**5. Methods for measuring the antioxidant activity of anthocyanins**

effect, have the capacity to inhibit in vitro the growth of cells that cause tumours in humans and are even able to act as modulators of the macrophages in the immune response [89, 112]. Anthocyanins are effective against cytotoxicity, lipidic peroxidation, and as protectors of DNA, by forming co‐pigments of DNA‐anthocyanins. Moreover, anthocyanins have cellular antioxidant mechanisms comparable to or greater than other micronutrients, such as vitamin E. The capacity of the anthocyanins for stabilizing triple‐helical complexes of DNA [136] by

Pharmacokinetics of anthocyanins has recently reviewed [85, 113, 138, 139]. The most recent papers published on the subject are summarized in **Table 2** [96, 101, 140–151]. Anthocyanins are metabolized to a structurally diverse range of metabolites that exhibit dynamic kinetic profiles. A multicompartmental (theoretical physiologically based) pharmacokinetic (PBMK) model has been proposed [138] in order to describe the anthocyanins fate in vivo. Understanding the elimination kinetics of these metabolites is key to the design of future studies [152] concerning with their utility in dietary intervention or as therapeutics for disease risk reduction.

**Comments References**

[96]

[141]

[143]

[144]

[145]

[146]

[101]

[147]

[149]

Pharmacokinetic trial to evaluate the bioavailability of anthocyanins and colonic polyphenol metabolites

Evaluation of the protective effects of protocatechuic acid [140] Effects of black raspberry extract and protocatechuic acid on DNA adduct formation and mutagenesis in

Influence of ethanol on the bioavailability and pharmacokinetics of blackberry anthocyanins [142]

Pharmacokinetic characterization of anthocyanins in overweight adults on the basis of meal timing [97]

Pharmacokinetic trial to evaluate the of nanoencapsulation of a phenol extract from grape pomace on

Determination of cyanidin 3‐glucoside in rat brain, liver and kidneys: a short‐term pharmacokinetic

Pharmacokinetics, bioavailability and regional brain distribution of polyphenols from apple‐grape seed

Evaluation of changes in metabolic parameters, and in cardiovascular and liver structure and function in

Bioavailability and uptake of anthocyanins and their metabolites from an anthocyanins‐rich grape/

Effects of anthocyanins and their corresponding anthocyanidins on the expression levels of organic

Anthocyanin pharmacokinetics and dose‐dependent plasma antioxidant pharmacodynamics by intake

Effect of flavan‐3‐ols and anthocyanins against inflammatory‐related diseases [148]

Pharmacokinetics of the metabolites of cyanidin‐3‐glucoside [150] Abundance and persistence of metabolites of anthocyanins in human urine [151]

rat due to administration of either cyanidin 3‐glucoside or Queen Garnet plum juice

**Table 2.** Selected papers on pharmacokinetics of anthocyanins in the 2014–2016 period.

forming complexes of anthocyanins‐DNA [137] is well established.

after consumption of aronia berry extract in plasma and urine

214 Flavonoids - From Biosynthesis to Human Health

rat oral fibroblasts

human plasma

extract mixture and bilberry extract

blueberry juice and smoothie in vivo and in vitro

of Montmorency tart cherries in healthy humans

anion transporting polypeptides in primary human hepatocytes

study

Although a plethora of biological actions has been ascribed to flavonoids, their antioxidant activity, in particular, has recently attracted much attention. Anthocyanins behave as antioxidants by a variety of ways, including direct trapping of ROS, inhibition of enzymes responsible for superoxide anion production, chelation of transition metals involved in processes forming radicals and prevention of the peroxidation process by reducing alkoxy and peroxy radicals.

There are a variety of methods for measuring antioxidant activity, either in vitro or in vivo (greater complexity involved) or a combination of both. The number of reviews published on the matter reflects the transcendence of this hot topic and its richness. Selected reviews found in the literature from 2000 up to the present time are summarized in **Table 3** [154–204]. The most common chemical methods used for measurement in vitro of antioxidant activity of polyphenolic compounds (e.g. anthocyanins) are shown in **Table 4** [197–241]. Methodological contributions are preferably cited in **Table 4** instead of specific practical applications. Both conceptual and technical problems limiting the use and validity of three commonly used [119] assays TEAC/ABTS\*+, DPPH and ORAC have been subject of recent revision. Some reviews dealing with the DPPH [208, 212, 214], ORAC [223] and CUPRAC [239, 240] assays have also been the subject of recent treatments. However, the aspects concerning with the assay chemistry, standardization and report of the antioxidants determination have not been solved after 25 years of intense study [199].

Antioxidant activity is always measured in an indirect way as a response (of the antioxidants present in the sample) to induced oxidation [192, 160, 173]. For foodstuffs, there is a range of methods for determining antioxidant activity. These can vary from those that evaluate the inhibition of lipidic peroxidation by the antioxidants and quantify the products as peroxides, hydroperoxides and products resulting from decomposition measured by the thiobarbituric acid reactive substances (TBARS) assay [171], to methods that determine the content of free fatty acids, polymer content, viscosity, absorptivity at 232 and 268 nm, colour and physiological measurements in vivo, such as measuring the products from oxidation of the LDLs, or indirect indicators of lipidic oxidation. Alternatively, antioxidant activity can be evaluated by measuring the immunological response to antigens (the products of lipidic oxidation). Though solvent effect is a vital parameter [203] exerting an influence on the chemical behaviour of antioxidant compounds, the information concerning about its role on the antioxidant capacity is relatively scarce.

