SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Abstract:  In recent years, much interest has been observed in the field of phenol-based antioxidants. As a result of this, many analytical methods have been developed for the purpose of the quantification of phenolic and polyphenolic antioxidant capacities in biological materials. Many of these methods have been altered for application toward the in vitro assessment of antioxidant activities in animal and human model systems as well as in vivo. Due to the varied applicability and usage, methods for the assessment of phenol antioxidant capacities have become so widespread that they are often misused. It is the intent of this work to review the chemistry behind the antioxidant activity of phenolics as well as summarize the many methods applicable for the measurement of in vitro phenolic antioxidant capacity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Due to the growing popularity of phenolic antioxidants over the past 2 decades, new scientific methods have been developed to measure either the content or antioxidant capacity of phenolics present in plants, foods, and food components (Naczk and Shahidi 2004; Stratil and others 2006; Moon and Shibamoto 2009; Deng and others 2011). Methods have been established to determine phenolics’ antioxidant efficacy in lipid and food model systems (Becker and others 2004; Decker and others 2005; Laguerre and others 2007), as well as gauge relative antioxidative activities of phenolic compounds in vitro (Llesuy and others 2001; Schlesier and others 2002; Apak and others 2007; Yoo and others 2007) and in vivo (Halliwell 2008; Jensen and others 2008; Larrosa and others 2008). Perhaps of particular interest is research directed toward the elucidation of structure-activity relationships of different classes of phenolic antioxidants (Rice-Evans and others 1996; Lien and others 1999; Lemańska and others 2001; Nijveldt and others 2001; Kim and Lee 2004; Hoelz and others 2010). Given the presence of many different antioxidants in biological systems, methods involving the quantification of “total antioxidant capacity” (TAC) are commonplace in today's scientific and medical laboratories. Antioxidant capacity corresponds to the total radical-scavenging capability of a test solution, independent of individual antioxidant activity constants (Ghiselli and others 2000).

Many publications have involved the use of very few antioxidant evaluation methods per source, and there exist notable inconsistencies in the methods employed. The lack of uniformity in the standards used for calibration, assay modifications employed, and the basis for the expression of determinations contributes to inconsistent data and impairs the ability to compare the results reported in the literature. This concern was illustrated in a publication by Bhagwat and others (2007) who showed that the ORAC values of common foods from two different laboratories varied wildly (in reference to the published values of Ou and others 2002; and Wu and others 2004a).

The wide variety of modifications employed in assays measuring antioxidants is undoubtedly due to these assays’ inherent versatility. Many in vitro antioxidant assays can be modified for the production/quenching of several radical species based on the use of a variety of azo-initiator compounds, metal-ion catalysts, and thermal or photodegradative processes. For example, the ORAC assay (Huang and others 2002b; Prior and others 2003) typically measures peroxyl-radical scavenging; but it has been successfully modified for the screening of hydroxyl-radical; a radical species of much greater reaction rate (Ou and others 2002). Given the different chemistries involved in each group of antioxidants and the different rates of reactions in the varying radical-scavenging reactions, the choice of assay can have great effect upon the results obtained. Not all methods and antioxidant sources are compatible, and the same antioxidant species can yield dissimilar results in different assays. This suggests a need to complete a multitude of antioxidant assessment assays on potential phenolic sources, bearing in mind the chemistry involved and the important factors regarding the assays.

Another concern of many of the assays employed is that of external relevance. Though some assays can be quick and easy to perform, they may yield data with little biological significance. For instance, despite being two of the most widely used antioxidant methods, the mixed-mode assays of DPPH (Sánchez-Moreno and others 1998) and TEAC (Re and others 1999) do not involve reactive oxygen species (ROS), but instead organic nitro-radicals. Furthermore, the radical substrates ABTS•+ and DPPH are very large compounds and steric issues may apply when considering the potential docking of incoming reactants.

When chosen and performed properly, antioxidant capacity measurements can produce valuable in vitro data involving the potential capabilities of antioxidant compounds in vivo (although still only in the case of screening; bioavailability, and bioactivity are other issues entirely). Two excellent reviews discussing the strengths and weaknesses of antioxidant methods/techniques have recently been published (Schaich 2006; Frankel and Finley 2008), as well as a review specifically examining the evidences of the in vivo relevance of in vitro phenolic antioxidant assessments (Fernandez-Panchon and others 2008). Though standardization of methods has been suggested (Prior and others 2005), no official antioxidant capacity assays exist to date. However, a review of the scientific literature reveals that certain in vitro methods have emerged as being commonly implemented for the measurement of phenolic antioxidant content and efficacy. The purpose of this work is to discuss these assays, summarize the chemistry of their processes, and address the benefits and drawbacks of each.

Oxidation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Oxidation can be defined as a chemical reaction involving the transfer of electrons between molecules or an electron-rich species to an oxidizing agent (which undergoes a simultaneous reduction). This transfer of electrons between entities can give rise to radicals. In nature, molecules are made of protons, neutrons, and electrons. While protons and neutrons comprise the nucleus of atoms, electrons are left to occupy regions of space outside of the nucleus known as orbitals. In compounds, each molecular orbital can contain a maximum of two paired electrons with opposite spins. Orbital shape (s-1, p-3, d-5, f-7) and orientation (x, y, and z dimensions) will differ from molecule to molecule depending on its composition. A free radical is an atom or molecule that can exist independently with one or more unpaired electrons in its outermost shell. These shells can be atomic or compounded. Given that molecules are most stable in the ground state, radicals are highly reactive species that often do not last long in a given form.

Among the most important oxidation mechanisms in food systems is that of autoxidation of lipids, which will be discussed here as a descriptive example of biological oxidative reactions. These free-radical reactions involve the following mechanisms: (1) initiation reactions in which the number of free radicals increases; (2) propagation reactions in which the total number of radicals remains constant (although the number of radical species may change); and (3) termination reactions in which the number of free radicals decreases. The following reaction schemes (1–5) illustrate these processes:

  • image(1)
  • image(2)
  • image(3)
  • image(4)
  • image(5)

The generated radical (1) can interact with molecular oxygen (3O2) (2) and undergo many subsequent propagation reactions (3) with endogenous or exogenous substrates resulting in a variety of ROS. It is important to note that R (1) is relatively unreactive; however, once it propagates with 3O2 to form ROO, it becomes highly reactive.

The primary products of lipid autoxidation are lipid hydroperoxides (LOOHs), which are not sensorially active. LOOHs are very unstable and degrade to secondary oxidation or scission products, such as aldehydes, ketones, alcohols, and hydrocarbons that can impact food quality. Though many of the in vitro radical generation reactions discussed herein are initiated by chemical (metal-ion catalyst), thermal (heat), and electromagnetic (light) means; there are also important enzymatic radical-generation systems (Hodgson and Fridovich 1976).

Antioxidants

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Although the term antioxidant originally referred to molecules that prevent the consumption of oxygen by human tissues, it has evolved to refer to the prevention of oxidative systems as a whole. An antioxidant is a molecule or species that slows or prevents the oxidation of another molecule, and therefore can be considered as a reductant. It is important to note, however, that not all reductants are necessarily antioxidants. In explanation, “reductant” and “oxidant” are chemical (redox) terms, while “antioxidant” and “pro-oxidant” hold a specific reference to biological systems (Prior and Cao 1999).

Antioxidants can be classified either as “primary antioxidants” (those which actively inhibit oxidation reactions) or “secondary antioxidants” (those which inhibit oxidation indirectly, by mechanisms such as oxygen-scavenging, binding pro-oxidants, etc.) (Shahidi and Wanasundara 1992). Much of primary antioxidant chemistry reactions can be grouped into the categories of hydrogen-atom transfer (HAT) and single-electron transfer (SET), both of which are applicable to the discussion of phenolic antioxidant action. Phenolics are also considered to operate as secondary oxidants due to their ability to bind with potentially pro-oxidative metal ions (Rice-Evans and others 1997).

Hydrogen-Atom Transfer (HAT) Mechanism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

The HAT mechanism occurs when an antioxidant compound quenches free-radical species by donating hydrogen atoms. Reaction scheme (6) below illustrates HAT chemistry: an antioxidant component (abbreviated as an aromatic component [Ar] and a hydroxy component [OH]) donates an H-atom to an unstable free radical and in this process becomes a more stable free-radical species. This more stable species is then less likely to propagate further radical reactions with initiation substrates (Wright and others 2001):

  • image(6)

Figure 1 is an illustration of the conjugated resonance stabilization of phenoxyl radicals. Although the phenoxyl-radical electron (6) initially exists on the highly electronegative oxygen atom, it is likely that the electron is delocalized and shared throughout the aromatic ring. In reaction (6), n represents the stoichiometric factor for the reactant free radical and the resultant phenolic compound. Vitamin E has been shown to react with two peroxyl radicals per molecule (Burton and Ingold 1981). The weaker the hydrogen atom is held to the reactant hydroxy substituent of the antioxidant compound in reaction (6), the more likely and faster it will participate in HAT reactions with free-radical substrates. Therefore, the bond dissociation enthalpy (BDE) of an antioxidant species is a parameter when studying the capacity of a phenolic compound to undergo a HAT in free-radical reactions (Wright and others 2001). That is, the greater the BDE required, the less active a phenolic compound will be in participating in free-radical quenching reactions via the HAT mechanism.

image

Figure 1–. A mechanism of phenolic antioxidant efficacy; conjugative resonance stabilization.

