**3. Extraction techniques**

To measure antioxidant activity of food raw materials, the active molecules must be extracted from the food matrix. The processes of extraction of the phenolic compounds are affected by several factors such as the pH, the temperature, the solvent used. Thus, the optimization of this step requires a judicious choice of the set points of these factors. However, in the bibliog‐ raphy few studies have been devoted to the optimization of these factors.

Moreover, these factors need to be adjusted according to the matrix of the raw material and the quantity of antioxidant molecules. To help in the choice of the most suitable method of the extraction, the main processes described in the literature are summarized in table 4. The advantages and the drawbacks of each process are also reported.

Origin of the Variability of the Antioxidant Activity Determination of Food Material http://dx.doi.org/10.5772/60453 81


**ABTS FRAP ORAC**

Ultrasound extraction with methanol 6.7 3.5 7.8

Solvent extraction with methanol 40 \_ \_

Solvent extraction with methanol/ water (80%v/v) 4.62 \_ \_

These results indicates that for a given method, the extraction procedure has a great impact on antioxidant activity values. For example with the ABTS method, using an extraction with methanol as solvent and assisted by ultrasound, this leads to 6.7 µmol TE/g FW; while the extraction with a mixture of methanol and water (80%v/v) or with acetone furnishes only 0.94 µmol TE/g FW. The measurement of bioavailability directly in plasma gives a value of 8.3 µmol TE/g FW. Different values are also obtained depending on the method of the extraction

The analysis of the results of antioxidant activity of pure components and extracts from the food matrix indicates a broad variability in antioxidant values whatever the method used. This variability is also observed for a given method with the variety or the degree of maturation of the food raw material. This variability of the antioxidant activity determination can be attributed to three sources (i) factors related to food products such as the variety, and the growth method. (ii) Factors related to the extraction method such as pH, temperature, solvent, presence of an accelerator and (iii) factors related to the method used for the antioxidant

To measure antioxidant activity of food raw materials, the active molecules must be extracted from the food matrix. The processes of extraction of the phenolic compounds are affected by several factors such as the pH, the temperature, the solvent used. Thus, the optimization of this step requires a judicious choice of the set points of these factors. However, in the bibliog‐

Moreover, these factors need to be adjusted according to the matrix of the raw material and the quantity of antioxidant molecules. To help in the choice of the most suitable method of the extraction, the main processes described in the literature are summarized in table 4. The

raphy few studies have been devoted to the optimization of these factors.

advantages and the drawbacks of each process are also reported.

**Table 3.** Total antioxidant capacity of golden delicious apples (in µmol TE/ g FW) according to [4-7].

Solvent extraction with acetone (70%), water (28%), and

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used with FRAP or ORAC protocols.

activity determination.

**3. Extraction techniques**

Extraction in plasma 8.3 4.4 9.15

acetic acid (2%) \_ \_ 26.47


**Table 4.** Advantages and drawbacks of the main extraction methods used

Different processes of extraction of active compounds are available. However, the effectiveness of these processes is affected by several factors such as the nature of the solvent, the temper‐ ature or the extraction time. The presence of an accelerator of extraction such as microwaves or ultrasounds also plays significant role. The availability of the active molecule will be also taken into account. The analysis of the efficiency of the different processes described above indicates that the use of accelerators provides higher yields than the solid-liquid extraction (SLE) while allowing a low temperature to be maintained. The least advantageous method is the solid-liquid extraction due to the toxicity of solvent and the long time extraction in the majority of cases. The use of microwaves (MAE) as accelerator is highly acclaimed as an alternative method. The use of ultrasounds (UAE) also allows an enhancing of the extraction of active compounds at low temperatures but it leads to lower yields than with microwaves. Other accelerators can be used (ASE, PLE) but they need to increase the pressure and/ or the temperature, which can damage target molecules or alter their properties. Supercritical fluid extraction (SFE) does not use drastic conditions but the molecules extracted must to be soluble in liquid CO2. The use of a co-solvent may be necessary if antioxidants are poorly soluble in CO2. So, the difference in the efficiency of the different extraction methods used for antioxidant activity determination could be at the origin of the variability observed in the bibliography. Thus, the choice of a method of extraction needs to take into account the nature of the food matrix and the structure of the molecule to be extracted. The physico-chemical factors of the extraction must be also adjusted carefully. In conclusion there is a great need to standardize the methods of extraction by establishing different protocols and pay attention to different conditions.