There are some drawbacks to assays in vivo. The interpretation of changes in the antioxidant activity of the plasma can be complicated because of the possibility of producing adaptability in response to an increase in oxidative stress. However, assays in vitro can also have their drawbacks, such as the interactions between samples and reagents.



**Table 3.** Published reviews on used methods for measurement of antioxidant activity.

**Content References**

[154]

[155]

[156]

[159]

[162]

[167]

[169]

[174]

[180]

Antioxidant activity/capacity measurement: classification, physicochemical principles, mechanisms and

Antioxidant activity/capacity measurement: hydrogen atom transfer‐based, mixed‐mode and lipid

Antioxidant activity/capacity measurement: reactive oxygen and nitrogen species scavenging assays,

Capacity of antioxidants to scavenge multiple reactive oxidants and to inhibit plasma lipid oxidation

A comprehensive overview on the biology behind some reactive molecules and the means for their

Synthetic and natural phenolic antioxidants: mode of action, health effects, degradation products and

Use of metallic nanoparticles and quantum dots as novel tools for reliable assessment of antioxidant

Application of both stationary and flow electrochemical methods for analysis of antioxidant properties

Overview of the importance and mechanism of action of antioxidants, as well as of the methods of

Recent applications for in vitro antioxidant activity assay [157] Evaluation of procedures for assessing anti‐ and pro‐oxidants in plant samples [158]

Analytical methods applied to antioxidant and antioxidant capacity assessment in plant‐derived products [160] Advantages and limitations of commons testing methods for antioxidants [161]

Potentiometric study of antioxidant activity: development and prospects [163] Methods for determining the efficacy of radical‐trapping antioxidants [164] Electrochemical methods for total antioxidant capacity [165] The role of consumption of dietary bioactives on the prevention of adverse health [166]

Up‐to‐date overview of methods available for measuring antioxidant activity [168]

Review on in vivo and in vitro methods evaluation of antioxidant activity [170] IUPAC technical report: methods of measurement and evaluation of natural antioxidant capacity/activity [171] Evaluating the antioxidant capacity of natural products: a review on chemical and cellular‐based assays [172] Application of free radical diphenylpicrylhydrazyl to estimate the antioxidant capacity of food samples [173]

Phenol‐based antioxidants and the in vitro methods used for their assessment [175] Main components in the foodstuffs and beverages: antioxidant methods, chemical and kinetic basis [176] Estimation of antiradical properties of antioxidants using DPPH assay [177] Evaluation of antioxidants: scope, limitations and relevance of assays [178] A comprehensive review of cupric reducing antioxidant capacity methodology [179]

Methods for evaluating the potency and efficacy of antioxidants [181] A comprehensive review of chemical methods to evaluate antioxidant ability [182] Assessment of antioxidant capacity in vitro and in vivo [183]

oxidative stress biomarkers and chromatographic/chemometric assays

electron transfer‐based assays

216 Flavonoids - From Biosynthesis to Human Health

induced by different biological oxidants

activity in food and biological samples

of plant and clinical samples

assessment of the antioxidant capacity

peroxidation assays

detection

toxicology

The in vivo antioxidant potential of anthocyanins can be measured by reducing the serum concentration of the reactive substance to thiobarbituric acid (TBARS assay) or by increasing the resistance to oxidation in the plasma of the lipidic peroxidation caused by 2,2′‐azobis (2‐amidinopropane) hydrochloride (AAPH) or by Cu2+.

Most in vitro measurements of the antioxidant activity of anthocyanins involve the following factors: calculating the rate and range of the decrease of the substance in assay or the oxygen consumption, the formation of products from oxidation and the formation or decline of the number of FR. Detection can be carried out by inhibition of fluorescence, chemoluminiscence, oxygen consumption or absorbance, the evolution of which is related to the end product.


*Abbreviations*: ABTS (2,2′‐azino‐bis (3‐ethylbenzothiazolinine‐6‐sulfonic acid); DMPD (N,N‐dimethyl‐p‐phenylenediamine dihydrochloride); DPPH (2,2‐diphenyl‐1‐picrylhydrazyl); FRAP (ferric reducing ability of plasma); ORAC‐PE (oxygen radical absorbance capacity) with β‐phycoerythrin; ORAC‐FL (oxygen radical absorbance capacity) using fluorescein (3′6′‐dihydroxyspiro[isobenzofuran‐1[3H], 9′[9H]‐xanthen]‐3‐one), ORAC‐PGR (oxygen radical absorbance capacity) with pyrogallol red (pyrogallol sulphone phthalein); TROLOX (6‐hydroxy‐2,5,7,8‐tetramethilcroman‐2‐carboxylic acid).