Download figure to PowerPoint

Antioxidant size, chemistry, and polarity play a role in their capacity and speed in HAT reactions (Silva and others 2000). HAT reactions may be hindered by the presence of electron withdrawing groups in the 3- and 5-positions (meta) via deactivation of the aromatic ring (Streitwieser and Heathcock 1981). HAT reactions increase with the presence of t-butyl groups at the 2- and 6-positions (ortho), and methoxy constituents in the 4-position (para) by inductive donation of electron density to help in the resonance stabilization of the generated phenoxyl radical (Howard and Ingold 1963). The p-type lone pair orbital of an oxygen-containing substituent located in the 4-position on a phenolic ring is thought to overlap with the semi-occupied molecular orbital (SOMO) of the generated radical species upon hydrogen abstraction (Burton and Ingold 1986). If, however, the 4-methoxy substituent is forced out of the plane by neighboring alkyl groups, as in the case of TMMP (4-methoxy-2,3,5,6-tetramethylphenol), its p-type lone pair electrons are no longer available to participate in resonance structures with the aromatic ring (Burton and Ingold 1981). Generally, the presence of large substituents on the aromatic ring reduces the capability of free radicals to dimerize with the phenolic hydroxy group by steric crowding (Mahoney 1969), thereby increasing the likelihood of HAT. These parameters give a possible explanation for the strong antioxidant activity observed for the food preservatives butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).

Figure 2 is an example of two sequential hydrogen abstractions incurred by peroxyl radicals resulting in the conversion of l-ascorbic acid (vitamin C) to dehydroascorbic acid, and the resultant creation of two hydroperoxides. Although ascorbic acid lacks phenol chemistry, it does contain two hydroxy functional groups located on a conjugated furan ring, which renders it sufficiently stable to participate in free-radical redox chemistry (Brand-Williams and others 1995). Although generated phenoxyl radicals (6) could terminate with each other (dimerization) or with substrate-radical initiators (complexation), the generated phenoxyl radicals are sufficiently stable to readily react with further substrate stoichiometrically, until fully oxidized (Blois 1958). Rice-Evans and others (1996) offer excellent insight into the structure-activity relationships of phenolic acid and flavonoid HAT reactions. Furthermore, Dangles and others (2000) have assessed the phenomenon exhibited by DPPH HAT mechanisms with 3′,4′,7-trihydroxyflavylium cation and (+)-catechin (as models for anthocyanins and proanthocyanidins [PACs], respectively).

image

Figure 2–. HAT conversion of L-ascorbic acid (vitamin C) to dehydroascorbic acid.

Download figure to PowerPoint

Single Electron Transfer (SET) Mechanism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

The SET mechanism describes the cases where an antioxidant transfers a single electron to aid in the reduction of potential target compounds. The following reaction schemes (7–9) illustrate a SET mechanism, in which an antioxidant transfers a single electron to a ROS. The resultant radical-cationic antioxidant compound is then deprotonated through interaction with water.

  • image(7)
  • image(8)
  • image(9)

The finality of a SET reaction (8) is the same as a HAT reaction (6) in terms of radical scavenging; however, the SET reaction (7) can be subject to further radical-propagation reactions with the extended life of [ArOH]•+ (Wright and others 2001). The resultant antioxidant species from reaction (7) [ArOH]•+ illustrates that even though the radical electron and formal charge do initially exist on the oxygen atom, it is likely that the electron is delocalized and distributed throughout the aromatic ring.

Given that reaction (7) involves the creation of ionic species, the ionization potential (IP) of an antioxidant compound becomes a parameter for predicting the capability of a phenolic species to scavenge free radicals via SET. The greater the ionization energy required, the more reluctant an antioxidant molecule will be to donate an electron (Wright and others 2001). IP decreases with increasing pH, so SET reactions are favored in alkaline environments.

Mixed HAT and SET Mechanisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Leopoldini and others (2004) and Wright and others (2001) assert that although many antioxidant reactions are characterized as following either HAT or SET chemical processes, these reaction mechanisms can, and do, simultaneously occur. Migliavacca and others (1997) assert that α-tocopherol undergoes simultaneous HAT and SET mechanisms with radical substrates, and that these processes are interrelated. Zhang and Ji (2006) corroborate this assertion through studies of the interaction of vitamin E with DPPH in polar protic media, in which both HAT and sequential proton-loss electron transfer (SPLET), also termed proton-coupled electron transfer (PCET) by Huang and others (2005), were found to be thermodynamically favorable reactions. Reaction schemes (10–12) illustrate a SPLET mechanism (Klein and Lukeš 2006).

  • image(10)
  • image(11)
  • image(12)

SPLET reactions represent one of the main mechanistic sources of error in falsely denoting SET reactions as HAT, because they can occur rapidly in certain environs. SET reactions are often slower than HAT ones; therefore, if the reaction kinetics between an antioxidant substrate and free radical are expeditious in a given system, HAT is often assumed to be the predominant mechanism. Figure 3 is an example of a SET mechanism between α-tocopherol and 4-methoxybenzoyloxyl radical, as suggested by Evans and others (1992). Although α-tocopherol can undergo a SET with radical substrates, its radical-scavenging behavior is still thought to be predominantly HAT (Burton and Ingold 1981; Nakanishi and others 2002; Zhang and Ji 2006).

image

Figure 3–. A SET mechanism between α-tocopherol (vitamin E) and 4-methoxybenzoyloxyl radical; adapted from Evans and others (1992). J Am Chem Soc 114:4589–93.

Download figure to PowerPoint

The most prevalent mechanism in any system will depend on antioxidant structure, properties, and medium of interaction (Huang and others 2005; Prior and others 2005). If bulky constituents are located adjacent to phenolic hydroxy groups, steric issues may hinder HAT or SET reactions. Similarly, if antioxidant reactions are carried out in hydrogen bond-accepting environments, HAT efficiency will be greatly reduced (Evans and others 1992; Barclay and others 1999).

Secondary Antioxidative Action by Metal-Ion Chelation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

The presence of ionic metals such as copper and iron in a system can accelerate the rate of oxidation in that system. This action can occur either by the promotion of the decomposition of hydroperoxides, or through the production of hydroxyl radicals by the Fenton reaction (Koppenol 1993; Fernandez and others 2002). The Fenton reaction is shown in reaction schemes (13) and (14).

  • image(13)
  • image(14)

Phenolic compounds have been shown to inhibit the pro-oxidative action of metals by a chelation process, in which the phenolics bind with the metal ions to form a complex incapable of promoting oxidation (Mira and others 2002). By this chelation process, phenolics operate as “secondary” or “preventive” antioxidants; effectively inhibiting oxidation without directly interacting with oxidative species. In phenolic compounds, the 5-OH and/or 3-OH moiety with a 4-oxo group in the A/C ring structure or a large number of hydroxyl groups are important for the binding/chelation of metal ions (Khokhar and Apenton 2003). In flavonoids, the presence of 3′-4′ and/or 7′-8′o-dihydroxyphenyl groups on the B- and A-rings also influences the chelation of metal ions, as does a double bond between the C2 and C3 of flavones (Mira and others 2002). Hydroxyl groups in conjugation with methyl groups or a carbohydrate moiety are not involved in the complexation of metal ions (Andjelković and others 2006). The propensity of transition metal ions to be chelated follows the order: Cu2+ > Fe2+ > Fe3+, which is to be expected according to their relative stabilization energies (Mira and others 2002).

The ability of phenolics to enact chelation in vivo is as of yet uncertain, but the use of phenolics has been suggested as treatment for metal-overload diseases such as hemochromatosis (iron overload) and Wilson's disease (copper overload) (Afanas'ev and others 1995). The extent to which the chelation process explains the observed antioxidative action of phenolics (be it in synergy with HAT and SET mechanisms or in the place of) is an area of ongoing research.

Reactive Oxygen Species (ROS) and the Human Body

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Oxygen is required for life; yet, its reaction products with biological substrates can generate compounds (such as reactive oxygen species or ROS) that can damage health if left unchecked. It has been stated that oxygen is the greatest paradox in biological science. ROS are of interest in biology and biological chemistry due to strong evidence relating them to the pathogenesis of many degenerative diseases and aging in humans (Halliwell and others 1992; Barber and Harris 1994; Halliwell 1996; Hiramatsu and others 1997). ROS have even been implicated in the disruption of cellular signaling pathways, and thus can affect gene expression (Palmer and Paulson 1997). Perhaps the best-known forms of ROS include certain oxygen radicals like the superoxide radical anion (O2•−), hydroxyl radical (HO), as well as alkoxyl and peroxyl radicals (RO & RO2, respectively). In addition to these, there are non-radical oxidizing agents such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), and ozone (O3) (Halliwell and others 1995). Reactive nitrogen species (RNS) such as nitroxyl radicals (NO, NO2) and peroxynitrite (ONOO) also exist and can have adverse effects on human health and disease (Halliwell and others 1992; Halliwell 1996).

