, ascorbic acid, coenzyme Q, melanin, melatonin, α-

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tocopherol and lipoic acid.5

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Besides these antioxidants produced during normal metabolism in the body, exogenic

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antioxidants can be provided from food materials. Many antioxidant compounds naturally

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occurring in plant sources have been identified as free radical or active oxygen scavengers.6

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Especially, fruits and vegetables are accepted as good sources of natural antioxidants, which

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provide protection against free radicals and have been associated with lower incidence and

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mortality rates of cancer and heart diseases in addition to a number of other health benefits.7-9

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Even though a wide range of studies on the antioxidant potential of plant foods have been

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performed, there are still conflicting results and lack of information as a result of unstandardized

51

assay techniques, and differences between the principles of the methods applied. So, several

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review articles have been published describing different antioxidant assays with different

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perspectives.3,10-13 On the other hand, databases on the antioxidant power of molecules, plant

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extracts and foodstuffs could be enhanced through better control, improvement and

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harmonization of current methods.4

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For extrapolating in vitro antioxidant assay results to in vivo conditions, the question of

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bioavailability and the fate of metabolites of the antioxidant components must be addressed.14

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Other than reactivity towards ROS/RNS, several factors such as concentration, distribution,

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localization, fate of antioxidant-derived radical, interaction with other antioxidants, and

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metabolism should be evaluated.15 Also, in vitro antioxidant assays carried out at unrealistic

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pH values (i.e., far from physiological pH, either in alkaline or acidic range) cannot mimic the

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in vivo conditions for the estimation of antioxidant action which makes the evaluation harder.

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As a result of the large differences among total antioxidant capacity (TAC) assays16,17

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in regard to variability of reaction times, end-point detection and media,18,19 several tests with

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different principles such as ROS/RNS scavenging methods, ET based or HAT based assays,

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etc. had to be implemented at the same time for full evaluation of antioxidant activities (AOA)

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of complex samples.20 An equally important requirement was to identify and quantify

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individual antioxidant constituents of complex mixtures such as plant-derived materials21 with

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the aid of chromatographic separations. Compared to traditional assays, these chromatographic

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methods proved to be fast and cost-effective for the identification of key antioxidant

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compounds.22

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On the other hand, some spectroscopic and chromatographic assays may lead to the

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over/underestimation of antioxidant contents of foods or biological and plant materials due to

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the interferences with other components such as sugars, ascorbic acid, proteins, etc. present in

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the food matrix.23,24 For this aspect, chemometric techniques are being used to analyze the

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spectra, such as partial least squares (PLS) or principal component analysis (PCA).25 Several

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recent articles highlight the potentiality and applicability of chemometric techniques in

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different antioxidant analyses as covered in this review. 26,27

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The aim of this comprehensive review is to present the mechanism, advantages and

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disadvantages of ROS/RNS scavenging assays including examples from their applications in

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food systems. Besides, in vivo AOA assays including oxidative stress biomarkers and cellular

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based assays as well as chromatographic and chemometric assays are covered within this

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review.

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2. ROS/RNS SCAVENGING METHODS

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2.1. Scavenging of Superoxide Anion Radical

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Oxygen, which is present in the atmosphere in its ground state as a stable triplet biradical (3O2),

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undergoes a step-wise reduction process.28,29 Molecular oxygen, in the ground state, has two

90

unpaired electrons with parallel spins in its two separate antibonding orbitals.30 Because of this

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spin restriction, molecular oxygen is reluctant to accept a pair of electrons (having antiparallel-

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spins) from an electron donor, and therefore, the univalent reduction of O2 to O2●– is a facile

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process.31 O2●– is well-documented to be a highly reactive free radical and can be generated in

94

a variety of biological systems either by auto-oxidative processes or by enzyme-catalyzed

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95

reactions (xanthine oxidase) involved in aerobic metabolism.32-34 These oxygen based free

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radicals cause cell damage by initiating the peroxidation of the membranal lipids29,35 thus,

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playing an important role in a wide variety of pathologies, such as aging and cancer.7,29,36

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Although aerobic organisms have various defense systems to protect themselves from such

99

oxidative stress, the capacity of these protective systems has been well-known to decrease

100

gradually with aging. Thus, it is strongly suggested to provide the body with a constant supply

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of antioxidant phytochemicals, in order to replenish this aging-induced loss.37,38

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Research on superoxide radical scavenging activity (SRSA) of antioxidants has gained

103

an increasing interest as being one of the most important ways of clarifying the mechanism of

104

antioxidant activity.39 On the other hand, detection and measurement of O2●– within cells is a

105

goal, which is as difficult as it is desirable, because of the instability of this ROS in aqueous

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solutions.31 The methodologies that are applied to determine the O2●– generation in biological

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systems involve the reaction of superoxide with an indicator that forms a stable product by

108

oxidation, reduction, or binding of superoxide to the indicator. The sensitivity, efficiency, and

109

specificity of detecting O2●– vary greatly depending on the different reaction pathways

110

involved.39

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Methods used to determine SRSA mostly rely on the measurement of the inhibition of

112

O2●– generation with the hypoxanthine-xanthine oxidase system (HX–XOD).34 XOD reduces

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oxygen while catalyzing the oxidation of its substrates, thus producing O2●– and H2O2.31,33,34,40

114

The working mechanism of a nano-interfaced superoxide biosensor was recently

115

described by Thandavan et al.41 A hydrothermal method was utilized to synthesize nano

116

Fe3O4 and superoxide dismutase (SOD) which was attached to it by covalent linking. SOD was

117

immobilized on iron oxide nanoparticles (nano- Fe3O4 ) coated on a Au-electrode surface. The

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cyclic voltammogram of the SOD/nano- Fe3O4 /Au bioelectrode exhibited an electrochemical

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reduction behavior for increasing concentrations of superoxide. Superoxide anion (O2•-) was

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generated from (DMSO+NaOH) and reacted (i.e. was dismutated) with the catalytic copper(II)

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center of the SOD enzyme to produce O2 and H2O2. The Fe3O4 coating helped this catalytic

122

degradation by providing the necessary conduction pathway for electron transfer between the

123

enzyme and the electrode. The final products of dismutation were electrochemically sensed.

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The amperometric response in the presence of O2•- scavengers (e.g., ascorbic and uric acid)

125

diminished slightly, enabling superoxide scavenging activity measurement of antioxidants.

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Also, the sensing capability of a flavonoid-metal ion complex has been investigated.

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The naringin-copper complex exhibits good sensitivity towards O2●– in a range of 0.2-4.2 µM.

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Cyclic voltammetric experiments revealed that the increase in the reduction current with

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increase in the scan rate can be attributed to a surface controlled process. Furthermore,

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flavonoid-metal ion complexes are generated due to their superior antioxidant activities.

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Detection of O2●– has been mainly based on enzymes and cytochromes. Even though the

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performance of the sensor is not affected by pH and common interferents, these sensors are

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limited by their poor structural stability and high cost.42 In a similar work investigated by

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Moridani et al.43, a number of flavonoids were selected along with one clinically available iron

135

chelator deferoxamine to investigate their superoxide radical scavenging properties and their

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abilities to prevent the hypoxic hepatocyte injury. This study suggests that iron complexes of

137

flavonoids readily scavenged superoxide radicals. The mechanism of superoxide radical

138

scavenging by luteolin-iron complexes is given as an example in Figure 1.

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Figure 1

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2.1.1. Luminol-Based Chemiluminescence

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In alkaline dimethyl sulfoxide, the luminol monoanion can be univalently oxidized by a variety

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of oxidants, in the presence of O2, and the luminol radical generated can add O2●– yielding an

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unstable endoperoxide,

whose decomposition leads to the electronically excited

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aminophthallate.44 This excited aminophthallate is the light-emitting species; an oxygen-

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dependent luminescence occurs when the excited aminophtallate returns to the ground state.40

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However, luminol was reported to be an unreliable detector of O2●–, since it can spontaneously

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reduce O2 to O2●–, in the presence of any univalent oxidant, acting as a source of O2●–.31,40,45,46

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The mechanism of the chemiluminescence of luminol is shown in Figure 2. In this reaction,

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luminol is converted in basic solution into the resonance-stabilized dianion (1), which is

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oxidized by hydrogen peroxide into the dicarboxylate ion (2), along with the loss of molecular

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nitrogen, N2. The formed molecule “2” is in an excited electronic state, and sheds its extra

153

energy by emitting a photon of light (hν), allowing the molecule to go to its ground state form

154

(3).44

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Figure 2

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2.1.2. Nitroblue Tetrazolium (NBT)-Based Chemiluminescence

158

O2●– is mostly produced enzymatically, and to a minor extent generated through a non-

159

enzymatic reaction of phenazine methosulphate in the presence of NADH and molecular

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oxygen. It reduces NBT to formazan (at pH 7.4, room temperature) which can be measured

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spectrophotometrically. Any added molecule that is capable of reacting with O2●– inhibits the

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production of formazan, so the SRSA is estimated by the attenuation of the absorbance,

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compared to the value of a reference solution without tested antioxidants.34 The most popular

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version of the assay spectrophotometrically measures the absorbance of the formazan product

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at 560 nm.47 In a modified version of this test48, the yellow-colored NBT is reduced by

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superoxide anion radicals to the blue formazan which is sparingly soluble (can be solubilized

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with aqueous NaOH+DMSO to enable colorimetric determination at 620 nm using a microplate

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reader).48

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Water insolubility of the diformazan, formed as the end product of NBT reduction, may

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be a drawback of this method causing irreproducibility in absorbance readings and restricting

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the use of a reliable microtiter plate assay. Other redox-active agents have also been used in

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several studies instead of NBT, including hydroxylammonium chloride, which was converted

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to nitrite with O2●– and determined by the Griess spectrophotometric method,34,49 and with

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ferricytochrome c.32

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The enzymatic method is also prone to artifacts, i.e. if the assay is performed in the

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presence of NADPH-cytochrome c reductase, this enzyme reduces NBT to a free radical

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intermediate that further generates O2●– in the presence of oxygen, leading to erroneous

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results.39,50

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2.1.3. Electron Spin Resonance (ESR) Spin Trapping Method

180

In this technique, a nitrone or a nitroso compound reacts with a short-lived free radical to

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produce a nitroxide (a spin-trapped adduct) whose lifetime is considerably greater than that of

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the parent free radical and therefore, detectable by conventional ESR spectroscopy.51,52 Due to

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the rapid decomposition of nitroxides that are derived from the reaction of nitroso compounds

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with oxygen-centered free radicals (i.e., O2●– and ●OH), nitrones have been well-documented

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as the principal spin traps.52,53 The most widely used nitrone, as a spin trap, is 5,5-dimethyl-1-

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pyrroline-1-oxide (DMPO)51,52,54,55 which produces spin-trapped adducts with characteristic

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ESR spectra as a result of its reaction with O2●–.52,56 However, this spin trap has also its own

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limitations: i.e. it is inefficient for the reaction with O2●–, having a rate constant no greater than

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10 M-1s-l ,57 its partition coefficient is only 0.09,40 indicating a preference for water over lipid

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environments; it is susceptible to metal ion-catalyzed air oxidation which may result in the

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formation of unwanted products; the cellular toxicities may be a serious and limiting problem

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since the concentration of DMPO required is so high.52 A demonstration of the mechanism of

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nitrone spin traps is given in Figure 3.58,59

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Figure 3

195 196

2.2. Scavenging of Hydrogen Peroxide

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H2O2, a biologically relevant and non-radical oxidizing species, may be formed in tissues

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through oxidative processes.60 Electron reduction of O2 initially generates the superoxide anion,

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which is then spontaneously or enzymatically converted to H2O2.61 H2O2 can induce the

200

degradation of biological macromolecules such as lipids, proteins or enzymes, carbohydrates

201

and nucleic acids through generation of ●OH.62 H2O2 is reduced to ●OH in the presence of

202

transition metal ions (Fe2+, Cu2+) by Haber-Weiss or Fenton reaction.63

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The measurement of H2O2 scavenging activity in biological fluids and foods is

204

important. A number of assays were developed for the determination of H2O2 scavenging

205

activity depending on the oxidation of a spectrophotometric, fluorogenic or chemiluminogenic

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probe (detector molecule) by H2O2 using horseradish peroxidase or transition metal ions as