**Table 4.** Commonly used methods for measurement in vitro of antioxidant activity.

FR can be generated by various chromogenic compounds, such as azo ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline)‐6‐sulphonic acid), DMPD (N,N‐dimethyl‐p‐phenylenediamine dihydro‐chloride), DPPH (2,2‐diphenyl‐1‐picrylhydrazyl), FRAP (ferric reducing ability of plasma) and DMPO (5,5‐ dimethyl‐1‐pyrroline N‐oxide). Inhibition of oxidation can be measured by the reduction in fluorescence by the ORAC method or by the TRAP (total radical‐trapping antioxidant parameter) assay.

Currently, ABTS is one of the methods most frequently used for assays of coloured compounds, like anthocyanins [197], as the radical generated has a maximum absorption at a wavelength of 734 nm, reducing the possibilities of interference of antioxidants that absorb in the red colour zone. The radical ABTS•+ can be generated by enzymes (peroxidase, myoglobin) or chemically (manganese dioxide, potassium persulphate or ABAP (2,2′‐azobis‐2‐ amidino‐propane hydrochloride). The radical, once generated, displays new characteristics with maximums of absorption at 414, 645, 734 and 815 nm.

Kuskoski et al. [51] found a maximum absorption of around 754 nm in an alcoholic medium, and this wavelength was used to determine the antioxidant activity of fruit extracts of baguaçu (*Eugenia umbelliflora* Berg) that are rich in anthocyanin pigments [242].

If compared with other methods of formation of FR, such as DPPH, DMPD and others, the capture reaction time of the radical ABTS•+ is fairly rapid, it can range from 1 to 7 min, although according to Re et al. 4 min is sufficient to complete the reaction. Antioxidant data based on ABTS assay are dependent on reaction time because the applied standard compounds (trolox) present a scavenging kinetic profile [200] different from that of polyphenol‐ rich foods. Studies have been carried out on the effects of molecular structure (molecular weight, number of ─OH groups, redox potential) on kinetics and dynamics of [201] the trolox equivalent antioxidant capacity assay with ABTS. Attempt has been made to standardize the method [202] by extrapolating to zero sample concentration.

The chromatic properties of the stable radical cation DPPH were first described [171] by Blois in 1958, who used the radical to measure the antioxidant activity of several natural compounds. Only much later did Brand‐Williams et al. develop a technique based on the reduction of the absorbance of the radical DPPH• at 517 nm. This technique has also been applied by other authors with modifications and measurement of absorbance at 515 nm. Results are expressed as IC50 [213], that is, the quantity of antioxidant required to reduce the initial concentration of DPPH to 50%, or as the percentage of interacted DPPH % DDPH = [(Absreferencia − Abse xtracto)/(Absreferencia)] × 100. DPPH assay on food additives and foods and beverages has been subject to interlaboratory study [211, 215]. The DMPH reaction has been revisited and re‐evaluated [216–218] and simplified in order to characterize samples of wine origin [210].

The influences of reaction time, DPPH concentration inference and kinetics parameters of bioactive molecules and plant extracts [209] in the reaction with the DPPH radical have been evaluated. A collaborative study on the DDPH assay [215] has been promoted as well as a kinetic‐matching approach to express antioxidant capacity in a more standardized way.

The spectrophotometric DMPD method, described by [171, 206, 207] Fogliano et al. in 1999, is similar to the ABTS method. In the presence of an adequate oxidant solution, the radical cation DMPD•+ generated has the ability to link hydrogen atoms, causing the discolouration of the solution, producing a reduction of absorbance measured at 515 nm. DMPD cannot be used with hydrophobic antioxidants, as it is only water soluble [171, 206]. DMPD method is not considered suitable for assays of coloured compounds, as interference can occur in the measurements, because they absorb in the same region of the spectrum.

FR can be generated by various chromogenic compounds, such as azo ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline)‐6‐sulphonic acid), DMPD (N,N‐dimethyl‐p‐phenylenediamine dihydro‐chloride), DPPH (2,2‐diphenyl‐1‐picrylhydrazyl), FRAP (ferric reducing ability of plasma) and DMPO (5,5‐ dimethyl‐1‐pyrroline N‐oxide). Inhibition of oxidation can be measured by the reduction in fluorescence by the ORAC method or by the TRAP (total radical‐trapping antioxidant parameter) assay. Currently, ABTS is one of the methods most frequently used for assays of coloured compounds, like anthocyanins [197], as the radical generated has a maximum absorption at a wavelength of 734 nm, reducing the possibilities of interference of antioxidants that absorb in the red colour zone. The radical ABTS•+ can be generated by enzymes (peroxidase, myoglobin) or chemically (manganese dioxide, potassium persulphate or ABAP (2,2′‐azobis‐2‐ amidino‐propane hydrochloride). The radical, once generated, displays new characteristics

*Abbreviations*: ABTS (2,2′‐azino‐bis (3‐ethylbenzothiazolinine‐6‐sulfonic acid); DMPD (N,N‐dimethyl‐p‐phenylenediamine dihydrochloride); DPPH (2,2‐diphenyl‐1‐picrylhydrazyl); FRAP (ferric reducing ability of plasma); ORAC‐PE (oxygen radical absorbance capacity) with β‐phycoerythrin; ORAC‐FL (oxygen radical absorbance capacity) using fluorescein (3′6′‐dihydroxyspiro[isobenzofuran‐1[3H], 9′[9H]‐xanthen]‐3‐one), ORAC‐PGR (oxygen radical absorbance capacity) with pyrogallol red (pyrogallol sulphone phthalein); TROLOX (6‐hydroxy‐2,5,7,8‐tetramethilcroman‐2‐carboxylic acid).