## **4. In vitro methods for antioxidant activity measurement**

An antioxidant is usually defined as a molecule which delays, prevents or removes oxidative damage to a target molecule [17], thus an antioxidant is assessed according to its ability to neutralize free radicals as for example in equation 1 to avoid oxidative degradations.

$$\text{AO} \vdash \text{FR} \cdot \rightarrow \text{AO} \cdot \text{+FR} \tag{1}$$

AO: antioxidant molecule, FR⋅: free radicals

**Technique Principle Tool Advantages Drawbacks Example of use**

Efficiency in solvent useRecycling possibility Prevention of oxidation reactions High product quality. Absence of solvent in solute phase. Flexible process.

High extraction capacity. Mild conditions. Low cost. Short process time without back extraction. The potential to achieve the desired purification and concentration of the product in a single step.

Different processes of extraction of active compounds are available. However, the effectiveness of these processes is affected by several factors such as the nature of the solvent, the temper‐ ature or the extraction time. The presence of an accelerator of extraction such as microwaves or ultrasounds also plays significant role. The availability of the active molecule will be also taken into account. The analysis of the efficiency of the different processes described above indicates that the use of accelerators provides higher yields than the solid-liquid extraction (SLE) while allowing a low temperature to be maintained. The least advantageous method is the solid-liquid extraction due to the toxicity of solvent and the long time extraction in the majority of cases. The use of microwaves (MAE) as accelerator is highly acclaimed as an alternative method. The use of ultrasounds (UAE) also allows an enhancing of the extraction of active compounds at low temperatures but it leads to lower yields than with microwaves. Other accelerators can be used (ASE, PLE) but they need to increase the pressure and/ or the temperature, which can damage target molecules or alter their properties. Supercritical fluid extraction (SFE) does not use drastic conditions but the molecules extracted must to be soluble in liquid CO2. The use of a co-solvent may be necessary if antioxidants are poorly soluble in CO2. So, the difference in the efficiency of the different extraction methods used for antioxidant activity determination could be at the origin of the variability observed in the bibliography.

No reports on the use of the ATPE to extract and purify anthocyanins.

Extraction of mulberry anthocyanins.

pressure with a supercritical fluid.

Extraction in an aqueous two-phase system.

Short chain alcohol/ hydrophilic organic solvents. Inorganic salts.

**Table 4.** Advantages and drawbacks of the main extraction methods used

Aqueous twophase extraction (ATPE) [16]

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$$\mathbf{A}\mathbf{H} + \mathbf{R} \cdot \to \mathbf{A} \cdot + \mathbf{R} \tag{2}$$

#### AH : antioxidant molecule, R⋅: free radicals

Free radicals are reactive oxygen species produced either through numerous biological reactions: mitochondrial respiratory chain or any inflammatory conditions, or from numerous environmental factors such as pollutants, U.V., alcohol, smoking, stress, drugs,... Free radicals are useful if they are in low quantity; they allow the elimination of old cells of the living organism by oxidation reactions or participating in the body's defense. However if they are too numerous, they attack other cells inducing a rapid aging of these cells which causes damage to living organisms. To avoid these reactions, antioxidants can neutralize free radicals and protect our cells. When antioxidant quantity is not enough to neutralize free radicals, it leads to the oxidative stress which has a great importance in the development of chronic degenera‐ tive diseases including coronary heart disease, cancer and the degenerative processes associ‐ ated with aging.

Antioxidants can neutralize radicals by two different mechanisms. The final product will be the same but reactions occurring are different. Radicals can be deactivated either by hydrogen donation (Hydrogen Atom Transfer HAT) or by electron transfer (Single Electron Transfer SET). HAT and SET mechanisms may occur in parallel, the predominant mechanism being determined according to antioxidant structure and properties, solubility, partition coefficient, and system solvent [18]. A wide variety of one-dimensional methods have been developed to measure antioxidant activity in vitro. The methodological diversity is due to the use of a broad range of conditions for antioxidant activity measurement. This diversity has led to widely conflicting results that are extremely difficult to interpret.