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oxidation catalysts.64 Spectrophotometric probes used in horseradish peroxidase coupled

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reactions include guaiacol and phenol red. Fluorogenic probes are p-hydroxyphenylacetic acid,

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homovanilic acid, scopoletin, 2,7-dichlorodihydrofluorescein, and amplex red,65 while the most

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extensively studied chemiluminogenic probes are luminol and peroxyoxalates.10 The

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horseradish peroxidase linked assays have some disadvantages. The assays lack specificity

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since peroxidase also oxidizes certain substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-

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6-sulphonic acid) (ABTS), diaminobenzidine, tetramethylbenzidine, etc. without addition of

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H2O2.64,66 Different biological compounds including thiols and ascorbate can serve as substrate

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for horseradish peroxidase and hence compete with the probe for oxidation, leading to

216

underestimation of H2O2 production. Furthermore, horseradish peroxidase is expensive and

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unstable in solution, and has strict requirements for experimental conditions.65,66

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Another common assay for assessing the scavenging capacity against H2O2 is based on

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the intrinsic absorption of this molecule in the UV region at 230 nm.67,68 As scavenger

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compounds decrease the concentration of H2O2, the absorbance value at 230 nm decreases. Yet,

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it is quite common that most plant and food samples also absorb at this wavelength, requiring

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the performance of a “blank” measurement. This situation can compromise both the precision

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and accuracy of this method.11 First of all, it may be hard to differentiate between small changes

224

when there is much larger background absorption. Secondly, the absorption of samples may

225

change after reaction with H2O2, and the blank measurement would not be valid.69 Additionally,

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the conventional UV-absorbance assay of H2O2 scavenging is carried out without a transition

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metal ion-based catalyst, which may cause insufficient reaction during the incubation time of

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the assay.69,70 This assay was performed to measure the ability of tea extracts,71 where

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scavenging of H2O2 is followed by decay in H2O2 concentration.

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It is widely considered that H2O2 is toxic in vivo and must be rapidly eliminated,

231

employing enzymes such as catalases, peroxidases and thioredoxin-linked systems.72 Thus,

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removing H2O2 as well as O2●– is very important for the protection of pharmaceuticals and food

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systems.69,72

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Generation of ●OH at a palladium oxide nanoparticles-modified electrode during the

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concomitant reduction of palladium oxide in the presence of H2O2 either added to the solution

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or produced in situ by oxygen reduction was investigated. It was found that this method may

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be more selective towards antioxidants than other similar methods exploiting electrochemical

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oxidation of antioxidants at anodic potentials, because palladium oxide reduction occurred at

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negative potentials where antioxidants are usually not oxidized.73

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On the other hand, nano-sized iron has been applied in the degradation of halogenated

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organic compounds and other persistent toxic substances due to its ability to catalyze redox

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processes.74 Catalytic amounts of iron are sufficient to yield ●OH from O2●– and H2O2.75,76

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In another study, for investigating the electrocatalytic reduction of H2O2, Au nanoparticles-

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modified electrodes were also examined.77,78 A gold electrode, modified with an amino and a

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thiol compound, Au nanoparticles, and Prussian blue, showed a wider pH adaptive range, better

246

electrochemical stability and larger response current to the reduction of H2O2.77

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2.3. Scavenging of Hydroxyl Radical

249 250

●

251

radical chemistry. Most notably ●OH is produced from the decomposition of hydroperoxides

252

(ROOH) or, in atmospheric chemistry, by the reaction of excited atomic oxygen with water.80

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●

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where trace amounts of transition metal ions in their lower oxidation states catalyze peroxide-

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mediated oxidations of organic compounds.80 Since ●OH attacks almost all organic compounds

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relatively non-selectively and oxidize them via H-atom abstraction or addition to double-bonds

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with second-order rate constants ranging in between 106-1010 M-1s-1,81 some antioxidant

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scientists consider ●OH scavenging assays rather irrelevant.82 Yet, in many recent antioxidant

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activity assays using novel techniques, antioxidants are tested for their defensive power against

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a Fenton-type oxidizing system, because these ROS generation systems generally produce a

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mixture of reactive species along with ●OH.82

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OH is highly reactive and consequently short-lived,79 however it forms an important part of

OH is also produced during UV-light dissociation of H2O2 and likely in Fenton chemistry,

●

OH scavenging can be conventionally determined using the “deoxyribose assay”.83

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This assay is based on {Fe(III)+EDTA+H2O2} plus ascorbic acid system to generate a constant

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flux of ●OH. These radicals attack the sugar deoxyribose (used as target), degrading it into a

265

series of fragments, some or all of which react upon heating with thiobarbituric acid (TBA) at

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low pH to give a pink chromogen.84 Hence the scavenging activity towards ●OH of a substance

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added to the reaction mixture is measured on the basis of the inhibition of deoxyribose

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degradation.34 This method was recently used to evaluate the antioxidant activity of wheat,

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bran, flour, shorts and feed flour85, raspberries, blackberries, red currants, gooseberries and

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Cornelian cherries86, ginger extract (Zingiber officinale)87, Ocimum basilicum L. and Origanum

271

vulgare L. extracts.88

272

●

OH may be identified by making use of their ability to form nitroxide adducts from the

273

commonly used DMPO spin trap.89,90 The adduct DMPO–OH radical exhibits a characteristic

274

ESR response.91 Furthermore, these adducts have relatively long half-lives, whereas the life

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span of ●OH is very short. Spin trapping has become a valuable tool to characterize and quantify

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oxygen radical.92 Using this method, scavenging effect towards ●OH was evaluated for phenolic

277

compounds of Castanea sativa Mill. leaf (sweet chestnut),89, apple pomace,91 and phlorotannins

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isolated from Ishige okamurae.90

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In another study performed by Cao et al.,93 a spin labeled fluorescence probe (Figure 4)

280

based on rhodamine B was developed to detect ●OH in vitro and in the cells under oxidative

281

stress condition induced by rotenone (an inhibitor of the mitochondrial respiratory chain

282

complex I). The product was detected by HPLC-MS, and according to the results the main

283

product of the reaction was found to be ortho-methylhydroxylamine. The product peak areas

284

measured by HPLC-UV/vis and HPLC-FLD were observed to increase proportionally with the

285

increase of ●OH concentration93.

286

Figure 4

287

An electrochemical method for the determination of antioxidant capacity via

288

nanoparticles was reported.94 In this study, photocatalytic oxidation of water by UV-illuminated

289

TiO2 nanoparticles was selected for the generation of ●OH, and 4-hydroxybenzoic acid (4-

290

HBA) was used as a trapping agent for the photogenerated ●OH, leading to the formation of

291

3,4-dihydroxybenzoic acid (3,4-DHBA) subsequently measured by voltammetry. According to

292

this method, the antioxidant scavenging ability could be correlated with the decrease of 3,4-

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DHBA peak current. Antioxidant capacity of standard substances were measured with the

294

proposed method and compared to the findings of a fluorometric method utilizing illuminated

295

TiO2 as the ●OH source and therephthalic acid as probe, producing the fluorescent 2-

296

hydroxyterephthalic acid upon hydroxylation.

297 298

2.4. Scavenging of Hypochlorous Acid

299 300

HOCl is an oxidizing agent generated by the H2O2-mediated oxidation of chloride, catalyzed

301

by myeloperoxidase. Oxidation of Cl- with H2O2 catalyzed by heme enzyme myeloperoxidase

302

(MPO) causes formation of HOCl.34,95 HOCl may tend to inactivate α1-antiprotease by

303

oxidizing methionine residue and trigger tissue damage resulting in a wide range of human

304

diseases96 although HOCl has antibacterial effect on microorganisms.97 Some compounds such

305

as thiols, thioethers,98 methionine and vitamin C99 might help to protect against tissue damage

306

by scavenging HOCl or inhibiting HOCl production by MPO.95 MPO can be assayed by

307

measuring HOCl production, which is one of the several ways to assay mylepreoxidase.100 In

308

this type of assay, an absorbance change is resulted by reaction of HOCl with

309

monochlorodimedon.95,100 For examination of scavenging of HOCl, methods are described with

310

different modifications in several publications. Basically, adjusting the NaOCl solution to pH

311

6.2 with sulfuric acid can produce HOCl and concentration of HOCl can be calculated by using

312

its molar absorptivity at 235 nm.101

313

In another study, possible mechanism for the yield enhancement of 8-oxo-7,8-dihydro-

314

2’-deoxyguanosine in the reaction of 2’-deoxyguanosine with HOCl was investigated in the

315

presence of (-)-epigallocatechin gallate. In this study a 20-fold increase of 8-oxo-7,8-dihydro-

316

2’-deoxyguanosine by (-)-epigallocatechin gallate was observed compared with the reaction

317

without (-)-epigallocatechin gallate.102

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The protection of scavengers against inactivation of α1-antiprotease is generally focused

319

on determining good scavenging activity of these compounds. Therefore, reactions with HOCl

320

were examined by using the elastase assay to determine the antioxidant activities of

321

compounds.103 This method was used in two publications to report antioxidant and prooxidant

322

effect of gallic acid, its esters, and constituents of rosemary extract.104,105

323

MPO/NaCl/H2O2 system was also used to determine antioxidant activity of tomato products. In

324

this system, the reaction mixture is prepared with phosphate buffer (pH 6.0), NaCl, H2O2, MPO,

325

1-aminocyclopropane-1-carboxylic acid (ACC), and extracts of tomato products with different

326

concentrations in phosphate buffer or acetone. Ethylene released from ACC was measured after

327

incubation at 37°C for 30 min.106,107 The reaction scheme for ethylene production from HOCl

328

mediated oxidation of aminocyclopropane-1-carboxylic acid (ACC) is provided in Figure 5108.

329 330

Figure 5

331

Garlic derivatives were examined for their HOCl scavenging capacities by catalase

332

protection assay in which elimination of the catalase peak due to the destruction of the heme

333

prosthetic group by HOCl was monitored spectrophotometrically.109 In another study, the

334

hydrophilic extract of mana cubiu, an Amazonia fruit, was found to be a potent HOCl

335

scavenger. HOCl scavenging capacity was measured with the increase in fluorescence intensity

336

due to the oxidation by HOCl of dihydrorhodamine (DHR) to rhodamine.110 The scavenging

337

effect of centaury (Centaurium erythraea Rafin.) infusion was exhibited with a different

338

method by measuring the inhibition of TNB (2-nitro-5-thiobenzoate) oxidation to DTNB (5,5’-

339

dithiobis(2-nitrobenzoic acid)) induced by HOCl (Figure 6).110,111 It was first shown by Von

340

Frijtag Drabbe Künzel et al.112 that the oxidizing properties of HOCl induced the conversion of

341

TNB (λmax = 412 nm) to DTNB (λmax = 325 nm). However, a deeper insight into the reaction

342

mechanism of thiol oxidation by HOCl revealed that the initial oxidation product was sulfenyl

15



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343

chloride which further reacted with the thiol to give disulfide as the predominant product.113 It

344

should be borne in mind that since DTNB (also known as Ellman’s reagent) is used to

345

colorimetrically determine thiols (utilizing thiol-disulfide exchange), this reaction cannot be

346

used to measure the HOCl scavenging activity of thiol-type antioxidants. Using the same

347

method, antioxidant activities of Cardoon (Cynara cardunculus L.)114 and tronchuda cabbage

348

(Brassica oleracea L. var. costata DC)115 were also reported. The potential of lycopene as a

349

scavenger of HOCl has been mentioned by emphasizing that the oxidation of lycopene by HOCl

350

gives metabolites and causes color change.97 Reaction of lycopene with increasing

351

concentrations of HOCl yielded a range of metabolites resulting from the oxidative cleavage of

352

one or more C = C double bonds, where HOCl may directly mediate the degradation of lycopene

353

by nonselective cleavage at any double bond position.97 Recently, a novel fluorometric method

354

has been developed to assess the scavenging activity of HOCl, in which chlorination of a

355

fluorogenic probe, resorcinol (1,3-dihydroxybenzene), with HOCl to non-fluorescent products

356

was inhibited by antioxidants acting as HOCl scavengers.116 The assay is based on the

357

chlorination of resorcinol to its nonfluorescent products in the presence of HOCl. Resorcinol

358

and HOCl scavengers compete for HOCl as it may react with both. The relative increase in the