**Method Detection Measurement/oxidant References**

TEAC value, antioxidant activity equivalent to trolox

Expressed in EC50 (quantity of antioxidant required to reduce to 50% of the initial concentration of DPPH) or

Expressed in μmol of equivalents of reduced ferric ion (Fe2+) by g of sample or a value equivalent to a pattern

μmol equivalent to trolox (TEAC) by g of sample

μmol equivalent to trolox (TEAC) by g of sample

μmol equivalent to trolox (TEAC) by g of sample

μmol equivalent to trolox (TEAC) by g of sample

Expressed in μmol equivalent to trolox (TEAC)

by g of sample

[197–205]

[206, 207]

[208–219]

[203, 204, 220]

[221–236]

[199, 237–241]

(μmol/g)

in TEAC

of the radical cation in an aqueous medium at 414 nm (or 645, 734 or 815 nm)

Radical ABTS•+ Reduction of absorbance

218 Flavonoids - From Biosynthesis to Human Health

Radical DMPD•+ Reduction of absorbance to 505 nm

Radical DPPH•+ Reduction of absorbance to 517 nm

FRAP Increase of the absorbance

ORAC‐PE Reduction of fluorescence

ORAC‐FL Reduction of fluorescence

ORAC‐PGR Reducon of fluorescence

CUPRAC Absorbance measurement

chelate

to 593 nm

(β‐phycoerythrin)

(fluorescein)

(pyrogallol red)

of the Cu(I)‐neocuproine

**Table 4.** Commonly used methods for measurement in vitro of antioxidant activity.

with maximums of absorption at 414, 645, 734 and 815 nm.

The ferric reducing ability of plasma (FRAP) assay measure the ability of antioxidant to reduce the ferric [Fe3+−(TPTZ)<sup>2</sup> ] 3+ complex to the ferrous [Fe2+−(TPTZ)<sup>2</sup> ] 2+ complex (blue coloured) in acidic medium. It is a simple, reproducible method that can not only be applied to the study of the antioxidant activity of plasma, or in foods and beverages, but also to the study of the antioxidant efficacy of pure compounds with results that are comparable to those of more complex methodologies. It is widely used to determine the antioxidant activity of anthocyanins in different samples. However, the FRAP assay is carried out at a very low pH (3.6), far from the pH found in biological fluids. Nevertheless, this method has the advantage of determining the activity of the antioxidant directly in plasma; it does not depend on an enzymatic or a nonenzymatic method for generating FR and evaluates the antiradical efficacy of plasma. It also does not need the isolation of plasma fragments as is required in LDL.

The assay by fluorescence spectrophotometry known as ORAC was first set up [25, 29, 171] by Cao et al. in 1993 and later modified by Cao et al. in 1995. The ORAC method is based on measuring the decrease of the fluorescence of the proteins β‐phycoerythrin and R‐phycoerythrin (PE). These proteins have a high fluorescence in the presence of peroxyl radicals generated by the thermic decomposition of the 2′2′azobis (2‐amidinopropan) dihydrochloride (AAPH); the decrease is recorded in the presence of antioxidants. It is considered to be a very sensitive method that evaluates the oxidation process from its beginning, although it has the drawbacks of being expensive and time‐consuming [194].

However, β‐phycoerythrin [219] is photo‐unstable and it forms complexes with polyphenols giving, therefore low values of ORAC‐PE. For this reason, it is substituted [234–236] by fluorescein (ORAC‐FL), which, in contrast, is much more stable, and does not react with polyphenols, making it a much more precise and more economic method. Two alternative solutions have been proposed to decrease a systematic error related to AAPH addition in the fluorescence‐based ORAC assay [221].

A simple mathematical model for conversion of ORAC values to mass units [229] has been proposed. ORAC standardization [227] and validation [230] have been attempted. The use of pyrogallol red as a probe [233, 225] for competitive antioxidant assay is a significant improvement. Pyrogallol reacts faster than fluorescein with RCOO\* radicals, and its consumption does not present induction times, even in the presence of very reactive oxidants, with the exception of ascorbic acid. First action ORAC assay has been reported both with fluorescein [226] (dextracts from tea, blueberry and grape skins) and pyrogallol red [228] (red wine, fruit juices and iced teas).

A proportional measurement of antioxidant activity is obtained using the ORAC assay, which is currently one of the most commonly used methods for measuring the antioxidant activity of the anthocyanins [218].

Cupric reducing antioxidant capacity (CUPRAC) test is conceptually similar to the FRAP test, but is based on the reduction of Cu2+ ions in the presence of neocuproine (2,9‐dimethyl‐1,10‐ phenatroline) at pH 7, which involves faster kinetics. The ammonium acetate buffer solution account for the liberated protons in reaction with polyphenols.