Though ROS are often viewed as exogenous and deleterious to mankind, these compounds are naturally present within humans and held in check by multiple defense systems found within the body. These defenses including the following: endogenous antioxidant enzymes (such as catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase), endogenous factors (such as glutathione, co-enzyme Q), metal-ion sequestration systems, and endogenously-generated primary and secondary antioxidants (including vitamin E, vitamin C, and carotenoids) (Machlin and Bendich 1987; Sies 1993; Halliwell 1996). In fact, all vascular cell types produce ROS enzymatically via membrane-associated NAD(P)H oxidase. ROS are involved in many stages of vascular function, including cell contraction/dilation, cell growth, programmed cell death, and inflammation (Touyz 2005; Park and others 2011), various stages of cellular respiration, including mobilization of the electron transport system, oxidative phosphorylation, and, consequently, in various redox signaling pathways (Adam-Vizi 2005).

ROS can become dangerous when present in excess in an animal body. They result in the overproduction of free radicals that can damage multiple components of cells including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), lipids, proteins, carbohydrates, and enzymes (Machlin and Bendich 1987; Aruoma 1994). Furthermore, cellular damage incurred by free radicals can cause further radical production, as well as an increased risk of inflammation, cardiovascular disease, cancer, diabetes, Alzheimer's disease, and age-related functional decline; even though it is perhaps not the primary cause of these diseases (Temple 2000). Of recent interest is the role of redox modulation in insulin signaling as it pertains to vascular endothelial function and possible links to diabetes (Christon and others 2005; Stevens 2005; Estivalet and others 2011). Even though some may think that free-radical research in biological systems is fairly recent, significant breakthroughs in free-radical theory have occurred since the work of D. Harman in the mid 1950s (Niki 1997).

As a recognized contributing factor to the overproduction of ROS, oxidative stress is thought to be linked to many degenerative diseases. For example, oxidative processes are involved in the formation of atherosclerotic plaques within arterial walls and, therefore, can lead to an increased incidence of cardiovascular disease in humans (Steinberg 1991). Oxidized nucleic acids in DNA can be mutagenic and lead to carcinogenesis (Nakabeppu and others 2006). Many inflammatory processes can lead to an increased incidence of oxidative stress; this often results from an overproduction of ROS, which the body's natural defense systems cannot overcome (Sies 1993). Greater nutritional intake of pro-oxidant food sources or prolonged nutrient deficiency can also result in oxidative overload within the body (Sies and others 2005). Although chromatographic and spectrophotometric assays are able to measure the extent of nucleic acid oxidation (Collins 2005), studies in oxidative/antioxidative research have tended toward in vitro antioxidant and radical-scavenging capacity assays, lipid oxidation model systems in foods, and in vivo assays of human biological fluids.

The Role of Antioxidants in Humans

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Antioxidants are important to humans because of the multiple beneficial interactions they may be able to have within our bodies. Often this protection is dependent upon case, type, and location. For example, an antioxidant generated to help protect against lipid peroxidation in human tissues may or may not be able to prevent oxidative stress caused to DNA, proteins, or other compounds. In some cases, they may in fact cause more damage than good (Halliwell 1996). Antioxidant effectiveness in vitro may not correlate with effectiveness in vivo. The human digestive tract can degrade or alter the chemical form of antioxidant compounds as they pass through the stomach, preventing them from being absorbed in the lower intestines, and, therefore, rendering them ineffective at preventing oxidation in the body (Scalbert and Williamson 2000). As a screening process, it is reasonable to assume that if an antioxidant has a poor capability of scavenging free radicals or preventing oxidative reactions in vitro, then it likely will also have poor efficacy in vivo. Given the cost of animal model systems and human intervention studies, cell culture models to directly assess antioxidant effectiveness in vivo have been attempted and hold promise (Liu and Finley 2005; Wolfe and Liu 2007; Araújo and others 2011).

Assessment of antioxidant profiles in human plasma (Polidori and others 2001) and other biological fluids is commonplace as an index of oxidative stress. Past research suggests that exogenously supplemented antioxidants may provide relief from multiple oxidative reactions within humans and act as anti-inflammatory, anticarcinogenic, anticancer, and antiradical agents (Rice-Evans and Diplock 1993; Diplock 1994, 1996). However, the role of such supplementation in eliciting a favorable response in the body is still under debate (Halliwell and others 2005). Some in vivo studies have suggested that supplementing the human diet with antioxidants may not be warranted given the possibility of pro-oxidative reactions. A pro-oxidant effect of supplemented vitamins C and E was observed in in vivo dietary trials (Kontush and others 1996; Paolini and others 1999; Abudu and others 2004). The possibility for pro-oxidative effect by phenolics has been demonstrated in vitro, but this effect been shown to lessen dramatically with decreases in copper ions present (Cao and others 1997; Fukumoto and Mazza 2000; Rufián-Henares and others 2006). To date, neither the pro-oxidative or antioxidative effect of phenol consumption has been adequately substantiated in vivo (Halliwell 2008).

The Role of Antioxidants in Foods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Another important application of antioxidants is their inclusion in food products as preservatives to extend shelf-life and to maintain quality. This has led to the attempted correlation of in vitro antioxidant capacity data with projected capabilities of antioxidants to perform in food systems. Even though antioxidant capacity assays can gauge the relative capabilities of antioxidant components, antioxidant activity in food systems depends on many factors including the antioxidant's physical location in the food, interaction(s) with other food constituents, and the overall conditions of the food environment (pH, ionic strength, hydrophilic/lipophilic balance, and so on) (Decker and others 2005). An antioxidant's efficacy at scavenging free radicals in the aqueous phase depends on its solubility in both the aqueous and lipid phases of a food or beverage. To this end, antioxidant model systems in vitro need to take into account the complex nature of foods if they are to achieve high relevance and accuracy.

Phenolic and Polyphenolic Antioxidants

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

One of the most well-known groups of antioxidant compounds in the scientific literature is the phenolics. Any compound that contains a hydroxy-substituted aromatic ring is a phenolic compound. Phenolics and polyphenolics (polymeric phenolics) can provide relief from certain physical ailments and degenerative diseases in humans, including the reduction of cardiovascular disease and certain cancers (Scalbert and others 2002, 2005; Arts and Hollman 2005). Therefore, it is not surprising that the extraction and analysis of phenolics from plants and other food sources have been extensively studied (Naczk and Shahidi 2004; Dai and Mumper 2010).

Occurrence of Phenolics in the Plant Kingdom

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

In plants, phenolic compounds are metabolized from the amino acid l-phenylalanine and, in some cases, l-tyrosine (Shahidi 2000, 2002). Figure 4 is an illustration of the pathways of production of phenylpropanoids including stilbenes, lignans, suberins, cutins, flavonoids, and tannins. Figures 5 and 6 are illustrations of the enzymatic reactions undergone in the synthesis of phenolic acids (trans-cinnamic and benzoic acids) and flavonoids from phenylalanine. Phenolic compounds exist as a monomeric aglycone or in various bound forms. They are also the building blocks of large polymeric compounds such as tannins (Shahidi and Naczk 2004; Cheynier 2005). Figure 7 is a summary of the current classification of dietary phenolics, including examples. Many phenolic compounds and mixtures thereof are prevalent in a wide variety of fruits, vegetables, grains, and other plant products (Madsen and Bertelsen 1995; Pietta and others 1998; Paganga and others 1999; Adom and Liu 2002; Chu and others 2002; Sun and others 2002; Shan and others 2005; Stratil and others 2006). Research has shown that diets rich in fruits, vegetables, whole grains, and other sources of phenolics can lead to an increased quantity of antioxidants in the human body (Cao and others 1998). Also, phenolics may work together synergistically to improve one's total health status (Liu 2004).

image

Figure 4–. Synthesis of phenylpropanoids from phenylalanine, the origin of phenolics.

Download figure to PowerPoint

image

Figure 5–. Formation of phenylpropanoids from phenylalanine and tyrosine; adapted from Shahidi (2000). Nahrung 44:158–63.

Download figure to PowerPoint

image

Figure 6–. Production of flavonoids and stilbenes from phenylpropanoid (p-coumaryl CoA) and malonyl CoA; adapted from Shahidi (2000). Nahrung 44:158–63.

Download figure to PowerPoint

image

Figure 7–. Classification of dietary phenolics; adapted from Liu (2004). J Nutr 134:3479S–85S.

Download figure to PowerPoint

Phenolic acids

As depicted in Figure 5, phenolic acids of the benzoic and trans-cinnamic acid families are synthesized from l-phenylalanine (and l-tyrosine) in plants. This process is commonly referred to as phenylpropanoid metabolism. Hydroxycinnamic acids are most widely distributed in plant tissues. They are often found in the form of hydroxyacid esters with quinic, shikimic, or tartaric acid residues (Herrmann and Nagel 1989).