359

fluorescence intensity of intact resorcinol is proportional to the antioxidative activity of HOCl

360

scavengers.116

361

Figure 6

362 363

2.5. Scavenging of Singlet Oxygen

364 365

1

366

blue, and safranine. Singlet oxygen may behave as a more selective oxidant than other ROS,

367

and can give rise to specific damage at selected sites of probes; 2,5-dimethylfuran was proposed

O2 can be generated in the laboratory by photosensitizer dyes such as rose bengal, methylene

16


Page 17 of 84


368

as an indicator of 1O2, and gave a thin layer chromatography (TLC)-detectable product upon

369

scavenging of 1O2.117 Another selective scavenger of 1O2 was p-nitrosodimethylaniline (RNO),

370

and it was observed that some 1O2 acceptors (like imidazole derivatives) induce the bleaching

371

of RNO as followed spectrophotometrically at 440 nm; since 1O2 does not react chemically

372

with RNO, this bleaching was a consequence of 1O2 capture by the imidazole ring which

373

resulted in the formation of a trans-annular peroxide intermediate [1O2] capable of inducing the

374

bleaching of RNO (-RNO).118 Singlet oxygen is reactive as a dienophile toward the furan ring,

375

where dicarbonyl compounds are produced through decomposition of the initially formed

376

endoperoxide adducts. For example, 1,3-diphenylisobenzofuran having the largest rate constant

377

for singlet oxygen quenching yielded o-dibenzoylbenzene via an endoperoxide.119

378

Photosensitizer dyes used in the generation of 1O2 were reported not to interfere with the

379

proposed RNO assay. A commercially available reagent (‘1O2 sensor green’, SOSG), which

380

was found to be selective for 1O2 and not responsive to other ROS, was applied to a range of

381

biological systems known to be 1O2 generators (including wounded leaves in the dark), and 1O2-

382

induced increases in SOSG fluorescence was noted to follow the increase in conjugated diene

383

levels.120

384

dihydroxypropyl)-9,10-anthracenedipropanamide, was found to be a chemical trap of singlet

385

molecular oxygen to be utilized in biological investigations.121 Recently, to increase the yield

386

of 1O2 trapping, diethyl-3,3′-(9,10-anthracenediyl) bisacrylate (DADB) was synthesized as a

387

lipophilic fluorescent probe, and was found to react with 1O2 at a rate of k = 1.69×106 M−1s−1

388

forming a stable endoperoxide (DADBO2), which was characterized by UV-Vis, fluorescence,

389

HPLC/MS and 1H and 13C NMR techniques122; this compound showed the potential for further

390

applications in biological systems.

A

hydrophilic

and

non-ionic

anthracene

derivative,

the

N,N’-di-(2,3-

391

A study on the measurement of 1O2 quenching rates and the relative 1O2 absorption

392

capacity values of rice bran extracts was performed. The concentrations of antioxidants were

17



Page 18 of 84

393

determined using HPLC-MS/MS, UV-HPLC, and UV-Vis absorption spectroscopy. It was

394

concluded that the 1O2 absorption capacity method is applicable to general food extracts to

395

evaluate their 1O2 quenching activity.123 Additionally, 1O2 absorption capacity method was used

396

to evaluate the 1O2-quenching activity of food extracts including tomato, spinach, red paprika,

397

carrot, Chinese leck, pumpkin, cucumber, broccoli, green pepper, okra, egg plant, cabbage,

398

onion, sweet potato, mandarin, orange melon, banana, strawberry, apple and green melon.124

399 400

2.6. Scavenging of Nitric Oxide Radical

401 402

NO

403

the Griess reagent, after conversion to nitrite,125,126 because nitrite is the only stable end product

404

of the autoxidation of NO in aqueous solution.127 The principal oxidation product of NO in

405

aerated or oxygenated sodium phosphate buffer at pH = 7.4 was NO2- .128 The overall reaction

406

(consisting of several steps) of oxidation of NO to NO2- in aqueous solution can be represented

407

by the equation (Eq. 1):

408

4NO + O2 + 2H2O → 4 NO2- + 4H+

generated from a sodium nitroprusside system can be spectrophotometrically measured by

(Eq. 1)

409 410

The NO scavenging ability of some flavonoids was measured, and anthocyanidins were found

411

to be the most effective class.129 A gold nanoparticle-based modification of the Griess reaction

412

system for colorimetric nitrite determination (using 4-aminothiophenol (4-ATP) modified gold

413

nanoparticles and naphthylethylenediamine as coupling agent for azo-dye formation) has been

414

described (Figure 7) elsewhere.130

415 416

Figure 7

417

18


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418

Recently, Wang et al.131 synthesized CdSe−ZnS nanocrystals as fluorophores and

419

surface bound tris(N-(dithiocarboxy)sarcosine)iron(III) as reactive centers for NO. The

420

fluorescence of the QDs was quenched by energy transfer between the excited QD cores and

421

the surface bound iron(III) dithiocarbamates, where NO selectively restored the fluorescence

422

of the QDs through reduction of the surface bound iron(III) complexes to iron(I)−NO

423

adducts.131

424

In another study, NO scavenging activity of Phyllanthus emblica was evaluated. After

425

the methanolic extraction of dried fruit rind of P. emblica, it was separated into hexane, ethyl

426

acetate, and water fractions. Among these, only the ethyl acetate phase showed strong NO

427

scavenging activity in vitro, when compared with water and hexane phases. Then, the ethyl

428

acetate fraction was subjected to separation and purification using Sephadex LH-20

429

chromatography. Spectral methods including 1H NMR, 13C NMR, and MS were used to identify

430

NO

431

corilagin, furosin, and geraniin have significant NO scavenging activity and geraniin showed

432

the highest activity among all the isolated compounds.132

scavenging activity of ethyl acetate fractions. It was found that gallic acid, methyl gallate,

433 434

2.7. Scavenging of Peroxynitrite Anion

435 436

ONOO− is a potent and relatively short-lived oxidant with a half-life of approximately 1 s under

437

physiological conditions (at pH 7.4 and 37 oC), and is highly diffusible across cell

438

membranes.133 The protonated form of ONOO− is peroxynitrous acid (ONOOH) which is a very

439

strong oxidant.134 ONOO− forms an adduct with carbon dioxide dissolved in body fluid under

440

physiological conditions, and the secondary radicals (oxo-metal complexes, lipid peroxyl

441

radicals, OH, NO2, etc.) derived from this adduct are responsible for the oxidative damage to

442

proteins.134-136 In addition to the generation of a prooxidant species, the formation of ONOO−

19



Page 20 of 84

443

results in decreased bioavailability of NO, therefore diminishing both its salutary physiological

444

functions.137

445

Characterization of herbs including witch hazel bark, rosemary, jasmine tea, sage,

446

slippery elm, black walnut leaf, Queen Anne’s lace and Linden flower was performed in terms

447

of their ONOO− scavenging activities with the use of a fluorometric method. The results of this

448

study indicated that witch hazel had the strongest effect for scavenging ONOO−. Moreover,

449

hamamelitannin was obtained as a major active component of witch hazel bark, shown to have

450

a strong ability to scavenge ONOO−.138

451

There are several fluorogenic compounds which have been used to determine ROS

452

concentrations at the single cell level. 2,7-Dichlorodihydrofluorescein (DCDHF), commonly

453

known as dichlorofluorescin, and dihydro-rhodamine 123 (DHR-123) are often used to detect

454

the production of ROS/RNS in cells via oxidation to their respective fluorescent products. To

455

determine which biological oxidants might be involved, DCDHF and DHR-123 were exposed

456

to a number of oxidants in vitro to determine which are capable of oxidizing these compounds.

457

These methods are based on the oxidation of the reduced nonfluorescent forms of fluorescent

458

dyes such as fluorescein and rhodamine by ONOO− to produce the parent dye molecule,

459

resulting in a dramatic increase in fluorescence response.139 Glebska and Koppenol140 showed

460

that the oxidations of dichlorodihydrofluorescein and dihydrorhodamine probes by

461

peroxynitrite were zero-order with respect to the concentrations of either probe in the pH range

462

of 3-10, and that the yield of the respective oxidation products, dichlorofluorescein and

463

rhodamine, significantly increased at pH values exceeding 7. The observation that oxidized

464

products were produced with higher efficiency at higher pH was an indication of adduct

465

formation of peroxynitrite anion with the probe, followed by protonation and oxidation of the

466

probe.

467

20


Page 21 of 84


468

The method of Kooy et al.141 was based on the inhibition of the oxidation of DHR 123

469

by ONOO−. The initial rate approach was used to quantify ONOO− scavenging capacity. This

470

approach is especially useful for demonstrating the effectiveness of fast-reacting

471

oxidants/antioxidants, and involves stopped-flow spectrophotometry at a fixed wavelength with

472

a mixing time of reactants less than 2 ms, where a high number of absorbance measurements

473

are recorded during the initial part of the reaction (generally within first 0.2 s) and evaluated by

474

kinetic analysis. In order to obtain information for the formation of peroxynitrite and its possible

475

fate in biological systems, it is relevant to understand and characterize the kinetics of its

476

reactions with biomolecules. Initial rate approach is a direct method for studying the reactivity

477

of peroxynitrite in which peroxynitrite decomposition is followed by the decrease of its

478

absorbance.142 Using the same method, Chung et al.143 studied the ONOO− scavenging and

479

cytoprotective capacity of a marine algae extract. On the other hand, Pannala et al.144

480

investigated the ability of hydroxycinnamate antioxidants to decrease ONOO−-mediated

481

nitration of tyrosine. According to their results, all compounds were able to inhibit nitration of

482

tyrosine. However, this method relies on the time-consuming HPLC separation and

483

quantification of nitrotyrosine.144

484

Folic acid and reduced folates can also act as ONOO− scavengers.145 Folic acid (FA-

485

OH) is thought to be oxidized through its 4-OH group to further products by peroxynitrite, in

486

the following sequence of reactions: FA-OH → FA-O• → final oxidation products.146 The final

487

oxidation products are possibly 10-nitro-folate and 12-nitro-folate.147 The analytical

488

significance of this reaction arises from the oxidative conversion of the reduced, low-

489

fluorescent folic acid by peroxynitrite to a high-fluorescent emission product.148 Using this

490

information, a fluorescent probe with folic acid was designed for a fluorometric method, based

491

on the oxidation of the reduced, low-fluorescent folic acid by ONOO− to produce a highly

492

fluorescent emission product.148 Compared to the commonly used probe DHR-123, the

21



Page 22 of 84

493

fluorescent probe with folic acid has some advantages including higher sensitivity that is

494

desirable for probing of ONOO− in lower concentrations, greater photostability, commercial

495

availability, and not being toxic to biological systems.12

496

Another fluorometric method for the determination of ONOO− was proposed by Liang

497

et al.149 together with possible reaction mechanisms. The method is based on a mimetic enzyme

498

catalyzed reaction with hemoglobin as the catalyst and L-tyrosine as the substrate, and was

499

reported to be a simple and highly sensitive method with a detection limit of

500

5.0 x 10−8 mol L−1 of peroxynitrite. Also, an electrochemical method for ONOO− was

501

proposed, based on direct voltammetric detection of ONOO− on a mercury film electrode

502

(MFE) at alkaline pH.150

503

2.8. Scavenging of Peroxyl Radical

504 505

ROO found in biological substrates is commonly used in antioxidant assays. It is less reactive

506

than OH and has an extended half-life of seconds instead of nanoseconds.33 Assays of

507

examining ROO• scavenging using azo-compounds as initiators (for peroxyl radical generation

508

at a relatively constant flux) were extensively used.151 A variety of foods were characterized in

509

terms of their ROO scavenging activity by oxygraphic method which was based on a rigorous

510

kinetic model. According to the Peroxyl Radical Trapping Efficiency (PRTE, the reciprocal of

511

the amount of food that reduces the steady-state concentration of peroxyl radicals to half152)

512

values, the potency of trapping ROO was in the following order: blueberry > red

513

chicory > coffee > pineapple ≈ red

514

chocolate ≈ apple ≥ tea > pomegranate.153

wine

≥

orange

≈

dark

515

Bentayeb et al.154 worked on the oxygen radical absorbance capacity (ORAC) assay to

516

better understand its properties regarding the possible synergistic or antagonist interactions

517

among the antioxidant constituents of real samples. Basil (Ocimum basillicum), cinnamon

22


Page 23 of 84


518

(leaves) (Cinnamomum zeylanicum), citronella grass (Cymbopogon winterianus), clove

519

(leaves) (Syzygium aromaticum), dill (seeds) (Anethum graveolens), oregano A (Origanum

520

vulgaris), oregano B (O. vulgaris), red thyme (Thymus vulgaris ct. thymol), rosemary

521

(Rosmarinus officinalis), thyme (T. vulgaris) essential oils were studied in this contribution.