The total radical‐trapping antioxidant parameter (TRAP) method [171] proposed by Wayner et al. (1985) is based on the measurement of oxygen consumption during a peroxidation reaction of lipids controlled and induced by the thermic decomposition of some substances, such as ABAP or AAPH, which produces a flow of peroxyl radicals at a constant rate that is temperature dependent. These peroxyl radicals initiate a chain of lipoperoxidations. The method has some problems, including being sensitive to temperature and to changes in pH. The storage conditions of the samples are also important due to the liability of some antioxidants; therefore, their immediate analysis is recommended. When this is not possible, it is advisable to rapidly collect plasma for blood samples, to store them at −80°C and to measure them within 3 days. The concentration of proteins or uric acid, because of their high antioxidant power, should be taken into account when describing the results.

It is interesting to mention the fact that electrochemical [243–249] and ESR [250] methods are increasingly being applied to the determination of antioxidant capacity. The kind of technology and free radical generator or oxidant influences the antioxidant capacity measurement. A key factor that helps researchers to choose a given method and to understand the results obtained is the comparison of different analytical methods. In order to gather comprehensible information about the total antioxidant capacity of a food [184], at least two of these tests, and preferably all, should be combined, if possible, taking into account both the arguments for and against, and its applicability. **Table 5** shows selected articles [20, 190, 198, 204, 231, 251–260] in which more than one criterion has been applied to real samples with practical purposes. Advantages and limitations of the most common chemical methods of determination of the antioxidant capacity are compiled in **Table 6** [160, 161, 167, 171–173, 176, 184, 231, 261–263].

antioxidant directly in plasma; it does not depend on an enzymatic or a nonenzymatic method for generating FR and evaluates the antiradical efficacy of plasma. It also does not need the isolation

The assay by fluorescence spectrophotometry known as ORAC was first set up [25, 29, 171] by Cao et al. in 1993 and later modified by Cao et al. in 1995. The ORAC method is based on measuring the decrease of the fluorescence of the proteins β‐phycoerythrin and R‐phycoerythrin (PE). These proteins have a high fluorescence in the presence of peroxyl radicals generated by the thermic decomposition of the 2′2′azobis (2‐amidinopropan) dihydrochloride (AAPH); the decrease is recorded in the presence of antioxidants. It is considered to be a very sensitive method that evaluates the oxidation process from its beginning, although it has the

However, β‐phycoerythrin [219] is photo‐unstable and it forms complexes with polyphenols giving, therefore low values of ORAC‐PE. For this reason, it is substituted [234–236] by fluorescein (ORAC‐FL), which, in contrast, is much more stable, and does not react with polyphenols, making it a much more precise and more economic method. Two alternative solutions have been proposed to decrease a systematic error related to AAPH addition in the fluores-

A simple mathematical model for conversion of ORAC values to mass units [229] has been proposed. ORAC standardization [227] and validation [230] have been attempted. The use of pyrogallol red as a probe [233, 225] for competitive antioxidant assay is a significant improvement. Pyrogallol reacts faster than fluorescein with RCOO\* radicals, and its consumption does not present induction times, even in the presence of very reactive oxidants, with the exception of ascorbic acid. First action ORAC assay has been reported both with fluorescein [226] (dextracts from tea, blueberry and grape skins) and pyrogallol red [228] (red wine, fruit juices and iced teas). A proportional measurement of antioxidant activity is obtained using the ORAC assay, which is currently one of the most commonly used methods for measuring the antioxidant activity

Cupric reducing antioxidant capacity (CUPRAC) test is conceptually similar to the FRAP test, but is based on the reduction of Cu2+ ions in the presence of neocuproine (2,9‐dimethyl‐1,10‐ phenatroline) at pH 7, which involves faster kinetics. The ammonium acetate buffer solution

The total radical‐trapping antioxidant parameter (TRAP) method [171] proposed by Wayner et al. (1985) is based on the measurement of oxygen consumption during a peroxidation reaction of lipids controlled and induced by the thermic decomposition of some substances, such as ABAP or AAPH, which produces a flow of peroxyl radicals at a constant rate that is temperature dependent. These peroxyl radicals initiate a chain of lipoperoxidations. The method has some problems, including being sensitive to temperature and to changes in pH. The storage conditions of the samples are also important due to the liability of some antioxidants; therefore, their immediate analysis is recommended. When this is not possible, it is advisable to rapidly collect plasma for blood samples, to store them at −80°C and to measure them within 3 days. The concentration of proteins or uric acid, because of their high antioxidant

of plasma fragments as is required in LDL.

220 Flavonoids - From Biosynthesis to Human Health

cence‐based ORAC assay [221].

of the anthocyanins [218].

drawbacks of being expensive and time‐consuming [194].

account for the liberated protons in reaction with polyphenols.

power, should be taken into account when describing the results.


**Table 5.** Antioxidant capacity of selected samples evaluated using more than one criterion.


**Table 6.** Advantages and disadvantages of the most commonly chemical methods used for testing the antioxidant activity [160, 161, 167, 171–173, 176, 184, 231, 261–263].