Phenolic acids have been associated with many aspects of food quality including color, flavor properties, and nutrition (Maga and Katz 1978). Of the many methods available for their selective separation, RP-HPLC methods with spectrophotometric detection are the overwhelming majority, with gas chromatography (GC) along with derivatization steps being employed to a lesser extent (Robbins 2003). Figures 8 and 9 are UV-spectral scans of phenolic acids of the benzoic acid and trans-cinnamic acid families. While benzoic acids typically yield their primary UV-maximum near 260 nm (especially p-hydroxybenzoic, vanillic, and protocatechuic acids), most trans-cinnamic acids absorb UV-radiation nearer to 320 nm. The inherent differences in UV-spectra exhibited by the two phenolic acid families provide for their selective chromatographic identification.

image

Figure 8–. UV-spectra of phenolic acids in the benzoic acid family.

Download figure to PowerPoint

image

Figure 9–. UV-spectra of phenolic acids in the trans-cinnamic acid family.

Download figure to PowerPoint

Lignans

Lignans consist of two phenylpropane units, which are joined by oxidative dimerization (Ignat and others 2011). Lignans typically exist in nature in their free form, with the glycoside derivatives occurring only in small quantities. Once consumed by humans, lignans are metabolized to enterodiol and enterolactone by the intestinal microflora (El Gharras 2009). It is speculated that there are additional precursors to these products that have not yet been identified (Heinonen and others 2001). Flax seed is considered to be by far the greatest dietary source of lignans, but lignans are also found in appreciable quantities in sesame seed and, to a lesser degree, in a variety of grains, seeds, fruits, and vegetables (Milder and others 2005). Table 1 shows a portion of the prominent dietary sources of lignans and their relative concentration levels. In addition to serving as antioxidants, lignans have been shown to be capable of inhibiting the cancer-promoting effects of estrogen on breast tissue by binding to estrogen receptors (Pianjing and others 2011). The potential effect of lignans upon a variety of cancer types has become a subject of ongoing research (Saleem and others 2005).

Table 1–.  Lignan content of solid foods.a
 Total lignan
Productconcentration (μg/100 g)
Oilseeds and nuts 
 Flaxseed301,129
 Sesame seed39,348
 Sunflower seed891
 Cashew629
 Peanut94
 Poppy seed10
Grain products 
 Multi-grain bread6,744
 Muesli764
 Rye bread (dark)320
Vegetables 
 Curly kale2,321
 Broccoli1,325
 Garlic536
 French bean273
Fruits 
 Apricot450
 Strawberry334
 Peach293

Stilbenes

Stilbenes are a family of hydrocarbons consisting of two phenyl groups joined via an ethene double bond (Leopoldini and others 2011). This double bond may occur naturally in either a cis or a trans configuration, and the stilbenes are generally present in glycosylated forms (Delmas and others 2006). The primary representatives of this family of compounds are pterostilbene, piceatannol, and resveratrol (Leopoldini and others 2011). Resveratrol has received particular attention for its potential health-promoting effects, including reports of possessing cardio-protective, neuro-protective, anticancer, antidiabetic, and antiaging capabilities (Pandey and Rizvi 2011). Resveratrol occurs in more than 70 plant species, including berries and peanuts, but is most commonly associated with red wine, in which resveratrol concentrations have been measured to be 0.3 to 7 mg aglycones/L and 15 mg glycosides/L (El Gharras 2009). The prevalence of resveratrol in red wine is often considered to play a major role in the so-called “French Paradox,” the observation that the people of France experience relatively low occurrences of cardiovascular disease despite high consumption of fats, high occurrence of cigarette smoking, and little exercise (Orallo 2006). Studies have suggested that this observed protection against heart disease could be a result of a combination of antioxidant activity, modulation of lipoprotein metabolism, and vasodilatory and platelet antiaggregatory properties.

Flavonoids

Flavonoids are the most common and widely distributed group of phenolic compounds in plants. As seen in Figure 6, their basic makeup is a diphenylpropane core structure that consists of two outer aromatic rings with a three-carbon bridge, which can be closed (as in flavones, flavanols, and anthocyanidins) or open (chalcones). Flavonoids most commonly occur as glycosides in plants, with some classes consisting of up to 380 variations in their chemical structure (Bravo 1998). In the case of flavonoids, their altered substitution and saturation patterns can result in the production of flavones, flavonols, flavanones, flavanonols, flavanols, and anthocyanidins. The chemical backbones of various flavonoids/isoflavonoids commonly found in plants are depicted in Figure 10.

image

Figure 10–. Chemical backbone of selected flavonoids and isoflavonoids found in plants.

Download figure to PowerPoint

Phenolic polymers

Phenolic polymers, or tannins, were named because of their capacity to bind to proteins in the transformation of animal hides to leather. Tannins can be subdivided into two classes based on their inherent chemical make-up: hydrolyzable and condensed tannins. Hydrolyzable tannins can be further segregated into gallotannins and ellagitannins. Gallotannins consist of gallic acid subunits esterified to glucose. Ellagitannins are simply polymers of ellagic acid and gallic acid. Figures 11 and 12 are examples of gallo- and ellagitannins, respectively. Hydrolyzable tannins are so named because they easily hydrolyze in weak acid or alkali to their individual monomeric units (Bravo 1998).

image

Figure 11–. A hydrolyzable gallotannin (tannic acid).

Download figure to PowerPoint

image

Figure 12–. A hydrolyzable ellagitannin (punicalagin).

Download figure to PowerPoint

Condensed tannins, often called proanthocyanidins or “PACs,” release anthocyanidin monomers when heated in the presence of acid (Cheynier and others 1999). In foods, PACs are usually classified as procyanidins or prodelphinidins according to the chemistry of their flavan-3-ol subunits. Procyanidins are comprised of (−)-epicatechin monomers, whereas prodelphinidins are comprised of epigallocatechin subunits. PACs are subdivided into A- and B-types according to their interflavonoid linkages. B-type PACs are most common and have a C4[RIGHTWARDS ARROW]D8 or C4[RIGHTWARDS ARROW]D6 interflavonoid linkage; whereas, A-type PACs have an additional ether linkage from C2[RIGHTWARDS ARROW]D7 (Ferreira and Li 2000) as seen in Figure 13. PACs can range from dimeric to oligomeric species with many subunits. In fact, decamers with a molecular mass greater than 30 kDa have been reported in cocoa and sorghum (Gu and others 2002).

image

Figure 13–. Condensed tannins, B-type (4[RIGHTWARDS ARROW]8) and A-type (4[RIGHTWARDS ARROW]8 2[RIGHTWARDS ARROW]7) procyanidin dimers (B2 and A2).

Download figure to PowerPoint

Extraction of Phenolics from Plants

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

In order for proper chromatographic analysis, phenolics must first be extracted from their respective plant or food matrices. Extraction efficiency is influenced by analyte particle size, extraction solvent(s), pH, time, temperature, and agitation, as well as the presence of potential interfering substances such as sugars (Naczk and Shahidi 2004). Solubility of targeted phenolic compounds in the selected extraction solvent is largely dependent on their relative polarities. If one is attempting to extract a wide variety of phenolic and polyphenolic constituents from a single plant or food source, the conditions for extraction should take into account the complex nature of the selected compounds. Often this is accomplished through the use of multiple extraction solvents and sequential liquid partitioning followed by the chromatographic analysis of the components in each fraction. Nevertheless, there is no assessment that “exhaustive” extraction of the analytes-of-interest has been achieved. The most common methods of phenolic extraction employed involve pH-buffered aqueous/organic mixtures of methanol, ethanol, acetone, and ethyl acetate (Naczk and Shahidi 2004).

Phenolics in Food

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Along with providing possible health benefits, ingredients rich in phenolics are employed as antioxidants in a variety of food systems (Andersen and others 2005; Brewer 2011). More recently, polyphenolics have been added to functional foods and nutraceuticals to bestow targeted health benefits to consumers. However, the inclusion of phenol-rich components in nutritive foods and beverages needs to be intelligently employed to ensure that the phenolics do not adversely affect sensory attributes of the food (Lesschaeve and Noble 2005) and that they are not significantly biodegraded before reaching their point of absorption in the human body. This is often accomplished by microencapsulation and other stabilization techniques.

Phenolic Bioavailability/Bioactivity after Ingestion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

As discussed, the human body contains a very complex system of chemical and enzymatic defense mechanisms. Once antioxidants enter the body, they do not necessarily pass through unaltered or reach their intended absorption site in the gastrointestinal (GI) tract; hence, bioavailability and bioactivity must be considered. The bioavailability of phenolics and polyphenolics has been studied extensively over the past 2 decades, whether by examining the kinetic patterns of polyphenol absorption in the bodily fluids of healthy volunteers (Manach and others 2005) or by epidemiological intervention studies in hospitals (Williamson and Manach 2005). These studies have, however, yielded conflicting results. Though much knowledge has been acquired involving the absorption of phenolic acids and flavonoids in the GI tract (Scalbert and Williamson 2000), more targeted investigations are warranted.