522

The results showed that 72–115% of the antioxidant capacity of essential oils corresponded to

523

the additive property of the antioxidant capacity of their constituents.

524

Wu et al.155 aimed to develop a reliable extraction procedure and assay to determine

525

antioxidant activity in meat products, and to assess the effect of beef finishing system (forage-

526

finished: alfalfa, pearl millet or mixed pastures vs. concentrate-finished) on longissimus muscle

527

antioxidant activity by the ORAC assay. Casettari et al.156 determined the capacity of

528

polysaccharides to scavenge ROO by ORAC, and synthesized four different grafted chitosan

529

derivatives in slightly acidic aqueous media. Ma et al.157 tested the antioxidant properties of

530

mango fruit extracts with ORAC and other assays for their total antioxidant capacity. ORAC-

531

fluorescein (ORAC-FL) was studied on human milk for determining its oxidative stability, and

532

the ORAC–FL assay was standardized for human milk through linearity, precision, and

533

accuracy to enable the measurement of AOA against thermal-induced ROO attack. ORAC-FL

534

was used because it offers advantages due to greater sensitivity and photostability and the

535

specificity for AOA against ROO.158 Carbonneau et al.159 aimed to synthesize red sorghum

536

anthocyanidins and compare their ORAC values. Atala et al.160 designed a work to develop an

537

ORAC assay capable of evaluating the antioxidant capacity of single phenolic compounds and

538

their complex mixtures (wines, fruit juices and teas) under stomach-like acidic conditions.

539

AAPH was used as the ROO source, and fluorescein, pyranine and pyrogallol red (PGR) were

540

employed as target molecules. Only PGR showed a behavior compatible with ORAC assay

541

under acidic conditions (ORAC-PGR). López-Alarcón and Lissi 161 have proposed a modified

542

ORAC-like methodology that employs pyrogallol red as target molecule, and this method

23



Page 24 of 84

543

(ORAC-PGR bleaching) was used to evaluate the scavenging activity of berry extracts

544

(blackberry, blueberry, and raspberry).160 López-Alarcon and Lissi161,162 showed that ORAC-

545

FL method can be used for single antioxidants and/or complex mixtures. In this regard, Alarcon

546

et al.163 used ORAC-FL and ORAC-PGR together in order to evaluate and compare the ORAC-

547

index of herbal infusions and tea extracts. Alternatively, plant samples may be shipped to

548

Brunswick Laboratories, Norton, MA, an independent contract laboratory specializing in

549

standardized testing of natural products, and analyzed for total antioxidant capacity in a panel

550

of ORAC assays, including hydrophilic and lipophilic ORAC, hydroxyl radical averting

551

capacity (HORAC), peroxynitrite radical averting capacity (NORAC), and superoxide radical

552

averting capacity (SORAC) assays.164

553 554

3. IN VIVO ANTIOXIDANT ACTIVITY/CAPACITY: OXIDATIVE STRESS

555

BIOMARKERS AND CELLULAR BASED ASSAYS

556 557

The term ‘in vivo antioxidant capacity’ generally refers to the antioxidant defenses of biological

558

fluids (such as plasma or serum), tissues or biomacromolecules (i.e. lipid, protein, and DNA)

559

against oxidative stress. The TAC increase in plasma or serum after consumption of certain

560

antioxidants may indicate an absorption/in vivo stability of the tested antioxidants and an

561

improved in vivo antioxidant defense status (e.g., free HCl-containing gastric juice may degrade

562

the condensed phenolics/tannins of black tea into simpler phenolic units).165 An increased

563

antioxidant capacity in plasma or serum may not necessarily be a desirable condition if it

564

reflects a response to increased oxidative stress (such as the observed increase in serum TAC

565

levels of patients with chronic renal failure, due to elevated uric acid content).166 Similarly, a

566

decrease in plasma or serum antioxidant capacity may not necessarily be an undesirable

567

condition if the measurement reflects decreased production of reactive species.165 The TAC of

24


Page 25 of 84


568

plasma/serum, resulting from either a radical overload or intake of dietary antioxidants, can be

569

regarded to provide a more representative picture of the in vivo balance between oxidizing

570

species and antioxidant compounds.167 While appropriate in vitro data is accepted to provide

571

important information for clinical studies, it is difficult to extrapolate from an in vitro assay to

572

an in vivo situation, and supporting evidence of in vivo antioxidant capacity is considered to be

573

the most helpful.168 It is difficult to transfer antioxidant mechanisms established in model

574

systems and in foods to the in vivo situation, because no simple relationship has been reported

575

between TAC determined for foods/beverages and health benefits for humans.169 Clinical

576

studies clearly demonstrate that the antioxidant status in vivo can be altered by diet, but the

577

response is dependent upon factors such as (i) TAC of food, (ii) amount of food consumed, (iii)

578

type of phytochemicals and their content, (iv) absorption/adsorption, bioavailability, stability

579

and metabolism of the dietary antioxidants in the body, (v) and the matrix of the food material

580

hence possibly the fructose content particularly of fruits and berries consumed in the diet.170

581

The capacity of antioxidants in vivo against oxidative stress is determined by several

582

factors such as bioavailability covering a wide range of physico-chemical phenomena, namely

583

absorption/adsorption, transportation, distribution between aqueous and lipid phases,

584

retainment/storage, stability, metabolic transformation, dietary interactions and excretion of

585

antioxidants. Thus, in vivo antioxidant testing should normally take into account all these

586

physico-chemical phenomena as well as the competitive actions of enzymes, endogenous

587

antioxidants, and possible oxidative/prooxidative cellular effects. In vivo measurements are

588

difficult owing to problems relating to cellular uptakes of the antioxidants and the transport

589

processes.20 Unfortunately, although in vivo testing methods are usually conducted on

590

biological substrates, most involve many other probes, indicators and reactions irrelevant to

591

actual conditions within the cell and thus may not reflect the true complexity of physiological

592

antioxidant defenses of an organism. Additional problems may arise from undesired side-

25



Page 26 of 84

593

reactions of the probes and from their non-homogeneous distribution in the studied system,

594

production of secondary ROS by the probes themselves, perturbation of the systems under

595

investigation by the probes, and artifacts due to the presence of ROS in the reaction medium.10

596

Novel in vivo assays are recommended to be designed with the use of suitable combinations of

597

oxidant and target/probe species (i.e. biological macromolecules and cell models) with relevant

598

biological significance and under realistic reaction conditions (with respect to time,

599

concentration and pH) as close as possible to those found in vivo.11

600

ESR and electron paramagnetic resonance (EPR) can detect unpaired electron spins of

601

free radicals. Short-lived radicals must be trapped with spin trapping agents, typically an

602

organic nitrone or nitroso compound to form relatively stable and detectable NO adducts. Most

603

probes (e.g., the spin trap agent DMPO: 5,5-dimethyl-1-pyrroline N-oxide, used for detecting

604

biologically relevant radicals) cannot be directly administered to humans because of unknown

605

toxicity at the high levels that would be required for radical trapping in vivo. It must also be

606

remembered that reaction products (such as DMPO- OH adduct) giving rise to an ESR signal

607

can be rapidly removed in a cell culture medium as a result of enzymic metabolism and

608

reductive action of endogenous antioxidants (e.g., ascorbate).171 One often overlooked

609

disadvantage is that many antioxidants react with the nitroxide spin adduct as well as the free

610

radical, making it difficult to distinguish if the decrease observed in the formation of spin adduct

611

is due to the scavenging of the free radical or the reduction of the spin adduct.3 Urate, being an

612

endogenous antioxidant, may be used as a nontoxic trap, where one of its oxidation products,

613

allantoin, can be measured in human body fluids such that elevated plasma allantoin levels may

614

be used as an indication of oxidative stress. Acetyl salicylate may also be used in vivo for a

615

similar purpose. During in vivo detection of OH, the produced hydroxylated compounds from

616

a salicylate probe can be measured directly, because in vivo radical metabolism of salicylic acid

617

produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids as DHBA

26


Page 27 of 84


618

isomers). While the 2,5-derivative can be also produced by enzymatic pathways (i.e. through

619

the cytochrome P-450 system), the 2,3-derivative is solely formed upon hydroxyl radical attack

620

to salicylate. Aromatic hydroxylation products of 2,3- and 2,5-dihydroxybenzoates (with 49%

621

and 40% yields, respectively), along with some catechol (11%), formed from a salicylate probe

622

upon the attack of hydroxyl radicals generated by a Fenton system were first demonstrated by

623

Grootveld and Halliwell.172 Later on, HPLC detection of 2,3-DHBA with an electrochemical

624

detector following oral administration of the drug acetyl salicylate was proposed for assessment

625

of oxidative stress in vivo.173

626

Antioxidant activity can be indirectly evaluated by monitoring levels of oxidative stress

627

with the use of biomarkers that can be used to assess oxidative damage to lipids (including F2-

628

isoprostanes, lipid hydroperoxides, malondialdehyde, 4-hydroxy-2-nonenal, and the

629

hydrocarbons, ethane and pentane), proteins (including protein carbonyls and nitrotyrosine) and

630

DNA (via 8-hydroxydeoxyguanosine assay).171,174 Biomarkers of oxidative stress measure the

631

oxidative conversion or hazard in the target biomacromolecule, and antioxidants, when present,

632

cause a decrease in this hazard, enabling an indirect determination of in vivo antioxidant

633

activity. Unfortunately in most cases, this inhibitive action of antioxidants is not directly

634

measurable (e.g., increasing levels of reactive species exposition does not directly correspond

635

to a decrease in antioxidant stock in cell cultures), and consequently there is a gap between the

636

concepts of antioxidant activity and oxidative conversion, though within a general frame they

637

are inversely related.175 Biomarkers of oxidative stress, generally working on lipid, protein and

638

DNA substrates, have been well summarized171 and tabulated in a recent comprehensive

639

review176.

640

The oxidative hazard on lipid substrates can be measured by means of ethane and

641

pentane in exhaled gas, lipid hydroperoxides usually measured by iodometry or ferric

642

thiocyanate test, aldehydes and ketones−covering malondialdehyde (MDA) usually measured

27



Page 28 of 84

643

by ‘thiobarbituric acid-reactive substances’ (TBARS) test, conjugated dienes, isoprostane and

644

neuroprostane176. Malondialdehyde (also known as the primary end product of the oxidative

645

degradation of unsaturated lipids) forms a 1:2 adduct with TBA that can be measured either

646

spectrophotometrically (at 532 nm) or fluorometrically (at 553 nm).47 The iodometric ‘peroxide

647

value’ test is almost 70 years old, and forms the basis of current methods, in which lipid

648

hydroperoxides (ROOH) and peroxides (ROOR’) oxidize aqueous iodide to the triodide

649

complex (I2 + I- → I3-) which is then titrated with standard thiosulfate solution using starch as

650

end-point indicator.20 Due to the possible addition of I2 to C=C double-bonds, oxidation of

651

iodide by dissolved oxygen and variable reaction kinetics of different peroxides, this test has

652

limited selectivity toward lipid peroxides. The ferric-thiocyanate test for ROOH makes use of

653

Fe(II)-Fe(III) oxidation by peroxides, which is then converted into a red-colored product

654

absorbing at λmax=500 nm (such as FeSCN2+ and other Fe(III)-SCN- complexes, depending on

655

ligand concentration) with the addition of acidic thiocyanate solution.177,178 This test is also

656

affected by iron(III)-complexing agents such as phenolic compounds which may drastically

657

change the Fe(II)-Fe(III) oxidation potential. The ferrous-xylenol orange (Fe(II)-XO) assay for

658

hydroperoxides, abbreviated as the ‘FOX’ assay, was originally developed to determine the

659

levels of lipid hydroperoxides in plant tissues.179 The FOX assay relies on the rapid peroxide-