Mechanisms involved in the corresponding chemical reactions are also shown in the table: hydrogen atom transfer, HAT, ability of an antioxidant to reduce radicals by hydrogen donation for ORAC and TRAP assays; single electron transfer, SET, ability of an antioxidant to transfer one electron to reduce any compounds, including metals, carbonyl and radicals for DMPD and FRAP assays. HAT and SET mechanisms may occur together as in ABTS and DPPH assays. The DPPH method is one of the oldest and most frequently used for determining the antioxidant activity of food extracts and single compounds. In comparison with DPPH assay, the ABTS assay estimates more accurately [183, 254] the antioxidant capacity of foods, especially for those contain lycophilic, lipophilic and highly pigmented compounds. However, it has been stated that methods using HAT reactions will be preferred to those with SET reactions because the peroxyl radicals used in the first are the main FR found in lipid oxidation and biological systems [259]. ORAC is the most commonly used total radical‐trapping antioxidant assay and the most widely used essay for evaluating antioxidant [172] both in the industry and in the academic institutions. The evaluation of total antioxidant capacity is preferable than the individual antioxidant measurements [74] due to the complexity of food composition and the possibility of synergic interactions among the antioxidant compounds.

### **6. Antioxidant activity of the anthocyanins**

*Mixed hydrogen atom transfer (HAT) and single electron transfer (SET)*

• Applicability in lipid and aqueous phase

• Complex mechanism of reaction • Extra step to generate free radical • Free radical not stable for long periods

• High price of ABTS reagent • Complex mechanism of reaction • DPPH colour can be lost

• Sensitive to acidic pH

available

• It is nonspecific

tion time

conditions

• Expensive equipment

equipment • pH sensitive

• Requiring an acidic pH

• Steric accessibility influences the reaction

• No data of its stoichiometry with antioxidant standard and radical stability are

• Not all antioxidants reduce Fe3+ at a rate fast enough to allows its measurement • Compounds that absorbs at the wavelength of the determination may interfere

• FRAP and CUPRAC depend on the reac-

• The antioxidant which reduce metal ions may exert pro‐oxidant effect under certain

• Low correlation between the capacity measured by FRAP or CUPRAC method with that for radical scavenging measured by competition method such as ORAC

• Data variability can be large across

• Requires long times to quantifies results

• DMPD is only soluble in water

of time • Not standardised

• Can be automated and adapted for use with

• It just needs a UV‐vis spectrophotometer to

• It does not require specialized equipment • It can be performed using automated, semi‐ automated, or manual methods

• Fast enough to oxidize thiol‐type antioxidants

• Applicable to both hydrophilic and lipophilic antioxidants It is carried out at nearly physi-

• Integrates both degree and time of antioxidant

• May be performed in thermostated microplates

**Table 6.** Advantages and disadvantages of the most commonly chemical methods used for testing the antioxidant

• Determine the capacity of hydrophilic and hydrophobic samples simply

• No sample separation is needed

DMPD • Simpler, more productive and less expensive and compared ABTS test

ABTS • Inexpensive and easy to use

222 Flavonoids - From Biosynthesis to Human Health

• Stable to pH • Fast reaction

microplates

DPPH • Simple and highly sensitive • Can be automated

perform

FRAP • Simplicity, speed and robustness

CUPRAC • Rapid way to study plant extract profiles

ological pH values

ORAC • Uses biologically relevant free radicals • Simple and standardised

reaction

activity [160, 161, 167, 171–173, 176, 184, 231, 261–263].

• Stable and accessible reagents

• Selective

*Hydrogen atom transfer (HAT)*

*Single electron transfer (SET)*

The capacity of phenolic compounds to trap FR depends upon their structure, in particular, of the hydrogen atoms of the aromatic group that can be transferred to the FR [5, 10, 20, 24, 63, 113] and of the capacity of the aromatic compound to cope with the uncoupling of electrons as a result of the surrounding displacement of the electrons‐π system. As compared to other antioxidants, research on their health effects started more recently. This late interest in polyphenols is largely explained by the complexity [264] of their chemical structures. The anthocyanin and anthocyanidin health properties are due to their peculiar chemical structure, as they are very reactive towards ROS because of their electron deficiency [265–269]. The antioxidative properties of anthocyanidins have been recently explored; most of the widely distributed anthocyanidins and anthocyanins show more scavenging activity than that of the well‐known strong antioxidants trolox and catechol [20]. The physicochemical characteristics of anthocyanins [83, 90, 91], that is structure and size of the molecules (number and position of hydroxyl and methoxyl groups), water solubility and acidity constants, can control their ability to cross biological barriers. Results of antioxidant activity of foods are commonly expressed in TEAC (mmol or μmol/g sample), a capacity equivalent to trolox (a hydrosoluble synthetic antioxidant similar to vitamin E). However, some authors suggest [270] that the results should be expressed in vitamin C equivalent antioxidant capacity (VCEAC in mg/100 g), given that vitamin C is found naturally in some foods, whereas trolox is a synthetic compound.

Quantum chemical computations have recently been performed to study [265] the antioxidative properties of anthocyanidins, quantitative structure activity relationships (QSAR) and mechanisms of action involved such as HAT, SET and SPLET (sequential proton loss electron transfer). Construction and evaluation of QSAR for predicting anthocyanin activity radical scavenging using quantum chemical descriptor have been developed [271] with good prediction efficiency. 3D‐QSAR models from 21 anthocyanins based on their ORAC values have been used [272] with prediction (eggplant and radish) purposes. 3D‐QASR models have also been developed in a series [273] of anthocyanin derivatives of CYP3AH inhibitors (cytochrome P450).