## **4.1. Systems based on SET**

SET-based methods involve two components in the reaction, i.e. the antioxidant and the oxidant. These methods measure the ability of an antioxidant to reduce any compound (metals, radicals) by electron transfer according to equations 3 and 4.

$$\text{R}\cdot + \text{AH} \rightarrow \text{X}\cdot + \text{AH} + \cdot \tag{3}$$

$$\text{M(III)} + \text{AH} \rightarrow \text{AH} + \text{M(II)}\tag{4}$$

SET reactions are pH dependent. Indeed, relative reactivity in SET methods is based primarily on deprotonation and the ionization potential of the reactive functional groups.. Ionization potential decreases when pH increases, so SET reactions are favored in alkaline environments. SET reactions are usually slow and can require a long time to reach their final state, so antioxidant capacity calculations are based on the decrease in product concentration rather than their kinetic.

#### **• ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay**

ABTS assay is a spectrophotometric method which measures the ability to an antioxidant to scavenge a free radical cation ABTS▪+. This method was developed by [19] and adapted by [20] to generate directly the radical ABTS▪+ through a reaction between ABTS solution (7mM) with potassium persulfate (2,45 mM) in water. The reaction mixture, which is allowed to stand at room temperature for 12-16 h before use, produces a dark blue solution. Thus, the mixture is diluted with ethanol or phosphate buffered saline (pH 7.4) to a final absorbance of 0.7 at 734 nm (wavelength the most used) and 37 °C. The assay is based on the discoloration of ABTS▪+ during its oxidation by antioxidant compounds, thus reflecting the amount of ABTS radicals that are scavenged within a fixed time period (generally 6 min). The absorbance of the reaction mixture between radicals and antioxidants is compared to that of the 6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid (Trolox). When Trolox is used as standard, this assay is also called Trolox Equivalent Antioxidant capacity (TEAC) assay.

The major advantages of this method are its simplicity to perform and its applicability in lipid and aqueous phases [21]. Thus this method has been widely used in testing antioxidant capacity in food samples. Moreover, the ABTS radical is stable over a wide pH range and can be used to study pH effects on antioxidant mechanisms [22]. This method can be automated and adapted to the use with microplates which allows the carrying out of this measurement with better precision and time.

A major disadvantage of this method is that only the rapid oxidation reactions can be measured because incubation time is often short (6 min). Thus, antioxidants whose constant rates of radical scavenging are low can be undervalued in comparison with their real antioxidant capacity. Moreover, imprecisions on ABTS values can be increased by the fact that variations can occur according to the preparation of ABTS▪+, and the medium temperature which has to be controlled.

#### **• DPPH (2,2-diphenyl-1-picrylhydrazyl) assay**

**4.1. Systems based on SET**

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than their kinetic.

with better precision and time.

be controlled.

SET-based methods involve two components in the reaction, i.e. the antioxidant and the oxidant. These methods measure the ability of an antioxidant to reduce any compound (metals,

SET reactions are pH dependent. Indeed, relative reactivity in SET methods is based primarily on deprotonation and the ionization potential of the reactive functional groups.. Ionization potential decreases when pH increases, so SET reactions are favored in alkaline environments. SET reactions are usually slow and can require a long time to reach their final state, so antioxidant capacity calculations are based on the decrease in product concentration rather

ABTS assay is a spectrophotometric method which measures the ability to an antioxidant to scavenge a free radical cation ABTS▪+. This method was developed by [19] and adapted by [20] to generate directly the radical ABTS▪+ through a reaction between ABTS solution (7mM) with potassium persulfate (2,45 mM) in water. The reaction mixture, which is allowed to stand at room temperature for 12-16 h before use, produces a dark blue solution. Thus, the mixture is diluted with ethanol or phosphate buffered saline (pH 7.4) to a final absorbance of 0.7 at 734 nm (wavelength the most used) and 37 °C. The assay is based on the discoloration of ABTS▪+ during its oxidation by antioxidant compounds, thus reflecting the amount of ABTS radicals that are scavenged within a fixed time period (generally 6 min). The absorbance of the reaction mixture between radicals and antioxidants is compared to that of the 6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid (Trolox). When Trolox is used as standard, this assay