660

mediated oxidation of ferrous to ferric ions in acidic medium, with subsequent formation of

661

Fe(III)-xylenol orange complex absorbing at 560 nm. Hydrogen peroxide as well as lipid

662

hydroperoxides (emerging as initial products of lipid oxidation) are capable of responding to

663

the FOX assay.180 In a linoleic acid model system, Fe(II) is oxidized by lipid hydroperoxides

664

to Fe(III), which forms a blue-purple colored complex with XO, having maximal absorption at

665

550 nm. Antioxidants in biological systems limit hydroperoxide formation, and attenuate the

666

ferric-XO absorbance. The absorbance maximum shifts to longer wavelengths in a membrane

667

phosphatidylcholine hydroperoxide solution containing 60% MeOH - 40% H2O with an

28


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668

increase in molar absorptivity.181 The procedure has been adapted for determination of

669

lipoxygenase activity in plant extracts.182 The secondary oxidation products of lipids, such as

670

aldehydes and ketones represented by MDA, are usually determined by the spectrophotometric

671

TBARS test,183 in which the MDA end-product forms a pink-colored complex (λmax=532-535

672

nm) with thiobarbituric acid (TBA). A significant improvement to the TBA test was made by

673

using HPLC to isolate the (MDA-TBA) chromogen before analysis.184 However, due to its low

674

specificity for lipid peroxides, the simple colorimetric TBA test was regarded as unacceptable

675

in modern research on antioxidants and oxidative stress, simply because most TBA-reactive

676

material in human body fluids is not related to lipid peroxidation.171 Our own experience with

677

the simple TBARS test confirmed that the decrease in chromophore formation was not

678

significantly related to either the antioxidant capacity or quantity of antioxidants used for

679

protecting lipids (in a linoleic acid emulsion model system) from oxidation.184 On the other

680

hand, isoprostanes, usually quantified by chromatographic and mass spectrometric techniques,

681

are considered by Halliwell and Whiteman171 as specific end products and therefore good

682

biomarkers of lipid peroxidation.185,186

683

Protein oxidation by ROS/RNS results in modifications such as loss of certain parent

684

amino acid residues, formation of unstable intermediates, and generation of stable products;

685

these modifications can be utilized to quantify protein damage,187 and hence to measure

686

antioxidant activity preventing this damage. The oxidative hazard on protein substrates can be

687

measured with the aid of protein carbonyls, hydroperoxide- and aldehyde-modified and cross-

688

linked proteins, disulfides (-SS-), sulfinic (-SO2H) or sulfonic (-SO3H) acids through sulfenic

689

acid (-SOH) intermediates, albumin dimers and other cleavage products.176 The most frequently

690

used biomarker of oxidative protein hazard is probably the carbonyl assay,188,189 formed by

691

protein glycation with sugars, protein binding of aldehydes (including lipid oxidation products),

692

and oxidation of amino-acid side chains by ROS/RNS, however only a small selection of

29



Page 30 of 84

693

proteins ‒mostly on fibrinogen‒ is oxidized, and carbonyl formation is not regarded as a

694

specific marker of protein oxidation, mainly because bound aldehydes and glycated protein are

695

also measured.171 Several antibody techniques exist to detect proteins modified by unsaturated

696

aldehydes emerging as lipid peroxidation products (such as 4-hydroxynonenal).190 The possible

697

oxidation products of proteins that can be used as stable biomarkers of oxidative damage were

698

excellently reviewed by researchers who showed that oxidative modification can involve direct

699

fragmentation or may provide denatured substrates for intracellular proteolysis.191-193

700

Denaturation involves protein unfolding and increased accessibility of peptide bonds (to

701

proteases). All amino acids in bovine serum albumin (BSA) were susceptible to modification

702

by both OH and O2−, though tryptophan, tyrosine, histidine, and cysteine residues were

703

particularly sensitive.192 Endogenous levels of free or protein-bound ortho- or meta-tyrosines

704

have also been used to implicate hydroxyl radical formation in vivo; likewise, nitrotyrosine is

705

often thought to be a specific marker for the attack of ONOO− upon proteins.171 In general, it

706

can be concluded that developing biomarkers of oxidative stress via measurement of protein

707

damage is more complex than similar efforts on lipids and DNA, and reliable novel methods

708

are required for the quantification of oxidation products formed from amino acids, peptides,

709

and proteins, which can be applied to complex biological systems.187

710

Mechanisms of oxidative damage to DNA involve abstractions and addition reactions

711

by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of

712

heterocyclic bases, thereby yielding a wide variety of base and sugar modification products that

713

can be quantified by GC-MS (with selected ion monitoring) or LC-MS techniques. The

714

measurement of multiple products (including oxidized purines and pyrimidines, strand breaks,

715

and C-8 hydroxylation products of guanine, frequently estimated as the oxidized

716

deoxynucleoside, 8-oxo-7,8-dihydro-2’-deoxyguanosine or 8-oxodG) should be preferred over

717

that of a single product, because product levels vary depending on reaction conditions and the

30


Page 31 of 84


718

redox status of cells.194 Although the most commonly measured marker of oxidative DNA

719

damage is 8-oxodG, or its deoxyribonucleoside (8-oxodGuo), LC or GC estimates with

720

electrochemical or MS detection of the concentration of either markers in DNA of normal

721

human cells vary over a range of three orders of magnitude, probably due to adventitious

722

oxidation of guanine during isolation of DNA, sample preparation or the analysis itself.195

723

Effects of dietary antioxidant supplementation on levels of 8-oxodG or other base damage

724

products in vivo seem limited171, questioning the validity of in vivo assays of antioxidant activity

725

based on inhibition of the oxidation of DNA bases.

726

On the other hand, new biomarkers of oxidative stress examination were assessed for

727

the effect of endotoxin lipopolysaccharide on various antioxidants in plasma in an animal model

728

(Göttingen mini pigs). Time and dose effect of endotoxin lipopolysaccharide on blood plasma

729

levels of total antioxidant capacity was investigated to detect whether it results in a loss of

730

antioxidants from plasma. It was found that compared with the controls, significant losses in

731

total antioxidant capacities were not found for animals that were injected with two doses of

732

endotoxin lipopolysaccharide at multiple time points.196

733

Cellular-based antioxidant activity assays (CAA) are performed within the cell medium,

734

and are claimed to be biologically more relevant than the corresponding chemical ‘test tube’

735

assays due to their better consideration of certain physico-chemical aspects of the medium such

736

as uptake, distribution (e.g., cell permeability) and metabolism of antioxidants within cells.197

737

López-Alarcóna and Denicola198 considered that antioxidant action is not limited to quenching

738

reactive species but includes upregulation of antioxidant and detoxifying enzymes, modulation

739

of redox cell signaling and gene expression, and therefore recommend to move to cellular

740

assays in order to assess the antioxidant activity of a compound or extract. Wolfe and Liu199

741

used the non-fluorescent probe 2’,7’-dichlorofluorescin (DCFH) entrapped in human

742

hepatocarcinoma HepG2 cells, and this probe was easily oxidized to the fluorescent product

31



Page 32 of 84

743

dichlorofluorescein (DCF) by ROO derived from ABAP decomposition. Antioxidants prevent

744

the oxidation of the probe and subsequently attenuate the cellular fluorescence, which enables

745

the determination of their concentration by an area under curve (AUC) approach to the

746

decreased fluorescence curve compared to that of the control cells. The authors reported a

747

number of reserves to avoid misinterpretations of CAA results, such as photoreduction of DCF

748

under visible light in the presence of reducing agents, redox cycling by newly generated radicals

749

in the presence of O2, incomplete trapping of probe at second exposition of cells, leakage of

750

DCFH from certain cells, and decreased DCFH oxidation with increasing endogenous

751

antioxidants such as reduced glutathione (GSH).197 It is also surprising that, contrary to the

752

TAC findings of other known assays, important antioxidant compounds such as ascorbic acid,

753

gallic acid, caffeic acid, and catechin had less than 10% of the activity of quercetin in the CAA

754

assay, while phloretin, resveratrol, and taxifolin had activity only at doses much higher than

755

their cytotoxic concentrations.197,199 Isoflavones had no cellular antioxidant activity, and CAA

756

results did not correlate with those of ORAC.199 The general criticisms of Halliwell &

757

Whiteman171 directed to CAA are:

758

(i) possible interference of enzymic and non-enzymic endogenous antioxidants to the

759

measurement procedure,

760

(ii) the intrinsic nature of cell culture process itself imposing oxidative stress, both by

761

facilitating the generation of reactive species and by preventing adaptive upregulation of

762

cellular antioxidants,

763

(iii) the inability of total fluorescence measurements (e.g., plate readers) to distinguish between

764

intracellular and extracellular fluorescence from chemical reactions in the culture medium,

765

(iv) the inability of DCF fluorescence measurement to specifically differentiate several reactive

766

species. In order to minimize erroneous interpretations of CAA results, one should always

767

consider ‘what, how, and how much’ reactive species are trapped and measured.171

32


Page 33 of 84


768

Rational use of cellular probes requires understanding and quantitation of the

769

mechanistic pathways involved, of environmental factors such as oxygen and pH, of the

770

reactivity, distribution and possible intermediary products of the probe, together with

771

photochemical stability, instrumental artefacts, and the fact whether the measured antioxidants

772

actually compete with the probe for reactive species.200 In another study, total antioxidant

773

activity of lipophilic and hydrophilic tomato extracts using cell-based assay was determined by

774

targeting on synergistic actions between tomato antioxidants. For the evaluation of total

775

antioxidant activity, extracts were assayed either alone or in combination with in vitro chemical

776

tests (trolox-equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power

777

(FRAP)) and cell-based assays using human hepatoma (HepG2) and human histiocytic

778

lymphoma (U937) cells. While ferric reducing antioxidant power (FRAP) assay detected

779

additive action between lypophilic and hydrophilic extracts, a slight synergistic action was

780

found in total antioxidant activity that was measured by the TEAC assay. Synergistic action

781

was better determined using U937 and HepG2 cells.201 Recently, López-Alarcón and

782

Denicola198 have reviewed cellular-based assays in comparison with chemical assays of

783

antioxidant activity.

784

4.

785

CHEMOMETRIC ANALYSIS)

MISCELLANEOUS

METHODS

(CHROMATOGRAPHIC

ASSAYS

AND

786 787

4.1. Chromatographic Methods

788 789

In view of the large differences among TAC assays16,17 including variability of reaction times,

790

end-point detection and media,18,19 several tests had to be implemented at the same time for full

791

evaluation of antioxidant activities of complex samples.20 On the other hand, identification and

792

quantification of individual antioxidants with the aid of chromatographic techniques are of

33



Page 34 of 84

793

critical importance and have been used for many years.21 The most frequently used method

794

includes liquid chromatography (LC) with diode array (DAD) and/or mass spectrometric (MS)

795

detection. UV/vis absorption (DAD) is used primarily for quantification but can also be used

796

for the identification of flavonoid subclasses. MS, tandem MS (MS2), and ion trap MS (MSn),

797

with electrospray (ESI) or atmospheric pressure chemical ionization, are usually used for

798

identification and structural characterization.202 On the other hand, in many research studies the

799

correlation between the content of phenolic compounds and their antioxidant capacity was

800

investigated. For example, according to Aaby et al.,203 the highest correlations between

801

electrochemical characteristics and antioxidant activities were found between electrochemical

802

responses and antioxidant activities obtained in the FRAP assay and in the DPPH (1,1-

803

diphenylpicryl-2-hydrazyl radical) assay after short reaction periods. Lower correlations were

804

observed between electrochemical responses and antioxidant activities obtained in the ORAC

805

assay. Indeed, a positive linear correlation between antioxidant activity and phenolic content of

806

several food materials including olive oil,204 barley,205 some herbs,206 and herbal plants75 was

807

reported. Moreover, according to Piluzza & Bullitta207 phenolic content could be used as an

808

indicator of antioxidant properties.