#### **7. Structure of the anthocyanins**

The chemical structure of anthocyanins is appropriate for acting as antioxidants, as they can donate hydrogen or electrons to the FR or trap them and delocalize them in their aromatic structure [5, 10, 20, 265–269]. The structural differences among anthocyanins are related [5, 14, 274, 275] to the number of hydroxyl or methoxyl groups in the anthocyanidin skeleton, the position and the number of bonded sugar residues as well as by the aliphatic or aromatic carboxylates bonded to them. The hydroxylation pattern influences [276, 277] physiological properties such as light absorption and antioxidative activity, which is the base for many beneficial health effects of flavonoids. The hydroxyl groups in positions 3′ and 4′ provide a high stability to the radical formed by trapping FR and displacing the electrons in ring B, as well as the free hydroxyl groups in position 3 of ring C, and in position 5 of ring A, together with the carbonyl group in position 4 (**Figure 4**).

There are three important structural criteria for evaluating the antiradical effectiveness of a compound: (1) the presence of neighbouring hydroxyl groups, that is, in the position of ring B; (2) double bonds at conjugation 4‐oxo of ring C; and (3) hydroxyl groups in positions 3 and 5 of ring A.

The aglucons with identical hydroxylation in rings A and C, and a single OH group in ring B (4′‐ OH), including pelargonidin, malvidin and peonidin (**Figure 4**), have lower antioxidant activity when compared to compounds with groups 3′, 4′ di‐OH substituted (e.g. cyanidin) (**Figure 5**).

**Figure 4.** Chemical structure of pelargonidin (top left), malvidin (top right) and peonidin (bottom).

3D‐QSAR models from 21 anthocyanins based on their ORAC values have been used [272] with prediction (eggplant and radish) purposes. 3D‐QASR models have also been developed in a

The chemical structure of anthocyanins is appropriate for acting as antioxidants, as they can donate hydrogen or electrons to the FR or trap them and delocalize them in their aromatic structure [5, 10, 20, 265–269]. The structural differences among anthocyanins are related [5, 14, 274, 275] to the number of hydroxyl or methoxyl groups in the anthocyanidin skeleton, the position and the number of bonded sugar residues as well as by the aliphatic or aromatic carboxylates bonded to them. The hydroxylation pattern influences [276, 277] physiological properties such as light absorption and antioxidative activity, which is the base for many beneficial health effects of flavonoids. The hydroxyl groups in positions 3′ and 4′ provide a high stability to the radical formed by trapping FR and displacing the electrons in ring B, as well as the free hydroxyl groups in position 3 of ring C, and in position 5 of ring A, together with the carbonyl group in position 4 (**Figure 4**). There are three important structural criteria for evaluating the antiradical effectiveness of a compound: (1) the presence of neighbouring hydroxyl groups, that is, in the position of ring B; (2) double bonds at conjugation 4‐oxo of ring C; and (3) hydroxyl groups in positions 3 and 5 of ring A. The aglucons with identical hydroxylation in rings A and C, and a single OH group in ring B (4′‐ OH), including pelargonidin, malvidin and peonidin (**Figure 4**), have lower antioxidant activity when compared to compounds with groups 3′, 4′ di‐OH substituted (e.g. cyanidin) (**Figure 5**).

series [273] of anthocyanin derivatives of CYP3AH inhibitors (cytochrome P450).

**Figure 4.** Chemical structure of pelargonidin (top left), malvidin (top right) and peonidin (bottom).

**7. Structure of the anthocyanins**

224 Flavonoids - From Biosynthesis to Human Health

**Figure 5.** Chemical structures of pelargonidin, cyaniding and delphinidin, their spots on TLC and the colour of plant tissues [276].

Apparently, the OH groups in position 3′ and 4′ of ring B (catechol) are determinants of the antioxidant activity of saturated flavonoids. However, delphinidin is an exception to this principle as it has groups 3′ and 4′ di‐OH substituted (**Figure 5**) and still has a low antioxidant activity. The importance of the hydroxyl groups in position 3′ and 4′ of ring B contributes to the high antioxidant capacity also found for flavones [276, 277].

Most flavonoids are found naturally in a glycosylate form, and glycosylation changes the antioxidant activity [5, 278]: for cyanidin, there is an increase; for malvidin, a decrease; and for pelargonidin, no significant effect was shown [137]. Different sugars can have distinct effects on antioxidant activity. For example, in ORAC assays for cyanidin, glycosylation in position 3 of ring C with glucose or rhamnose increases the antioxidant activity, but with galactose, it declines.