The major advantages of this method are its simplicity to perform and its applicability in lipid and aqueous phases [21]. Thus this method has been widely used in testing antioxidant capacity in food samples. Moreover, the ABTS radical is stable over a wide pH range and can be used to study pH effects on antioxidant mechanisms [22]. This method can be automated and adapted to the use with microplates which allows the carrying out of this measurement

A major disadvantage of this method is that only the rapid oxidation reactions can be measured because incubation time is often short (6 min). Thus, antioxidants whose constant rates of radical scavenging are low can be undervalued in comparison with their real antioxidant capacity. Moreover, imprecisions on ABTS values can be increased by the fact that variations can occur according to the preparation of ABTS▪+, and the medium temperature which has to

R + AH X- + AH+ ×® × (3)

M III + AH AH+ + M II ( ) ® ( ) (4)

radicals) by electron transfer according to equations 3 and 4.

**• ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay**

is also called Trolox Equivalent Antioxidant capacity (TEAC) assay.

DPPH is one of the oldest and most popular technique used to measure the antioxidant activity of a compound. This method was first described by [23] and subsequently modified by numerous researchers. This method measures the reducing ability of antioxidants toward DPPH▪. DPPH▪ is commercially available and does not have to be generated as for ABTS assay. The antioxidant effect is proportional to the disappearance of DPPH▪ in a methanolic solution. DPPH solution being purple, the absorbance of the mixture can be followed by spectopho‐ tometry at 515 nm. Assay time may vary from 10±20min up to 6h. Other techniques such as electron spin resonance (EPR) can be used [18].

Like ABTS, this method is simple and can be automated. However, values found by the DPPH method have to be considered as apparent antioxidant activities because (i) DPPH color can be lost via either radical reaction (HAT) or reduction (SET) as well as unrelated reactions, (ii) steric accessibility also influences the reaction, thus small molecules are favored because they have a better access to the radical site and other compounds such as carotenoids can interfer in the measurement of the antioxidant activity [24].

#### **• Ferric reducing ability of plasma (FRAP)**

The FRAP assay is different from the others as there are no free radicals involved but the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) is monitored. FRAP assay was initially described by [25] for measuring reducing power in plasma and subsequently adapted and modified by numerous researchers to measure antioxidant power of botanical extracts [26]. When an Fe3+-TPTZ (2,4,6-tripyridyl-s-triazine) complex is reduced to Fe2+ by an antioxidant under acidic conditions, it forms an intense blue color with absorption maximum at 593nm. Thus the antioxidant effect can be followed by a spectrophotometer.

A major advantage of the FRAP assay is its simplicity, speed and robustness. The validity of this assay was proved in order to quantify samples with hydrophilic and lipophilic antioxi‐ dants. As for ABTS assay, only rapid reactions will be taken into account until the incubation time in this method is short (4-6 min). The FRAP assay measures only reactions following the SET mechanism, antioxidant hydrogen donator may go unmeasured by this assay. This method is thus used in parallel with others to determine the action mechanisms of antioxidants. Protein and thiol antioxidants, such as glutathione cannot be measured by the FRAP assay.

#### **• CUPric Reducing Antioxidant Capacity (CUPRAC) assay**

The CUPRAC assay has many similarities to FRAP, Cu is used instead of Fe. This assay is based on the reduction of Cu (II) to Cu (I) by the antioxidants present in the sample. Cu (I) forms a complex with neocuproine (2,9-dimethyl-1,10-phenanthroline) with a maximum absorbance at 450 nm. A dilution curve generated by uric acid standard is used to convert sample absorbance to uric acid equivalents [18]. Phenanthroline complexes have very limited water solubility and must be dissolved in organic solvents. Cuprac values are comparable to TEAC values, whereas FRAP values are lower. The CUPRAC assay has many advantages [27]. Indeed, the CUPRAC assay is more selective due to its lower redox potential. Sugars and citric acid cannot interfere in the assay because they are not oxidized in CUPRAC. The CUPRAC reagent is much more stable than other radicals such as DPPH, ABTS. The redox reaction giving rise to a coloured chelate of Cu(I)-Nc is relatively insensitive to a number of parameters such as air, sunlight, humidity, and pH. The CUPRAC reagent can be adsorbed on a membrane to build an optical antioxidant sensor.