809

On the other hand, there are some methods which report the direct measurement of

810

antioxidant activity by using chromatographic techniques. Bertelsen et al.208 established a

811

convenient

812

chromatographically purified fractions of plant extracts. The assay electrochemically

813

determines the myoglobin-catalyzed oxygen consumption following addition of the fractions

814

to methyl linoleate. Since the oxygen consumption rate decreases with increasing antioxidant

815

activity, the method was used for systematic screening of naturally occurring antioxidants.

and

fast

method

for

detecting

the

relative

antioxidant

activity of

816

In another study, Ronowicz et al.209 investigated the antioxidant activity of Ginkgo

817

biloba extract based on their chromatograms. Chemometric analysis of the samples was carried

34


Page 35 of 84


818

out using hierarchical and non-hierarchical segmentation algorithms. The antioxidant activity

819

was successfully predicted based on the chromatographic description using a regression

820

method. The correlation between the predicted and experimental values was 0.949.

821

Lucio-Gutiérrez et al.210 predicted the antioxidant activity of Turnera diffusa using PLS

822

regression on chromatographic data. In this study, chromatograms were recorded with a diode

823

array detector and an enhanced fingerprint of each sample was constructed by compiling into a

824

single data vector at different wavelengths. It was reported that the proposed regression model

825

with four latent variables could be applied to an external prediction set, retrieving a relative

826

standard error of 7.8% for prediction. The authors indicated that antioxidant activity could be

827

related to chromatographic peaks at the selected wavelengths.

828

Similarly, Zhang et al.211 investigated the antioxidant activity of Epimedium from multi-

829

wavelength chromatographic fingerprints. Different statistical methods were used to construct

830

fingerprint matrix. A calibration model was formed between fingerprints and their antioxidant

831

activities by applying variable selection and PLSR. It was suggested that chromatographic

832

fingerprints can be used for predicting the antioxidant activity of Epimedium at the wavelengths

833

studied.

834

Şahin et al.212 predicted the antioxidant activity of Prunella L. samples from

835

chromatograms by employing PLS or a combination of orthogonal signal correction and partial

836

least squares methods. It was reported that the models developed were able to predict the total

837

antioxidant activity of samples with a precision comparable to that of the reference ABTS and

838

DPPH methods.

839

Ma et al.213 investigated the antioxidant marker compounds in North American and

840

neotropical blueberry species by applying multivariate statistics to data from LC-TOF-MS

841

analysis and antioxidant assays. It was reported that 44 marker compounds including 16

842

anthocyanins, 15 flavonoids, 7 hydroxycinnamic acid derivatives, 5 triterpene glycosides, and

35



Page 36 of 84

843

1 iridoid glycoside were identified. The authors suggested that application of multivariate

844

analysis to bioactivity and mass data could be useful in identification of pharmacologically

845

active natural products.

846

Mnatsakanyan et al.22 investigated the antioxidant profiles of espresso coffee samples

847

using HPLC with UV-absorbance detection and two simultaneous, online chemical assays that

848

enabled the relative reactivity of sample components to be screened. In the study, online DPPH

849

decolorisation and acidic potassium permanganate chemiluminescence assays were found to be

850

similar, while differences in selectivity reflected the complex array of antioxidant species

851

present in the samples. Chromatograms generated with the chemiluminescence assay contained

852

more peaks, which was ascribed to the greater sensitivity of the reagent towards minor, readily

853

oxidizable sample components.

854

On the other hand, Moon & Shibamoto214 reviewed different antioxidant assays for plant

855

and food components including malondialdehyde/high performance liquid chromatography

856

(MDA/HPLC) assay, and malondialdehyde/gas chromatography (MDA/GC) assay. The

857

principle of these methods was based on the determination of the exact amount of MDA formed

858

from lipid peroxidation by using an HPLC or a GC system.

859 860

4.1.1. Online Chromatographic Antioxidant Capacity Measurement

861 862

Over the past two decades, a number of analytical methods measuring antioxidant activity have

863

been developed, most of which are based on the ability of an antioxidant to quench free radicals

864

by hydrogen atom donation. The same chemical reactions have recently been used for online

865

HPLC-coupled methods, which not only intend the rapid measurement of antioxidant activity

866

but also allow profiling of antioxidants in complex mixtures following their chromatographic

867

separation from the matrix. As in batch colorimetric assays, the reduction reaction leads to a

36


Page 37 of 84


868

significant shift in the UV-Vis spectrum, and the change in absorption of a compound can serve

869

as a quantitative measure of antioxidant potential.215 This approach has been applied to the

870

characterization of several antioxidant phytochemicals. Among many TAC assays reported in

871

literature, only electron transfer (ET)- and mixed-mode assays (but not hydrogen atom transfer

872

(HAT)-based assays) were found to be compatible with online HPLC-post column techniques.

873

The most widely used online assays are free radical scavenging assays based on the

874

stable free radicals ABTS+ and DPPH. A schematic lay-out of the online antioxidant assays

875

using HPLC is presented in Figure 8. All these antioxidant activity assays online with liquid

876

chromatography were reviewed by Niederländer et al.216 The use of ABTS+ for online detection

877

of radical scavengers was introduced for the first time by Koleva et al.217 This method was

878

adapted for online determination of the antioxidant activities of separate components of fruit

879

juices.218 Same method was also used for detecting radical scavengers in different plant extracts

880

such as Potentilla fruticosa.219 Online ABTS assay was further used to characterize antioxidant

881

compounds in raspberry,220 green and black tea,221 coffee,222 and tomatoes.16,223 Moreover,

882

Exarchou et al.224 used both ABTS and DPPH online assays to detect radical scavenging

883

compounds in four plant extracts. Furthermore, Li et al.225 reported an online ABTS assay

884

combining DAD-MS for identification of antioxidants in Radix Angelicae sinensis, a

885

commonly used traditional Chinese medicine. In recent years, online antioxidant detection with

886

ABTS+ has been used for various other materials such as strawberry,226 black bamboo

887

leaves,227 black currents, blueberries, cranberries, red currents,228 South African herbal tea

888

rooibos,229 Athrixia phylicoides (another South African herbal tea),230 blue-berried honey

889

suckles, bilberry,215 and sour cherry.231

890 891

Figure 8

892

37



Page 38 of 84

893

Koleva et al.232 published the first paper on the use of the DPPH for detecting radical

894

scavenging compounds online after chromatographic separation using HPLC. The method was

895

applied to several pure natural antioxidants including eugenol, isoeugenol, carnosic acid,

896

kaempferol, quercetin, rutin, rosmarinic acid, α-tocopherol and Trolox®. The same group

897

improved the sensitivity of this method233 and the improved method was used to detect active

898

constituents in thyme leaves234 and sweet grass.235 The group of Bandoniene and Murkovic has

899

performed the online DPPH method to detect antioxidants in apples,236 borage,236 and sage.237

900

Later, the method was further improved for the screening of free radical scavenging compounds

901

in Lamiaceae plants.238 Bartasiute et al.239 also established a modified method to improve the

902

in vivo predictability of an online HPLC stable free radical decoloration assay for antioxidant

903

activity in methanol–buffer medium. The researchers modified the widely used DPPH online

904

decoloration assay. In this study, a medium which includes an aqueous buffer at physiological

905

pH was applied, resulting in the rapid establishment of equilibrium. The results obtained in an

906

aqueous medium at physiological pH are expected to be more relevant for extrapolation to in

907

vivo circumstances than previously published findings.

908

Combination of DPPH assay with mass spectrometry was applied for the rapid

909

identification of antioxidants in the Thai medicinal plant Butea superba.240 Similarly, combined

910

HPLC-DAD-SPE-NMR system with DPPH method was used to investigate the antioxidants in

911

rosemary extracts.241 Moreover, changes in radical scavenging activity and components of

912

mulberry during maturation were studied using online DPPH assay.242 Furthermore, online

913

HPLC-DPPH method was employed for the detection of antioxidants in Acacia confusa243 and

914

Balanophora laxiflora,244 which are traditional medicinal plants in Taiwan. Recently, online

915

measurement of antioxidant capacity using DPPH was also carried out for several other

916

antioxidant rich substances including olive leaves,245 flower buds of Lonicera species,246

38


Page 39 of 84


917

Hibiscus rosa-sinensis L. Flowers,247 blueberry,248 Livistona chinensis fruits,249 coffee,250

918

Sonchus oleraceus leaf251 and tea.252

919

An adapted post-column FRAP assay which was modified from Benzie & Strain253 was

920

performed by Raudonis et al.254 for screening the antioxidants in strawberries. In the study,

921

ABTS and FRAP post-column techniques were evaluated and compared according to the

922

validation parameters including specificity, precision, limit of detection, limit of quantification

923

and linearity. Both assays were performed under the same experimental conditions, and

924

therefore comparable results were obtained. According to the results, ABTS and FRAP post-

925

column assays were specific, repeatable and sensitive and thus could be used for the evaluation

926

of antioxidants in complex mixtures. On the other hand, it was reported that the precision values

927

are influenced by the instability of the baseline. Baseline stability is critical for the ABTS post-

928

column assay, since ABTS is a colored reagent. However, for the FRAP post-column assay,

929

this is not a problem as Fe(III)-TPTZ is converted into a colored Fe(II)-complex after reacting

930

with antioxidant compounds, and therefore its baseline is stable. Similarly, FRAP assay was

931

reported to have advantages for the LOD and LOQ values which define the sensitivity of the

932

assays, since the instability of the baseline might have a negative effect for the post-column

933

ABTS method. It should be remembered that the short residence time in the post-column reactor

934

may pose a problem for the slow-reacting phenolic acids (and especially for thiols) in the online

935

HPLC-FRAP assay.

936

Recently, a novel online HPLC- Cupric Reducing Antioxidant Capacity (CUPRAC)

937

method was developed for the selective determination of polyphenols (flavonoids, simple

938

phenolic and hydroxycinnamic acids) in complex plant matrices.255 Off-line HPLC-CUPRAC

939

procedure was applied firstly to parsley, celery leaves, nettle,256 then to apple juice257 and apple

940

peels.258 In these off-line assays, a post-column reactor was not used, and the contribution of

941

HPLC-identified constituents of complex samples to the observed TAC were calculated by

39



Page 40 of 84

942

means of their TEAC coefficients and concentrations. On the other hand, online HPLC-

943

CUPRAC was applied (with the use of a post-column reactor enabling the CUPRAC reaction

944

with antioxidants) to Camellia sinensis, Origanum marjorana, Mentha,255 and to elderflower.259

945

Like online HPLC-FRAP, online HPLC-CUPRAC made use of an increase in chromophore

946

absorption in the post-column eluate (instead of an absorbance decrease seen in ABTS and

947

DPPH versions of the combined assay), and therefore baseline stability did not pose a problem.

948

Since the solvent dependency of the CUPRAC assay is considerably smaller than those of other

949

similar assays, a greater variety of flavonoid glycosides could be measured with the online

950

method.259

951

Another less common type of antioxidant assay is based on indirect luminol

952

chemiluminescence detection. In this method, syringe pumps are used to merge streams of

953

luminol and an oxidant (typically H2O2) at a T-piece to generate chemiluminescence. This

954

mixture is then combined with the HPLC eluate at a second T-piece and the resulting solution

955

passes through a reaction coil to a photo-detector. Similar to the online radical discoloration

956

assays, antioxidants are detected as negative peaks as they inhibit the reaction between luminol

957

and oxidants causing quenching of the background chemiluminescence signal.216,260,261

958

A recent research on the online high-performance liquid chromatography−diode-array

959

detector−electrospray ionization−ion-trap−time-of-flight−mass spectrometry−TAC detection

960

(HPLC−DAD−ESI−IT−TOF−MS−TACD) system for the detection of antioxidants

961

in Prunus (P.) mume flowers was performed. DPPH scavenging activity and FRAP value of

962

identified samples were evaluated. According to the results, 78 compounds were identified in P.

963

mume flowers, 21 of which showed DPPH scavenging activity and 32 of which showed ferric

964

reducing activity. Thus, this system was referred to be a promising tool for quality control

965

analysis and antioxidant screening of different food and medicinal matrices.262 In summary,

966

online HPLC-coupled antioxidant measurement assays are simple, have broad applicability,

40


Page 41 of 84


967

and use common instruments, inexpensive and stable reagents, time-saving and non-laborious

968

experimental protocols.