Dietary origins of polyphenolics have been established including PACs in dark chocolate and ellagitannins in pomegranate, but methods for screening daily intake of these compounds have only recently been developed (Prior and Gu 2005). PACs have gained considerable attention as of late and are quickly becoming the most popular ingredient for natural in vivo antioxidant therapy (Dixon and others 2005). Much of this attention is due to their capability of binding to proteins and surviving passage through the human GI tract. Tannins can also survive certain thermal processes and greatly retard lipid oxidation in foods (Pegg and Amarowicz 2004; Amarowicz 2007). Whether or not the large-scale addition of phenolics to the human diet in the form of supplements or formulated foods is safe and/or of potential benefit is still a matter of debate (Pokorný 2007; Martin and Appel 2010).

Relationship between Phenolic Structure and Antioxidant Activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Another key point of study regarding the antioxidant activity of phenolic compounds is that of identifying and defining the relationships between the structures of phenolic compounds and their relative ability to perform as antioxidants. Within real systems, this relationship will be highly dependent upon the conditions of the system such as substrates, temperature, light, oxygen pressure, relative physical characteristics, polarity, and metals (Chen and Ho 1997; Chaiyasit and others 2007; Shahidi and Zhong 2011). Still, studies have successfully determined generalized relationships between phenolic structures and their relative antioxidant activities when assessed independent of real systems.

In a recent study, Hoelz and others (2010) compared the structural characteristics of 15 common phenolic antioxidants and related those characteristics to their determined antioxidant activity; the comparison of which was used for the creation of a predictive model. In this case, antioxidant potential was measured according to their relative capacity to inhibit peroxide formation in accelerated conditions (Zhiyong and others 2003). They found the structural variables OH bond homolytic dissociation enthalpy (BDE−OH) and ionization potential (IP) to be sufficient to form a successful model, while lipophilicity and relative lipophilicity required no consideration. The best determined equation [pIC50= 6.682 – 0.023(BDE−OH) − 0.0036(IP)] attained an adjusted R2 value of 0.866 signifying a strong predictive power. The result shows that increases in either OH bond homolytic dissociation enthalpy or ionization potential negatively affect a phenolic compound's antioxidant activity, both of which are in agreement with our discussion of HAT and SET mechanisms above. The authors assert their results suggest that the best phenolic antioxidants are compounds, which contain electron donor groups directly attached to an aromatic ring. A summary of their results is shown in Table 2.

Table 2–.  Comparison of physical attributes of certain phenolic compounds with their observed and predicted antioxidant activities.a
CompoundOH bond homolytic dissociation enthalpyIonization potentialpIC50CalcbpIC50Expc% Error
  1. aHoelz and others (2010); bpIC50= 6.682 – 0.023(BDE-OH)−0.0036(IP); cZhiyong and others (2003), measured by lipid peroxide inhibition assay.

o-Coumaric acid84.4188.84.104.140.98
p-Coumaric acid84.9184.94.104.100.00
Ferulic acid84.5177.64.134.150.48
Caffeic acid74.9181.64.344.213.09
Catechol76.4184.54.294.290.00
Pyrogallol77.7183.24.274.310.94
Phloroglucinol87.7188.24.023.981.00
Resorcinol86.1186.94.064.021.00
Hydroquinone80.6178.44.224.281.42
p-Aminophenol76.8163.24.364.421.38
Protocatechuic acid79.6228.24.064.090.74
Gallic acid79.8228.64.064.080.49
Salicylic acid93.0196.63.883.941.55
m-Hydroxybenzoic acid88.9199.03.963.921.02
p-Hydroxybenzoic acid89.2200.33.953.901.28

Another study (Kim and Lee 2004) examined the antioxidant potential of a representative variety of phenolic antioxidants using the Vitamin C Equivalent Antioxidant Capacity (VCEAC) assay (a method conceptually similar to the TEAC assay), and compared the results to structural characteristics. The study was able to determine key emerging patterns. Specifically, antioxidant activity generally increased with increasing number of phenolic rings (meaning polyphenolics are generally more effective than monophenolics), cinnamic acid derivatives generally showed greater antioxidant activity than benzoic acid derivatives, the substitution of sugars into flavonoids resulted in impaired antioxidant activity (speculated to be due to steric hindrance), and antioxidant activity of flavonoids increased in a linear fashion with an increase in free OH groups around the flavonoid framework. The observation of positive correlation with free OH groups was also made by Lien and others (1999), who measured antioxidant potential according to TEAC.

Additional studies investigating such findings, implementing different analytical techniques, and incorporating different assay conditions may still be required for more comprehensive conclusions regarding the structure-activity relationships of phenolic compounds.

Correlation between Phenolics Content and Antioxidant Activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Total phenolics content can have a strong association with the antioxidant activity observed within a system, but this will certainly not always be the case. As addressed previously, the mode of action of antioxidants is complex and may be highly dependent upon a wide range of variables within a system. Many studies have been conducted with specific food sources evaluating the level of correlation between total phenolics content and observed antioxidant activity; the results of which have shown great variation (for example, Di Majo and others 2008; Hu and others 2010; Yosefi and others 2010; Sulaiman and others 2011).

In sources in which a strong correlation is observed, it is typically concluded that phenolics are largely responsible for the antioxidant activities seen within the samples. In sources in which strong correlations are not observed, it is commonly concluded that there are significant amounts of antioxidants other than the measured phenolics present in the system, or that the specific phenolic species present in the system cannot be quantified properly through the total phenol assay. Another key point of consideration here may be the possible synergistic and antagonistic effects that can occur within the system based on additional components, as well as interactions between the phenolic compounds and their physical environment of the food matrix.

Although the phenolics content assay may in many cases provide indication of the potential antioxidant capacity of an extract, it should not be confused with an accurate assessment thereof. This must be evaluated by more direct and specific means.

Quantification of Antioxidant Content and Capacity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Most phenolic antioxidant assessment assays can be grouped according to the chemistry of the reactions involved; meaning it may specifically pertain to the mechanisms of either HAT or SET, or may be a mixed-mode method pertaining to both (Schaich 2006). Also of importance is the TPC, which is commonly used to directly quantify inherent phenol content, as well as methods that evaluate chelation activity. Examples of antioxidant assays are shown according to their categorization in Table 3, and Figure 14 demonstrates the changes in the respective rates of citation for some of the most commonly used assays. A more comprehensive list of in vitro antioxidant assays is beyond the scope of this work.

Table 3–.  Examples of antioxidant assessment assays.
AssayRadical/ion measuredDescriptive reference (listed in order of relevance for each assay)
HAT assays  
 Azo-initiated chemiluminescence (CL)RO2Alho and Leinonen (1999)
 Photochemiluminescence (PCL)O2•−Popov and Lewin (1999a); Pegg and others (2007)
 Total antioxidant reactivity (TAR)RO2Campos and others (1996); Lissi and others (1995)
 ORACFLRO2Huang and others (2002b); Prior and others (2003); Wu and other (2004b)
 TRAPRO2Wayner and others (1985); Wayner (1987); Lussignoli and others (1999)
 Crocin or β-carotene bleaching assaysRO2 
UV-Vis Miller (1971); Kampa and others (2002); Tanizawa and others (1983); Tubaro and others (1998)
 Microtiter plate Mikami and others (2009)
 Total oxyradical-scavenging capacity (TOSC)RO2 and HORegoli and Winston (1999); Winston and others (1998)
 Liposome model systemsRO2 and HORoberts and Gordon (2003)
 Low-density lipoprotein (LDL) oxidation modelsRO2 and HOEsterbauer and others (1992); Frankel and others (1995)
SET assays  
 Cupric reducing antioxidant capacity (CUPRAC)Cu2+[RIGHTWARDS ARROW] Cu+[complexed] 
UV-Vis Apak and others (2004); Moffet and others (1985)
 Microtiter plate Ribeiro and others (2011)
 Ferric reducing antioxidant power (FRAP)Fe3+– TPTZ [RIGHTWARDS ARROW] Fe2+– TPTZ 
UV-Vis Benzie and Strain (1996); Pulido and others (2000); Amarowicz and others (2004)
 Microtiter plate Firuzi and others (2005)
Mixed-mode assays  
 TEACABTS•+ 
UV-Vis Miller and others (1993); Re and others (1999)
 Microtiter plate Kambayashi and others (2009)
 DPPHDPPH 
UV-Vis Hatano and others (1988), Sánchez-Moreno and others (1998)
 Microtiter plate Fukumoto and Mazza (2000)
Chelation assays  
 FerrozineDivalent metal cationsDinis and others (1994)
 Tetramethylmurexide (TMM)Divalent metal cationsShimada and others (1992)
Quantification assay  
 Total phenolics content (TPC)Mo6+[yellow][RIGHTWARDS ARROW] Mo5+[blue]) 
UV-Vis Singleton and Rossi (1965); Folin and Ciocalteu (1927); Singleton and others (1999)
Microtiter plate Zhang and others (2006)
image

Figure 14–. Frequency of citation of popular antioxidant assays.aDetermined according to queries within SciFinder® on August 16, 2011 (see http://cas.org/products/scifindr&index.html). All searches used full names of assays (no acronyms or abbreviations), and results were filtered according to the keyword “antioxidant.” Results were refined to timeframes of 5 years (or 1 year and 7.5 months in the case of 2010–2011) and the number of hits was then divided according to the number of years within the search (1.625 in the case of 2010–2011).