The glycosylation (site, type and number of the glycosyl, glycosidic bond type) generally enhances [269] the stability, results in the hypsochromic effect and blueing, decreases the bioavailability and anticancer activity, and decreases, increases, or does not change the antioxidant activity of the anthocyanidins or anthocyanins. Note the diverse and complex chemistry of acyl groups and that their stabilizing effect exerted may be either independent or synergic. However, the acylation decreases the polarity of anthocyanins and creates steric hindrance effects (changing molecular size and spatial structure) to decrease the sensitivity of the anthocyanins to nucleophilic attack [274] and increasing the *in vitro* and *in vivo* chemical stability (though it lowers their apparent absorption) [113]. Nonacylated monoglycosylated anthocyanins have a greater inhibitory effect on human colorectal adenocarcinoma (HT29) cell proliferation [279]; anthocyanins with pelargonidin, triglycoside and/or acylation with cinnamic acid have a lesser effect.

Anthocyanins are more than flavylium cations [280]. In aqueous solutions, equilibrium of at least four other species determined by pH (and temperature) exists [281–285]. Above about pH 2.5, the coloured flavylium cation (only stable at pH ≤ 1, rare in natural environments) form typically hydrates (pH 4–5) to form the colourless hemiacetal (carbinol pseudo‐base), followed by ring‐opening tautomerization to the light yellow (E)‐chalcone, which can isomerize to the (Z)‐chalcone. At pH values of 7–8, blue‐purple quinoidal anions (which fades in several minutes) are formed. **Figure 6** shows a sample of wine (moderately acid pH 3.5–4.0) at different pH values and corresponds to the graphical abstracts of reference [280]. The state of ionization of the anthocyanins can be an important factor in relation to their antiradical activity. This is corroborated by the fact that the pseudo‐base and the quinoidal base of malvidin 3‐glucoside, generated at pH 4.0 and pH 7.0, respectively, have differences in antioxidant activity. It is possible play with the colour of anthocyanins [286, 287], for example, complexation with metal ions or with colourless organic molecules (co‐pigments) such as hydroxylated benzoic or cinnamic acids. Experiments undertaken with synthetic colourants (Ponceau 4R) have shown that they do not have antioxidant activity, whereas anthocyanin pigments confer an antioxidant activity far greater than that of the synthetic colourants available on the market. This shows that natural pigments besides providing a good source of colour have considerable antioxidant potential. Public concern about synthetic food dyes (suspected to cause adverse effect on health) has increased recently. For this reason, consumers and food manufacturers (i.e. beverage industry) increasingly demand "cleaner" colourants from natural sources [13, 48, 49, 54, 57, 79]. **Table 7** compares [288] the characteristics of both synthetic and natural colourants. Interesting alternatives in food systems to synthetic colourants are acylated anthocyanins [289–293]. A huge variety of hues can be achieved as a function of anthocyanin structure and pH of food matrix. The increasing interest in foods that help to prevent diseases has boosted the market for nutraceutical and/or medicinal food [294]. The term functional food appeared in Japan in the 1980s associated with processed food containing ingredients that affect physiological functions. Identification of health effects provoked by anthocyanins will increase their demanding what would open new perspectives [295] for their use in the food market.

**Figure 6.** Red wine at various pH values: graphical abstracts of Ref. [285].


pH 2.5, the coloured flavylium cation (only stable at pH ≤ 1, rare in natural environments) form typically hydrates (pH 4–5) to form the colourless hemiacetal (carbinol pseudo‐base), followed by ring‐opening tautomerization to the light yellow (E)‐chalcone, which can isomerize to the (Z)‐chalcone. At pH values of 7–8, blue‐purple quinoidal anions (which fades in several minutes) are formed. **Figure 6** shows a sample of wine (moderately acid pH 3.5–4.0) at different pH values and corresponds to the graphical abstracts of reference [280]. The state of ionization of the anthocyanins can be an important factor in relation to their antiradical activity. This is corroborated by the fact that the pseudo‐base and the quinoidal base of malvidin 3‐glucoside, generated at pH 4.0 and pH 7.0, respectively, have differences in antioxidant activity. It is possible play with the colour of anthocyanins [286, 287], for example, complexation with metal ions or with colourless organic molecules (co‐pigments) such as hydroxylated benzoic or cinnamic acids. Experiments undertaken with synthetic colourants (Ponceau 4R) have shown that they do not have antioxidant activity, whereas anthocyanin pigments confer an antioxidant activity far greater than that of the synthetic colourants available on the market. This shows that natural pigments besides providing a good source of colour have considerable antioxidant potential. Public concern about synthetic food dyes (suspected to cause adverse effect on health) has increased recently. For this reason, consumers and food manufacturers (i.e. beverage industry) increasingly demand "cleaner" colourants from natural sources [13, 48, 49, 54, 57, 79]. **Table 7** compares [288] the characteristics of both synthetic and natural colourants. Interesting alternatives in food systems to synthetic colourants are acylated anthocyanins [289–293]. A huge variety of hues can be achieved as a function of anthocyanin structure and pH of food matrix. The increasing interest in foods that help to prevent diseases has boosted the market for nutraceutical and/or medicinal food [294]. The term functional food appeared in Japan in the 1980s associated with processed food containing ingredients that affect physiological functions. Identification of health effects provoked by anthocyanins will increase their demanding what would open new perspectives [295] for

their use in the food market.

226 Flavonoids - From Biosynthesis to Human Health

**Figure 6.** Red wine at various pH values: graphical abstracts of Ref. [285].

**Table 7.** Advantages and disadvantages of natural and synthetic antioxidants commonly used for food protections [288].