969 970

4.2. Chemometric Methods

971 972

Traditional spectroscopic and chromatographic assays may lead to over/underestimation of

973

antioxidant contents of foods or biological and plant materials due to the interferences with

974

other components such as sugars, ascorbic acid, proteins, etc. present in the food matrix. These

975

problems can be overcome by using chemometric techniques to analyze the spectra, such as

976

PLS, PCA, etc.25 Indeed, the overlapped signals may lead to invalid quantification of analytes,

977

and PLS can be employed to overcome this problem. On the other hand, principal component

978

regression (PCR) which is the combination of PCA and least-squares regression is a

979

multivariate data analysis approach that can be employed to eliminate the influences of

980

background on quantification.263,264 Besides, problems like co-elution/overlapping, as well as

981

retention time shifts and baseline drifts have been overcome by some other chemometric

982

methods including parallel factor analysis (PARAFAC), multivariate curve resolution (MCR),

983

and alternating trilinear decomposition (ATLD). By the help of these techniques, detection of

984

minor components, baseline modeling, resolution of co-eluting peaks, further classification can

985

be performed.265 It was also indicated that these methods retain the second-order advantage that

986

calibration in the presence of unknown interferents can be performed to provide satisfactory

987

concentration estimates.266

988

Chemometrics has been defined as “the application of mathematical and statistical

989

methods to chemical measurements”. Chemometric technique uses information (i.e., spectrum,

990

chromatogram) and chemical values (such as concentration of a component) and establishes a

991

mathematical relationship between the two. It assumes that the chemical parameter (e.g.,

41



Page 42 of 84

992

concentration) is correct and attributes weighings of the spectral information accordingly. The

993

setting up of the model by correlating the information with a chemical index is known as

994

‘calibration’.267

995

Multivariate calibration methods are being successfully applied to instrumental data of

996

a variety of sources (such as spectroscopic and chromatographic) in order to construct

997

predictive models for selected analytes in complex plants and biological materials. Linear

998

calibration models are generally preferred, because these models are simple to apply and

999

amenable to straightforward physico-chemical interpretation.268 PLS model is a multivariate

1000

regression technique commonly used to establish a relationship between reference values for

1001

attributes such as concentration of a certain analyte, and predicted values for that attribute in a

1002

tested sample based upon its spectral or chromatographic features.269 It is also an efficient

1003

statistical prediction technique, especially suitable to small sample data with some correlated

1004

variables.270 To establish a PLS model, the first step is to choose the optimal number of latent

1005

variables.271 Loading plots in the PLS technique can be developed to justify a selection of a

1006

small number of orthogonal factors for construction of a PLS model. These loadings correlate

1007

to the principal components within a defined wavenumber region that account for the greatest

1008

difference between samples in a data set. PLS shows some important advantages:268

1009

(i) PLS model employs full spectral or chromatographic data, a feature critical for the resolution

1010

of complex multi-analyte mixtures;

1011

(ii) Analytical methods can be carried out in a short time, usually with no sample clean-up or

1012

physical separation;

1013

(iii) Calibration techniques ignore the concentrations of all other constituents except a selected

1014

analyte in the tested complex samples.

42


Page 43 of 84


1015

For all the above reasons, PLS is especially suitable for antioxidant determination in complex

1016

matrices, which often presents a complex, high absorbing background, severely overlapped

1017

with those from the analytes.268

1018

However, there are disadvantages to spectral analyses in PLS model.269 This model must

1019

be built based upon tested samples that contain a range of analyte concentrations. These samples

1020

are often difficult to obtain, and as the analyte concentration changes, spectral or

1021

chromatographic features of the matrix may also change in a manner that is not necessarily easy

1022

to predict and which may be difficult to compensate for in the predictive model. The predictive

1023

model needs to be checked regularly to account for optical shifts with spectral calibrations

1024

updated frequently to keep the model accurate. It is time consuming and laborious, and requires

1025

a continuous source of biological material with a wide range of analyte concentration; however,

1026

it is critical if chemometric models are to remain reliable. During the establishment of the PLS

1027

model, operational conditions and the parameter settings are required to be standardized and

1028

kept constant since these factors will affect the reliability of the spectral or chromatographic

1029

data and the resulting rigor of the analytical model.269

1030

Figure 9 shows the flowchart of steps of PLS technique. The reference value collection

1031

and the spectral feature collection need to be performed at the same time for the calibration and

1032

cross validation in PLS regression model. After this model is established and tested, new

1033

samples can be directly analyzed by spectroscopic techniques.

1034 1035

Figure 9

1036 1037

PCA as the most preferred technique is a linear dimensionality reduction technique

1038

which identifies orthogonal directions of maximum variance in the data set and projects the

1039

data into a lower-dimensionality space formed of a sub-set of the highest-variance

43



Page 44 of 84

1040

components.272 This technique reduces the dimensionality of the original data set by retaining

1041

the maximum variability of a large number of variables by few underlying factors (principal

1042

components) without losing the important information. Upon application of PCA technique,

1043

the number of variables in a data set is reduced by finding linear combinations of those variables

1044

which explain some of the data variability.273 Nowadays, some software has been developed to

1045

perform the PCA analysis to reduce the time for data processing. As a result, the obtained

1046

principal components became useful tools for examining the relationships between tested

1047

compounds and properties, looking for similarities or sorting out the outliers.272

1048

Several recent articles highlight the potentiality and applicability of chemometric

1049

techniques in different antioxidant analysis. 1H and 13C NMR-based sugar profiling with PCA

1050

and PLS-discriminant analysis (DA) was performed for 25 samples of herbhoneys and Polish

1051

honeys.274 The 1D and 2D NMR spectra of an artificial honey enabled reliable assignment of

1052

signals originating from mono- and disaccharides. Although no distinct clustering between

1053

honeys and herbhoneys was observed in either analysis, some tendency appeared regarding the

1054

β-glucopyranose and β-fructopyranose content. PLS-DA confirmed the results obtained with

1055

PCA, with only a little more distinct clustering on honey and herbhoney samples. In both

1056

methods, there was a clustering in relation to sucrose content, allowing for fast detection of the

1057

adulterated honey samples.

1058

The HPLC-TOF-MS data of the blueberry species (North American and neotropical

1059

blueberries) and their DPPH scavenging and iron chelating activities were analyzed by PCA

1060

to predict marker compounds contributing to the antioxidant activities of the blueberry samples.

1061

The compositional differences between North American and neotropical blueberries were

1062

determined by chemometric analysis, and 44 marker compounds including 16 anthocyanins, 15

1063

flavonoids, 7 hydroxycinnamic acid derivatives, 5 triterpene glycosides, and 1 iridoid glycoside

1064

were identified.213

44


Page 45 of 84


1065

Using a chemometric approach, the chemical composition, color, and in vitro

1066

antioxidant activity measured using ORAC and DPPH provided a suitable method to

1067

differentiate Brazilian lager and brown ale beers. The highest content of total phenolics and

1068

flavonoids were found for the latter group, and these species could be a source of bioactive

1069

compounds with suitable free radical scavenging properties. Chemometric approachs were

1070

efficiently used to discriminate the type of beer based on instrumental color and total phenolic

1071

compounds, while PCA permitted the evaluation of correlation among the 13 variables and 29

1072

samples simultaneously.275

1073 1074

5. ADVANTAGES AND DISADVANTAGES OF THE METHODS

1075 1076

There are many published techniques for assessing antioxidant capacities of food samples.

1077

Wide variations in analytical techniques make comparisons between various studies harder and

1078

also raise the question whether apparently conflicting results are associated with non-

1079

standardized assay techniques. As a result, it is not expected that a single method can determine

1080

all the antioxidant compounds and it is apparent that each method may have its own advantages

1081

and disadvantages. The principles of the methods such as the radical that is generated, the end-

1082

point of detection, or the required reaction time, vary greatly. Even the methods based on the

1083

same principle, such as ABTS and DPPH, can show several important differences in their

1084

response to antioxidants under certain conditions. The formation of radicals, or their solubility

1085

in different solvent systems, also varies.18,19 The measurement of antioxidant activities,

1086

especially in case of mixtures, multifunctional or complex multiphase systems, cannot be

1087

evaluated satisfactorily using a simple antioxidant test due to the many variables influencing

1088

the results. So it is highly recommended to apply several test procedures to evaluate antioxidant

45



Page 46 of 84

1089

activities.17 In this section, main advantages and disadvantages of the methods described in this

1090

review will be discussed.

1091 1092

5.1. Critical Evaluation of ROS/RNS Methods

1093 1094

The activity of an antioxidant may depend on its reactivity towards particular radicals, its ability

1095

to concentrate near the critical target in the cell or its inhibitory action on radical formation.

1096

However, investigations using a single oxidative agent affecting a single biological endpoint

1097

may give misleading results.276

1098

Most of the assays used to measure scavenging capacity of ROO, including water-

1099

soluble 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) and the lipid-soluble 2,2’-

1100

azobis(2,4-dimethylvaleronitrile) (AMVN), are time-consuming and their application requires

1101

a significant expertise and experience in chemical kinetics. On the other hand, scavenging

1102

capacity of H2O2 requires the performance of a blank measurement that can compromise

1103

precision and accuracy of this method.11 This situation may lead to controversy for the precision

1104

and the accuracy of this method as it might be difficult to differentiate small changes when

1105

there is much larger background absorption. Further, the absorption of samples may change

1106

after reacting with H2O2 and the blank measurement would not be valid.277

1107

DCFH and DHR-123 are fluorogenic probes, which are used to monitor ONOO− that is

1108

considered to be ideal. Rhodamine derivatives have advantages, including photostability, pH

1109

insensitivity over a broad range, giving high quantum yield in aqueous solution and being

1110

excitable at long wavelength. Nevertheless, there are some controversies that criticize the

1111

mechanism of ONOO−-mediated oxidation of fluorescent indicators. Moreover, these

1112

indicators are less suitable for ONOO− formation in vivo and the synthesis of these probes is

1113

difficult. Another fluorescent probe is folic acid that can act as a ONOO− scavenger. This

46


Page 47 of 84


1114

fluorometric method has advantages such as high sensitivity in lower concentrations, greater

1115

photostability, availability, and non-toxicity to biological systems.12

1116

Of the ROS/RNS scavenging methods, O2− quenching measurement may be

1117

inappropriate for the reactions with slow rate constants, and needs convenient equipment. The

1118

appropriate ratio of substrate to enzyme is necessary to form optimum amounts of O2−.

1119

Additionally, this method is not suitable for non-enzymatic antioxidants.168 Although ESR

1120

technique has limited applications to biological tissues due to their high water content, this

1121

problem can be overcome by the use of the spin trapping technique, which involves the

1122

conversion of highly reactive free radicals to relatively inert radicals, followed by ESR

1123

analysis.278

1124

Pulse radiolysis is the most often used technique for measuring the reaction of OH and

1125

antioxidants. This technique requires the specialized equipment and could be expensive.

1126

However, approximations of rate constants can be more easily obtained.168

1127

The method for the quantification of NO scavenging capacity of sulfur-containing

1128

compounds in aqueous solution using an amperometric NO sensor is a relatively simple

1129

method. ESR method is also used for the determination of NO scavenging capacity. The Griess

1130

reaction is frequently used for the assessment of NO production by whole cells or enzymes.