Download figure to PowerPoint

One should not expect the results of antioxidant contents or capacity assessed by a HAT assay to necessarily be compatible (either quantitatively or qualitatively) to that obtained by a SET assay. In fact, it has been reported that they do not directly compare (Bhagwat and others 2007); Table 4 serves to illustrate this phenomenon. One possible explanation of these discrepancies is that different mechanisms of measurement may present different determinations of antioxidant activity according to the particular antioxidant composition of the sample. For example, the antioxidant capacities of foods rich in ascorbic acid have been shown to be underrepresented by ORAC (Ou and others 2001). Another explanation is that different foods, when analyzed, may enact interferences, which have different magnitudes of effect upon different assays. Prior and others (2005) suggest that assessment of SET reactions may be more sensitive to potential interferences than those of HAT reactions. SET reactions often take long periods of time to reach completion, and interfering substances (such as trace components, metal contaminants, and uric acid) can exert a great effect on their measurement. Nevertheless, each assay—be it HAT, SET, or mixed—involves the correlation of an antioxidant's capability to perform in relation to a standard antioxidant compound.

Table 4–.  Comparison of the total phenolics content and antioxidant capacities of fruits and vegetables as measured by the TPC, ORACFL, TEAC, and FRAP assays.
 TPCa,bORACFLc,d,eTEACa,fFRAPa,f
 (mg GAE/100 g FW) ± SD(μmol TE/100 g FW) ± SD(μmol TE/100 g FW) ± SD(μmol Fe2+/100 g FW) ± SD
Fruit/vegetable[Column rank][Column rank][Column rank][Column rank]
Lettuce14±1 [14]1550 [10]171±12 [14]124±7 [14]
Red cabbage158±4 [4]300 ± 30 [14]1377±49 [4]1870±18 [4]
Spinach72±1 [9]2640 [7]757±54 [6]1009±35 [7]
Broccoli128±4 [6]1590 [9]648±25 [8]833±16 [8]
Onion88±1 [8]1029 [11]532±29 [9]369±13 [10]
Tomato30±1 [13]460 [13]255±14 [12]344±7 [11]
Apple48±1 [11]2936 [6]343±13 [10]394±8 [9]
Pear60±3 [10]5235 [2]282±19 [11]315±24 [12]
Orange126±6 [7]1814 [8]849±25 [5]1181±6 [6]
Banana38±4 [12]879 [12]181±39 [13]164±32 [13]
Red plum320±12 [2]6239 [1]1825±28 [3]2057±25 [3]
Blueberry151±19 [5]4848 [4]743 [7]1861 [5]
Strawberry330±4 [1]3577 [5]2591±68 [1]3352±38 [1]
Raspberry228±6 [3]4925 [3]1846±10 [2]2325±53 [2]

Also of relevance to this review are assays, which monitor the effect upon oxidation rates within a system (peroxide value, conjugated dienes, and so on), and these will be discussed.

HAT Assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Oxygen radical absorbance capacity (ORACFL) assay

The ORAC assay was developed by Dr. Alexander N. Glazer in the early 1990s for the determination of ROS in biological systems. The original assay was based on the fluorescence of photosynthetic phycobiliproteins from cyanobacteria (blue-green algae) and two groups of eukaryotic algae (red algae and cryptomonads) (Glazer 1990). The ORAC assay was adapted by Cao and others (1993) for the assessment of antioxidant species in human plasma. It was later automated on the Cobas Fara II centrifugal analyzer (Cao and others 1995) and used to determine the TRAP of human plasma (Ghiselli and others 1995). After the application of the phycoerythrin-based assay to tea, vegetables, and biological fluids (Cao and others 1996; Cao and others 1998), Dr. Ronald L. Prior and his colleagues modified the method using fluorescein (FL) (3′6′-dihydroxy-spiro[isobenzofuran-1[3H],9′[9H]-xanthen]-3-one) as a more stable and reproducible fluorescent probe (namely, the ORACFL assay) (Ou and others 2001). Over the following years, the ORACFL assay was adapted to a multi-channel liquid handling system coupled with a microplate fluorescence reader (Huang and others 2002b) and applied to both hydrophilic and lipophilic systems (Huang and others 2002a; Prior and others 2003). Dr. Prior's laboratory further modified the ORACFL assay for the controlled generation and scavenging of HO (Ou and others 2002). More recently, a derivative of fluorescein (namely, dichlorofluorescein) (Adom and Liu 2005) has been applied as the fluorescent probe in the ORAC assay, but fluorescein still remains the probe of choice for the majority of applications.

Despite the series of modifications discussed, the principles of the initial assay remain the same and include the following: azo-initiation of RO2via thermal degradation of AAPH followed by the competitive HAT reaction between antioxidant samples (or standard Trolox) and the generated peroxyl radicals with the fluorescent probe. Fluorescence gives off a real-time signal registered by the plate reader at an excitation/emission wavelength pair of 493/515 nm and declines rapidly as it undergoes a HAT reaction with the azide-generated peroxyl radicals. The following reaction scheme (15) illustrates this process:

  • image(15)

Any antioxidant species present in the reaction mixture will undergo HAT with the peroxyl radicals (15) and delay the reduction of the fluorescent signal. Figure 15 is a proposed mechanism by which FL (pictured in its free acid form) interacts with peroxyl radicals resulting in the loss of fluorescence at λEm= 515 nm.

image

Figure 15–. Proposed mechanism for the ORAC HAT FL(H) [RIGHTWARDS ARROW] FL (loss of signal).

Download figure to PowerPoint

Photochemiluminescent (PCL) detection of water- and lipid-soluble antioxidants

The capabilities of water- and lipid-soluble antioxidants to scavenge O2•− can be assessed using a Photochem® unit from Analytik Jena USA (The Woodlands, TX). The initial protocol and system upon which Photochem® was developed is the work of Drs. Igor Popov and Gudrun Lewin from 1987 to 1999. The span of their research covers the photochemiluminescent quantification of ascorbic acid and superoxide dismutase (SOD) in human plasma (Analytik Jena sells kits for these assays, PCLASC and PCLSOD, respectively) (Popov and others 1987, 2001; Lewin and Popov 1994; Popov and Lewin 1999b), as well as the measurement of antioxidant capacities of water- and lipid-soluble antioxidants (sold as PCLACW and PCLACL kits, respectively) (Popov and Lewin 1994, 1996, 1999a). Each assay involves the photodegradation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and results in the production/quenching of O2. From this work, Analytik Jena developed their testing kits for photochemiluminescent measurements as well as the Photochem® system. The simplified radical-generation reaction scheme (16) is as follows (Pegg and others 2007):

  • image(16)

In reaction (16), L* is an intermediate product of the photo-induced luminol and 3O2 is triplet oxygen (no 1O2 is involved in the reaction). Once the O2•− and luminol radicals are generated, they proceed through a series of reactions resulting in the production of blue luminescence (Merényi and others 1986; Popov and Lewin 1994; Schneider 1970). Though all the steps in the detection reaction are not known, an example of possible chemical intermediates in the chemiluminescence of luminol is illustrated in reaction scheme (17) (Pegg and others 2007):

  • image(17)

In reaction (17), AP*2− is an excited aminophthalate anion, and AP2− is the aminophthalate anion at the ground state. The chemical structure of luminol and the aminophthalate anion are discussed by Schneider (1970).

Once O2•− radicals are generated, any exogenous antioxidant species present in the reaction mixture will out-compete the luminol radical in the action of donating a hydrogen atom (a HAT reaction). This will halt the production of blue luminescence until the concentration is exhausted. The resultant lag/log relationships of antioxidant compounds performing in this closed system are then compared to the effectiveness of standards (ascorbic acid in ACW, and Trolox in ACL). The antioxidant capacity of compounds in the ACW and ACL assays gauge their relative antioxidant capacities in hydrophilic and lipophilic media.

SET Assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Ferric reducing antioxidant power (FRAP) assay

Benzie and Strain (1996) developed an assay to measure the ferric reducing power of human plasma. This method was later adapted to the quantification of ferric reducing antioxidant power (FRAP) of plant extracts (Pulido and others 2000). Recently, the FRAP assay was adapted to a microtiter plate reader in 96-well format (Dragsted and others 2004). The assay reaction involves the reduction of Fe3+−TPTZ (iron[III]-2,4,6-tripyridyl-s-triazine) to Fe2+−TPTZ through SET with an antioxidant compound. The result of this reaction is an intense blue color at λmax= 550 nm, as seen in reaction scheme (18):

  • image(18)

Figure 16 is a structural representation of the conversion of iron(III)−TPTZ to iron(II)−TPTZ. Reaction (18) can occur with antioxidant compounds with redox potentials lower than 0.7 V (the E° of Fe3+– TPTZ) and is, thus, comparable to ABTS•+ (E°= 0.68 V)-based assays (namely, TEAC) (Prior and others 2005). Furthermore, reducing power appears to be related to the extent of conjugation in phenols as well as the number of hydroxy constituents (Schaich 2006).

image

Figure 16–. FRAP color production reaction: SET reduction of iron(III)−TPTZ to iron(II)−TPTZ (blue at λmax= 595 nm).