A variant of CUPRAC assay is Bioxytech using bathocuproine instead of neocuproine [18].

### **4.2. Systems based on HAT**

The HAT-based methods involve a synthetic radical generator, oxidisable molecular probe and an antioxidant compound. This method measures the ability of an antioxidant to quench free radicals by hydrogen donation as in equation 2. Assays that are based on HAT mechanisms measure competitive kinetics [22].

Antioxidant with hydroxyl component OH donates an H atom to an unstable free radical to give a more stable radical. HAT reactions are solvent and pH independent and are usually quite rapid, typically completed in seconds to minutes. The presence of reducing agents, including metals, is a complication in HAT assays and can lead to erroneously high apparent reactivity [18].

#### **• Oxygen radical absorbance capacity (ORAC)**

The ORAC assay has been used widely in measuring the net resultant antioxidant capacity (or peroxyl radical absorbance capacity) of botanical and other biological samples.

The ORAC assay was developed by [28] for the determination of reactive oxygen species in biological systems. [29] modified the method using fluorescein (FL) as a more stable and reproducible fluorescent probe. This method exists under several adaptations but the principle always remains the same: using a fluorescent probe and AAPH (2,2'-azobis(2-amidinopro‐ pane) dihydrochloride) to generate peroxyl radicals. A HAT reaction occurs between antiox‐ idant samples (or standard) and the peroxyl radicals generated by thermal degradation of AAPH. These reactions lead to a loss of fluorescence measured at 515 nm.

The final results (ORAC values) were calculated using the differences between blank and sample areas under the quenching curves of fluorescein, and were expressed as micromoles of Trolox equivalents (TE).

The ORAC method is superior to similar methods because it combines inhibition time and inhibition degree of free radicals. The ORAC using fluorescein is specific for antioxidants and is sensitive, precise and robust. This assay can model reactions of antioxidants with lipids in both food and physiological systems and it can be adapted to detect both hydrophilic and hydrophobic antioxidants with minor modifications. However, the need of a fluorometer, which may be not routinely available, is considered as a disadvantage of this method. The long analysis time has also been a major criticism even if this assay can be automated.

#### **• β -carotene bleaching test**

This assay was developed by [30] and modified by other researchers. This assay is based on the generation of a stable β –carotene radical from β –carotene peroxyl radical; the latter coming from lipids (linoleic acid for example) in the presence of ROS and O2. Thus, the assay measures the ability to an antioxidant to quench β –carotene radical by donating hydrogen atoms. It results in the bleaching of the solution which can be followed with a spectrophotometer at 470 nm.

The main advantage of this assay is its applicability in both lipophilic and hydrophilic environments. Another advantage is that the carotenoid bleaching assay can detect either the antioxidant or pro-oxidant action of a compound under investigation. Lastly, the carotenoid bleaching assay can be automated by the use of microplates. However, a major limitation is that the discoloration of β-carotene at 470 nm can occur through multiple pathways, thereby complicating the interpretation of results. Other carotenoids such as crocin bleach only using the radical oxidation pathway but crocin is not commercially available. The use of molecules commercially available provide repeatable and reliable data between laboratories

#### **• Total peroxyl radical-trapping antioxidant parameter assay (TRAP)**

The total peroxyl radical-trapping antioxidant parameter (TRAP) assay was introduced by [31] to measure the total antioxidant status of human plasma. This assay monitors the ability of an antioxidant to interfere with the reaction between peroxyl radicals generated by AAPH (2,2' azobis (2-amidinopropane) dihydrochloride) and the target. The oxidation is monitored by oxygen uptake measurement. Results are expressed as time necessary to consume all radicals in comparison with a standard (Trolox). Many modifications were realized on this assay to react with lipids, or to be followed by fluorimetry or to take into account interference from lipids and proteins in plasma [32]. Despite its simplicity, the TRAP assay leads to imprecise results because of difficulties to maintain the endpoint over the period of time. Several modifications were developed by using chemiluminescence methods [33].