1131

Compared to other methods, this methodology is not simple, requiring the addition of several

1132

enzymatic reagents.11

1133

The methods developed for detecting HOCl and for measuring HOCl scavenging

1134

activity might also have certain disadvantages. In the widely used TNB method, scavengers

1135

containing free thiol groups react with DTNB, so excess of TNB can be found in samples.11

1136

Another method, protein carbonyl assay developed by Yan et al.279 examined inhibition of

1137

formation of carbonyl groups with HOCl; however, carbonyls can be formed by other oxidation

1138

mechanisms so this assay may give conflicting results. In methods using enzymes such as α1-

47



Page 48 of 84

1139

antiproteinase or elastase, potential scavengers can inhibit these enzymes instead of directly

1140

reacting with HOCl, thereby affecting the correctness of results. Enzymatic methods are also

1141

criticized for their labor requirement and time consumption.280

1142

On the other hand, the detection of ONOO− in biological systems was found to be

1143

complicated because of several reasons including; (i) direct isolation and detection of

1144

peroxynitrite is difficult because of the elusive nature of ONOO−, (ii) detector molecules that

1145

can efficiently outcompete the multiple reactions that ONOO− can undergo are required, (iii)

1146

footprints totally specific of ONOO− reactions are not present, (iv) discrimination between the

1147

biological effects of ONOO− versus that of its precursors (NO and O2−) and other NO -derived

1148

oxidants is difficult.137

1149

Chemiluminescence (CL) has gained great significance in many biochemical

1150

applications, due to its important analytical advantages such as higher sensitivity, lower

1151

detection limit, wider linear range, which can be achieved with simpler instruments.281-283

1152

However, several limitations apply to CL analysis, such as: the control of factors that affect the

1153

CL emission; the lack of selectivity because a CL reagent is not limited to just one analyte; and,

1154

finally, like other mass-flow-detection approaches, since CL emission is not constant but varies

1155

with time (light flash composed of a signal increase after reagent mixing, passing through a

1156

maximum, then declining to the baseline), and this emission versus time profile can widely vary

1157

in different CL systems, care must be taken to detect the signal in the flowing stream at strictly

1158

defined periods.284

1159

Total peroxyl radical trapping antioxidant parameter (TRAP) assay was introduced for

1160

assessing the time period where oxygen uptake was inhibited by the plasma during peroxidation

1161

reaction induced by the thermal decomposition of an azo-compound.285 Although the TRAP

1162

assay seems advantageous by initiating lipid peroxidation via producing water-soluble ROO,

1163

it also has some disadvantages. It is difficult to compare the results of this method between

48


Page 49 of 84


1164

laboratories, because too many different end-points have been used. TRAP assay is time-

1165

consuming, complicated, and requires experience to perform. Besides, the time period required

1166

for the colored radical to emerge in the reaction medium in the presence of antioxidants is not

1167

certain for all antioxidants.11,19 In addition, antioxidant capacity is often underestimated as the

1168

value after the lag phase is ignored.286,287 Moreover, oxidative deterioration and antioxidant

1169

preservation of fluorescent probe does not imitate a critical biological substrate.288 The probe

1170

must react with ROO at low concentrations. There should be an impressive spectroscopic

1171

change between the native and oxidized states of probe for sensitivity and there should not be

1172

radical chain reaction over probe oxidation for sensitivity of the TRAP assay.287 Another, more

1173

relevant problem in the TRAP assay originates from the high dilution of plasma required to

1174

produce a suitable lag phase. This dilution makes the propagation chain reaction between fatty

1175

acids “physically” difficult.289,290 Some researchers overcame this problem by adding a small

1176

amount of linoleic acid to the reaction mixture, potentially introducing an additional source of

1177

error.167,290,291.

1178

Among other HAT-based TAC methods, the ORAC assay has certain advantages that

1179

ROO are used as reactants with similar redox potential, pH, and reaction mechanism with

1180

physiological oxidants.292 In addition, the ORAC assay is also compatible with acidic

1181

conditions by using pyrogallol red as target molecule.293 Usage of relevant free radical

1182

generators or oxidants in the ORAC assay provides specificity to this assay.294,295 Furthermore,

1183

ORAC analysis can be applied in a wide range of samples such as fruits, tea, vegetables, dietary

1184

supplements, essential oils, botanicals, medicinal plants, drugs, biological samples.154,165,291,296-

1185

301

1186

inhibition time and inhibition degree of free radical action into a single quantity via AUC

1187

technique.294,296,302 Additionally, protection against oxidative damage from transition metals

1188

and hydroxyl radicals can be measured with the ORAC assay.298 Further, in the ORAC

Moreover, the ORAC method claims to differentiate from other methods on combination of

49



Page 50 of 84

1189

measurements, colored compounds were reported to cause less interference than in other

1190

methods, which may be useful in the analysis of fruits and vegetables.298 On the contrary, the

1191

ORAC assay requires expensive equipment and long time to complete oxidation reaction.295,303

1192

The probes of β-phycoerythrin and fluorescein successively used in ORAC assays caused

1193

problems, because the former interacts with phenolic compounds owing to nonspecific protein

1194

binding while the latter yields unexpectedly high TEAC values for certain compounds that have

1195

a lower ranking in other established methods. In addition, the classical ORAC assay is not

1196

suitable for antioxidant capacity of nonpolar components.304,305 However, lipophilic ORAC was

1197

measured by the addition of a solubility enhancer i.e. randomly methylated beta cyclodextrin

1198

(RMCD) to a dried hexane extract.286,305 On the other hand, perchloric acid and acetone were

1199

used to precipitate and remove proteins from plasma, which can interfere with measurement of

1200

antioxidant capacity.294

1201

The novel nanoparticles-based assays to evaluate antioxidant capacity in natural

1202

products have also been proposed as discussed above. The advantages of these new methods

1203

are the good linearity with polyphenol concentration and their nature of not being affected by

1204

the presence of reducing sugars, fruit acids, or amino acids present in the extracts.198

1205 1206

5.2. Critical Evaluation of In vivo Antioxidant Activity/Capacity:

1207 1208

To date, several different assays were reported to estimate the antioxidant activity of

1209

compounds against oxidative stress both in vitro and in vivo, which can be divided into chemical

1210

methods, biochemical assays (cell-based antioxidant methods), animal models and human

1211

studies. Among these methods, in vitro chemical methods are most widely used, but these

1212

methods seldom take into account the uptake, bioavailability and metabolism of the

50


Page 51 of 84


1213

antioxidants in the body3,306. Animal models and human studies are the best, but both are

1214

expensive, time consuming and not so suitable for initial antioxidant screening.306

1215

Molecular biomarkers are used to test oxidative damage in biomolecules and various

1216

aspects of oxidative stress by free radicals in experimental animals. The most widely used assay

1217

for lipid peroxidation is MDA formation. The method was greatly improved by combination

1218

with HPLC. Another analytical method for measurement of lipid peroxidation is the

1219

determination of diene conjugation from the polyunsaturated fatty acids. This technique is

1220

relatively insensitive to small changes.307 ROS-induced changes to gene expression may be

1221

measured simultaneously using microarray technology. However, the disadvantage of this

1222

method is that microarray technology is expensive. Thus, it is not clear if expression profiles of

1223

cells in biological samples reflect that in cardiovascular tissues.308

1224

Cultured cells have advantages such as several different stressors and cell types

1225

including model systems for some specific disease can be used for evaluation of the antioxidant

1226

effects. As the use of experimental animals will become more difficult in the future, cultured

1227

cells may become more important. The amount of antioxidants, especially lipophilic

1228

antioxidants, added into the culture medium should be chosen carefully to simulate the

1229

physiological conditions.3

1230

CAA is an important method for the evaluation of antioxidant activity in the extracts of

1231

natural products and expresses the potential to exert an antioxidant response at the cellular level,

1232

not just the capacity as a reducing agent.198 On the other hand, it is known that living tissues

1233

are under constant oxidative stress and as a result high sensitivity biomarkers should be used to

1234

measure changes in low background levels of damage. Additional problems are inherent in

1235

studies in vivo, among which is the existing level of antioxidants in the tissues and whether

1236

additional antioxidant is absorbed and accumulated to an extent which significantly alters the

1237

antioxidant status of the tissues.276 Another limitation regarding cell cultures is that they are

51



Page 52 of 84

1238

altered with time leading to the reaction between antioxidants and the medium or they are

1239

neutralized very quickly; thus causing incorrect results.309

1240 1241

5.3. Critical Evaluation of Miscellaneous Methods (Chromatographic and Chemometric

1242

Assays)

1243 1244

The methods based on electrochemical detection are more practical, but have still received only

1245

limited attention for practical screening purposes. The methods based on a single relatively

1246

stable reagent such as DPPH and ABTS+ have become most popular, because of their simple

1247

set-up and ease of control. The methods have been combined with online DAD, MS and NMR

1248

detection for rapid identification of active constituents.216 As is known, HPLC is extensively

1249

applied for quality control of drugs, foods, plants, etc. due to its sensitivity, superior precision,

1250

high resolution and extensive applicability. LC–MS, GC–MS, and LC–NMR have been

1251

increasingly used in complex chemical identification including antioxidants.310 This

1252

advancement in instrumentation is able to generate enormous amounts of data which record

1253

small differences between samples, enabling to provide large implications for discrimination.

1254

Electrochemical approaches are based on the measurement of chemical-physical properties

1255

which are considered as direct tests to evaluate antioxidant capacity because of the absence of

1256

reactive species. Unlike spectroscopic methods, turbid samples can be studied with

1257

electroanalytical techniques.310 On the other hand, chromatographic methods are generally

1258

time-consuming, require special reagents, and in some cases require quite expensive

1259

instrumentation. Therefore, novel, relatively simple and cheap analytical methods for the

1260

simultaneous determination of antioxidants are preferred.311

1261

The quality control of foods including the detection of adulteration by using

1262

chemometrics is also well established and increased in many fields of food science and

52


Page 53 of 84


1263

technology as these techniques are able to extract the maximum amount of information from

1264

chemical data, including chemical composition and antioxidant activity.312 Chemometric

1265

methods are very appropriate to give insight about structure-activity relationships313 and can be

1266

applied for in vitro, in vivo and ex vivo analyses.314 On the other hand, the use of chemometrics

1267

requires understanding of the principles of the method and of the meaning of the individual

1268

input parameters as well as a critical evaluation regarding the obtained results which might be

1269

complicated.33

1270 1271

ACKNOWLEDGMENTS

1272 1273

The authors would like to express their gratitude to Istanbul University-Application & Research

1274

Center for the Measurement of Food Antioxidants (Istanbul Universitesi Gida Antioksidanlari

1275

Olcumu Uygulama ve Arastirma Merkezi).

1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297

LIST OF ABBREVIATIONS AAPH = 2,2’azobis (2-methylpropionamidine) dihydrochloride ABTS = 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid AOA = antioxidant activity AUC = area under curve CL = Chemiluminescence CUPRAC = cupric reducing antioxidant capacity DCFH = dichlorofluorescein DHBA = dihydroxybenzoic acid DHR-123 = dihydro-rhodamine 123 DMPO = 5,5-dimethyl-1-pyrroline N-oxide DPPH = 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl DTNB = 5,5’-Dithio-bis(2-nitrobenzoic acid) EPR = electron paramagnetic resonance ESR = electron spin resonance ET = electron transfer FRAP = ferric reducing antioxidant power HAT = hydrogen atom transfer H2O2 = hydrogen peroxide HOCl = hypochlorous acid 53



1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329

HX = hypoxanthine MDA= malondialdehyde MPO = myeloperoxidase NBT = nitroblue tetrazolium NO = nitric oxide radical NO = nitrogen dioxide radical 2 1 O2 = singlet oxygen 3 O2 = oxygen O3 = ozon O2− = superoxide anion radical OH = hydroxyl radical ONOO− = peroxynitrite anion ORAC = oxygen radical absorbance capacity PCA = principal component analysis PGR = pyrogallol red PLS = partial least squares RO = alkoxyl radical ROO = peroxyl radical ROS = reactive oxygen species RNO = p-nitrosodimethylaniline RNS = reactive nitrogen species SOD = superoxide dismutase SOSG = 1O2 sensor green SRSA = superoxide radical scavenging activity TAC = total antioxidant capacity TBARS = thiobarbituric acid-reactive substances TEAC = trolox-equivalent antioxidant capacity TRAP = total peroxyl radical trapping antioxidant parameter XO = xylenol orange XOD = xanthine oxidase

1330

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2254

Figure Captions

2255

Figure 1. Mechanisms of superoxide radical scavenging by luteolin-iron complexes.

2256

Figure 2. The mechanism of the chemiluminescence of luminol.

2257

Figure 3. The structure of spin-trap (a) DMPO and its hydroxyl and superoxide adducts as

2258

well as (b) C-phenyl-N-tert-butylnitrone (PBN).

2259

Figure 4. The structure of the spin labeled fluorescence probe.

2260

Figure 5. Ethylene production from HOCl mediated oxidation of aminocyclopropane-1-

2261

carboxylic acid (ACC)

2262

Figure 6. The mechanism of HOCl mediated oxidation of DHR to rhodamine.

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Figure 7. Colorimetric sensing/determination of nitrite with 4-aminothiophenol-modified

2264

gold nanoparticles.

2265

Figure 8. Flow scheme for online antioxidant assays using HPLC.

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Figure 9. A quantitative PLS regression model.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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TOC Graphic

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RNS) Scavenging Assays, Oxidative Stress - ACS Publications

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