Download figure to PowerPoint

Because the FRAP assay solely measures reducing power, the activity of compounds that follow traditional hydrogen abstraction mechanisms may go unmeasured. This can in some instances serve as a shortcoming, but when used in conjunction with other assays this trait provides unique opportunities for determining the dominant mechanisms of antioxidant compounds. It is also important to note that the results of FRAP assays have been shown to produce considerably different results depending on the analysis time and the reaction medium used (Prior and others 2005). Further, the assay reaction must be carried out at acidic pH in order to maintain iron solubility, but this can lower the IP of the reactants and reduce the redox potential of the system.

Cupric reducing antioxidant capacity (CUPRAC) assay

In comparison with iron, copper has a greater potential to undergo redox reactions with antioxidant components (E° of copper[I] and [II] spectrophotometric-complexation reactions are generally lower than iron[II] and [III]) (Schaich 2006). Redox reactions with copper are often faster than with iron, thus reducing time constraints in the laboratory. Further, copper has more 3d-electrons than iron which may lend to its greater capability to coordinate with the π-electrons of incoming ligands during metal-ion chelation (Chatterjee and others 1983). Just like iron, copper ions coordinate with nitrogen-containing chelating agents such as 2,2′-bipyridine or 1,10-phenanthroline and its derivatives (Pilipenko and Falendysh 1972).

It follows from the reaction of iron(II) with 1,10 phenanthroline (the “ferroin” reaction) that the related copper(I) complexes with 1,10 phenanthroline derivatives began to carry the suffix “cuproine” (Smith and Wilkins 1953). Tütem and others (1991) sought to improve an existing bathocuproine (BC) (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) method for the selective spectrophotometric determination of copper(I) in the presence of copper(II) (Moffett and others 1985), by introducing the use of neocuproine (NC) (2,9-dimethyl-1,10-phenanthroline) as an alternative chelating agent. Later, Apak and others (2004) revised their copper(I)-NC method, applied it to the analysis of dietary polyphenols, and created the CUPRAC assay. The CUPRAC method involves the reduction of free copper(II) to copper(I) in the presence of NC, which results in the coordinated complex Cu(I)-NC at a ratio of 2:1 according to the following reaction scheme (19):

  • image(19)

Figure 17 is a structural representation of reaction (19), including NC and BC complexes. Reagents for the CUPRAC assay include a 0.1 M solution of copper(II) chloride (for free Cu2+), a 7.5 mM NC solution prepared in 95% (v/v) ethanol, ammonium acetate buffer (pH 7) for the reaction medium and diluent of samples, and a standard (usually uric acid). A variation of the old BC (copper[I]-BC, λmax= 490 nm) method for copper is sold as a Bioxytech® AOP-490™ assay kit by OXIS International, Inc. (Portland, OR). This kit consists of a ready-made dilution buffer, cupric sulfate solution, uric acid standard, and stop solution to halt the reaction.

image

Figure 17–. CuPRAC color production reaction: SET reduction of copper(II) to copper(I)-BC/NC (blue at λmax= 490 or 450 nm).

Download figure to PowerPoint

Mixed-Mode Assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Trolox equivalent antioxidant capacity (TEAC) assay

The Trolox equivalent antioxidant capacity (TEAC) assay is a spectrophotometric method based on the capability of an antioxidant to scavenge the free-radical cation ABTS•+. The TEAC assay was originally developed by Miller and others (1993) for the measurement of the antioxidant capacity of human plasma in infants. Re and others (1999) modified the assay for the direct generation of ABTS•+ without radical intermediates and applied it to hydrophilic and lipophilic antioxidants. Dragsted and others (2004) adapted the assay to a microplate reader for high throughput. Though the TEAC assay is generally accepted as a SET assay, ABTS•+ can be neutralized by SET and HAT mechanisms. The HAT and SET assay reaction schemes (20–21) are as follows:

  • image(20)
  • image(21)

Figure 18 serves as a structural representation of the ABTS•+ decolorization reaction (21). It is important to note that there are many points for careful consideration in the TEAC assay including the controlled generation of ABTS•+, pH, and temperature of the assay medium. Also, ABTS•+ is a nitro-radical; therefore, it may not correlate well with other antioxidant capacity assays that measure oxyl-radical scavenging. As with the FRAP and CUPRAC assays, the complex nature of ABTS•+ may render interaction with polyphenolics time-dependent, so time-curves are often prepared. Given the prevalence of both HAT and SET reactions with ABTS•+, the TEAC assay should be considered a mixed-mode assay.

image

Figure 18–. Conversion of ABTS•+ (green at λmax= 734 nm) to a colorless species ABTS(H) through a HAT mechanism with antioxidant compound ArOH.

Download figure to PowerPoint

DPPH (2,2′-diphenyl-1-picrylhydrazyl radical cation) Aassay

DPPH has been examined for its use as an organic colorimetric reagent since the 1950s (Blois 1958). Braude and others (1954) made the observation that DPPH undergoes a HAT mechanism with antioxidant compounds according to the following reaction scheme (22):

  • image(22)

Figure 19 is a structural representation of the reaction (22). Blois (1958) determined that if the phenolic compound under analysis contains more than one phenolic hydroxy functional group, the resultant ArO formed is sufficiently stable to undergo a second simultaneous HAT reaction with another molecule of DPPH, thereby preserving the stoichiometry of the reaction.

image

Figure 19–. HAT conversion of DPPH (purple at λmax= 517 nm) to colorless species DPPH(H).

Download figure to PowerPoint

Over the past two decades the DPPH assay has resurfaced as a method for the analysis of phenols in plants and plant-derived food products (Sánchez-Moreno and others 1998). The current version of the assay involves adaptation to a high-throughput 96-well microtiter plate system (Fukumoto and Mazza 2000). The assay is often run in-tube due to relative inexpensiveness. This renewed interest in the DPPH assay has resulted in a re-examination of the kinetics of its reaction with phenolics (Brand-Williams and others 1995; Bondet and others 1997; Silva and others 2000) and possible mechanisms of interaction, whether HAT (Brand-Williams and others 1995; Dangles and others 2000; Litwinienko and Ingold 2003), SET (Foti and others 2004; Huang and others 2005), or mixed (Schaich 2006). The following reaction scheme (23) is an example of a SET mechanism between DPPH and a phenolic antioxidant:

  • image(23)

As with the TEAC assay, the medium of interaction, size, polarity, and acidity of phenolic hydroxy groups play a role in whether SET or HAT mechanisms dominate. DPPH is known to react with a variety of compounds including aromatic amino acids, glutathione, α-tocopherol, ascorbic acid, and polyhydroxy aromatics (phenolics); therefore, the amount of potential interferences in the assay's progress is great.

Evaluation of Chelation Activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

The most commonly employed methods to determine the metal-ion chelating activity of phenolic compounds are the tetramethylmurexide (TMM) and the ferrozine assays, for ferrous and cuprous ions, respectively (Lee and others 2007). Both of these assays are spectrophotometric and easy to use. In both methods, a reagent (TMM or ferrozine) is used to form complexes with divalent metal cations; the result of which produces increases in absorbance readings at the measured wavelengths (485 and 562 nm, respectively). Absorbancies increase in a linear fashion with concentration according to Beer's Law, thereby allowing for the formation of a standard curve by linear regression. The standard curve can then be used for the comparative evaluation of the chelating properties of other compounds relative to reference compounds.

Although both assays have become commonplace, Karamać and Pegg (2009) reported the TMM assay to present limitations in the assessment of phenolic preparations rich in tannin constituents. Specifically, control samples not containing TMM produced high absorbance values, suggesting interference in the measurement. The authors suggest the use of the ferrozine method as the preferred one in such cases.

Methods to Evaluate Lipid Oxidation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Another strategy that may be employed in assessing antioxidants is to incorporate the antioxidant into a system and then monitor the affected oxidative stability (that is, the resistance to oxidation). This approach may offer a practical advantage in that it can successfully incorporate any multitude of variables (which may or may not be well understood) to give a relatively telling account of an antioxidant's capabilities within a particular system.

There are many methods used to evaluate oxidation rates within a system, most of which require the repeated performance of an assay over a period of time. The period of time may be abbreviated by use of radical initiators (Alsante and others 2007) or accelerated storage techniques like the Schaal oven storage stability test (Frankel 1993). Below is a very brief summary of the methods commonly used for the evaluation of oxidation.

Peroxide value

Given that the primary products of lipid oxidation are hydroperoxides (commonly referred to as peroxides), their quantification can provide a suitable measurement of the extent of oxidation present in a lipid sample. Protocols for the quantification of peroxide values (PVs) in foods can be iodometric or colorimetric methods, each with its strong and weak points (for example, iodometric titration endpoint) (Pegg 2005). Because the extent of oxidation of a lipid is related to its PV, the capability of an antioxidant compound to perform in a closed system can be gauged by the prevention of peroxide formation over time with respect to a control.

Conjugated dienes

The majority of polyunsaturated fatty acids (PUFAs) in nature have a 1,4-diene structure (their points of unsaturation are methylene-interrupted), so the occurrence/detection of conjugated dienes (CD) is an indication that fatty acids have undergone autoxidation (Corongiu and Banni 1994). The number of positional isomeric peroxides that can result from autoxidation of a lipid depends on the number of double bonds (n) contained and is equal to 2n-2 (Esterbauer 1993).

The premise of the CD assay, which has been in use since before the 1950s (Farmer and Sutton 1943), is the strong UV absorbance of the CD moiety at λmax= 234 nm. Conjugated dienes are the first indicator of oxidation in model lipid systems and are often retained in many secondary products, even after PVs decrease in later stages of oxidation. Given the lack of complicated reagents and preparative work required, the assay is a very attractive option for a quick assessment of lipid oxidation or the capability of an exogenous antioxidant to inhibit autoxidation. In some cases; however, absorbance of the diene moiety of an oxidized lipid is not easily related to the full extent of oxidation in a sample. The effects of autoxidation on lipids can vary (Gray 1978), and results are best explained if the composition of the lipid is known (Holman and Burr 1946).

Anisidine test

The p-anisidine value (PAV) measures secondary lipid oxidation products by an assessment of aldehyde concentration. Aldehyde carbonyl bonds react with the amine group of the p-anisidine reagent to form a Schiff base which absorbs radiation at 350 nm and can therefore be quantified using spectrophotometry (Laguerre and others 2007). The colorimetric response is affected by the level of aldehyde unsaturation (greater reactivity with higher degree of unsaturation), and therefore the results of the assay must be interpreted with a level of caution. The PAV is often combined with peroxide values to form a “totox value;” a simple summary of lipid oxidation that incorporates measures of both primary and secondary oxidation products (Nielsen 2010).

Thiobarbituric acid-reactive substances test

The thiobarbituric acid-reactive substances (TBARS) test quantifies malonaldehyde and malonaldehyde-type products (such as trans,trans-2,4-heptadienal, trans-2-heptenal, trans-2-hexenal, and hexanal), as well as secondary oxidation decomposition products of polyunsaturated fatty acids (Dahle and others 1962). These products react with 2-thiobarbituric acid (TBA) to form a stable pink chromophore, with a λmax of 532 nm, and can therefore be quantified spectrophotometrically (Dahle and others 1962; Frankel 1993; Nielsen 2010). Because the malonaldehyde-type compounds are highly reactive, the TBARS test only measures products temporarily occurring in the steps of oxidation.

Volatile organic compounds

Volatile organic compounds (hexanal, pentane, trans,trans-2,4-decadienal, and others) are common secondary oxidation products of foods containing linoleic acid, and their quantification with gas chromatography (GC) can be useful in the assessment of oxidation. This method can be of particular value in the case of foods because it allows not only for a strict assessment of oxidation, but also for the assessment of oxidation as it relates to the product's quality (Panseri and others 2011). The proliferation of volatile organic compounds is often the causative agent in the development of oxidized off-flavors. The introduction of solid-phase microextraction (SPME) has advanced the method and now allows for greater sensitivity (Nielsen 2010; Panseri and others 2011). Recent work has established successful methods by which the use of SPME/GC accurately assesses hexanal concentrations in systems at concentrations in the range of μg/g, thus allowing for detection of volatiles at early stages of formation (Giuffrida and others 2005).

Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

Another assay of relevance is the total phenolics content or TPC assay with Folin and Ciocalteu's phenol reagent, which quantifies the phenolic content of a sample. In addition to providing the obvious benefit of the assessment of quantities within food sources, this method may also be used in tandem with antioxidant assessment assays to determine antioxidant potential relative to quantity and/or concentration.

Phosphotungstic and phosphomolybdic heteropolyacids have been used as colorimetric reagents since the early 1900s (Folin and Macallum 1912). In 1912, a breakthrough in colorimetry occurred with the creation of a sensitive chromophoric complexing reagent for the quantification of tyrosine residues in protein hydrolysates (Folin and Denis 1912a, b). The Folin-Denis phenol reagent (as it was named then) was again reformulated, in 1927 by Folin and Ciocalteu (1927), with a greater incorporation of molybdenum for increased redox sensitivity. Singleton and Rossi (1965) applied Folin & Ciocalteu's phenol reagent to the assessment of antioxidant contents in wine, resulting in the well-known TPC assay. With the increased interest in phenolics over the past 2 decades, this assay has become a mainstay in laboratories the world over.

The TPC assay is often modified based on the antioxidant source under investigation. While the most up-to-date method involves automation on a 96-well microtiter plate reader (Zhang and others 2006), the original in-tube assay is most prevalent. Despite modifications, the bulk of the total phenol protocol has remained the same since Singleton and Rossi (1965). Briefly, the assay involves the mixture of excess phenol reagent and a diluted sample or standard (gallic acid). The mixture is then treated with alkali to a final pH of 10 to 11. The resultant color from interaction of phenolate anions with the phenol reagent complex is allowed to develop over 30 to 60 min and yields a λmax in the range of 745 to 765 nm, depending on the standard employed.

The mechanism behind the TPC assay involves reduction of the molybdenum component in the phosphotungstic-phosphomolybdic complexing reagent according to the following reaction scheme (24):

  • image(24)

Reaction (24) is subject to a great many interferences, particularly any readily reducible component present within the assay mixture. Ascorbic acid is the major interference in the case of wine analysis (Singleton and others 1999) and most fruits.

Although the phenol reagents of Folin-Denis and Folin-Ciocalteu have been around for 75+ years, the chemistry of the reagents and the phenol-reagent reactions are still not well understood. It is possible that the reaction product between phenolate anions and Folin and Ciocalteu's phenol reagent is a group of Keggin clusters: a common form of heteropolyacids comprised of a cage structure of oxygen-containing phosphomolybdic and phosphotungstic repeats bearing the common formula [XM12O40]n−, where X is the heteroatom (phosphorus) and M is an added metal atom (molybdenum or tungsten in this case) (Pope 1983). The inherent stability of Keggin clusters promotes the reduction of the metal ion contained and, thus, facilitates the utilization of such reagents for colorimetry. It is generally accepted that in alkaline media three competitive reactions are proceeding simultaneously, including the “destruction” of the yellow Folin-Denis/Ciocalteu phenol reagent, the reduction of the reagent by phenolate anions to produce the characteristic “molybdenum blue,” and the destruction of the blue pigment by alkali (fading). Increasing the alkalinity or temperature of the assay promotes the development of the color complex in a shorter period of time. However, precipitation of the phenol reagent can occur. The precipitate is a dense, white, crystalline material that can be formed by excessive heat (above 60 °C), alkalinity (above pH 10–11), or the quantity of reagent in the assay (above 5 mL/100 mL) (Rosenblatt and Peluso 1941).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References

During the dawn of the functional food and nutraceutical era, new antioxidant assessment methods are constantly being created for the fast and cost-efficient screening of extracts and food products with biological activities. Each of these assays includes a variety of critical steps that must be followed in order to maintain accuracy and precision. Furthermore, the chemistry behind the assays must be assessed throughout, in order to run the assays and troubleshoot in an effective manner. There has been a great need for the standardization of antioxidant methods to promote uniformity in the scientific literature for the better comparison between sources. Yet, even with the development of standard protocols for antioxidant evaluation, the complex nature of these methods and their compatibility with certain antioxidant sources must still be of concern for future research and, thus, great care should be exercised when formulating one's battery of methods for the measurement of antioxidant capabilities.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. Oxidation
  6. Antioxidants
  7. Hydrogen-Atom Transfer (HAT) Mechanism
  8. Single Electron Transfer (SET) Mechanism
  9. Mixed HAT and SET Mechanisms
  10. Secondary Antioxidative Action by Metal-Ion Chelation
  11. Reactive Oxygen Species (ROS) and the Human Body
  12. The Role of Antioxidants in Humans
  13. The Role of Antioxidants in Foods
  14. Phenolic and Polyphenolic Antioxidants
  15. Occurrence of Phenolics in the Plant Kingdom
  16. Extraction of Phenolics from Plants
  17. Phenolics in Food
  18. Phenolic Bioavailability/Bioactivity after Ingestion
  19. Relationship between Phenolic Structure and Antioxidant Activity
  20. Correlation between Phenolics Content and Antioxidant Activity
  21. Quantification of Antioxidant Content and Capacity
  22. HAT Assays
  23. SET Assays
  24. Mixed-Mode Assays
  25. Evaluation of Chelation Activity
  26. Methods to Evaluate Lipid Oxidation
  27. Total Phenolics Content (TPC) with Folin and Ciocalteu's Phenol Reagent
  28. Conclusions
  29. References