Antioxidant activity depends on the hydro-/lipophilicity of the antioxi- dant. The antioxidant activity of plant extract depends on the concentra- tion of antioxidant phytochemicals and the solvent used for extraction, and also on its form of preparation. Furthermore, the complex compos- itions of the extracts could intensify certain interactions such as syner- gistic, additive or antagonistic effects between their components and/ or medium (Koleva et al., 2002). Antioxidant capacity as measured by different in vitro assays differs. Different assays have been introduced to measure antioxidant capacity in order to assess its ability to counteract the effects of various radicals.
8.3.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay
The relatively stable organic radical DPPH has been used widely in deter- mination of the antioxidant activity of single compounds, as well as of
different plant extracts (Katalinic et al., 2006). DPPH assay uses a radical dissolved in organic media and is based on the reduction of the purple DPPH
∙
to 1,1-diphenyl-2-picrylhydrazine with discoloration, and is there- fore applicable to hydrophobic systems. DPPH∙
, a stable free radical with a characteristic absorption at 515 nm, is used to study the radical scav- enging effects of plant extracts. The degree of discoloration indicates the scavenging potential of the sample antioxidant. As antioxidants donate protons to this radical, absorption decreases (Fig. 8.1). The decrease in absorption is taken as a measure of the extent of radical scavenging. The lower absorbance at 515 nm represents the higher DPPH scavenging ac- tivity. The percentage of DPPH scavenging activity is expressed by the following equation:Percentage inhibition = [1 – (test sample absorbance/blank sample absorbance)] × 100.
The results are expressed in terms of EC50, which corresponds to the amount of sample needed in terms of antioxidant concentration to decrease the initial DPPH
∙
concentration by 50%. EC50 is calculated from a linear equa- tion obtained by plotting antioxidant concentration and average percentage antioxidant activity (Table 8.1). The antiradical power (ARP) is another way to express the results, and ARP of extract is calculated as (Arbos, 2004; Suja et al., 2005):ARP = (1/EC50).
High concentration of DPPH in the reaction mixture gives absorbance beyond the accuracy of the spectrophotometer measurement (Ayre, 1949; Sloane and William, 1977). As a result of widely different protocols being used by different research groups, the EC50 values for even the standard antioxidants like ascorbic acid and BHT vary. Light, oxygen and pH of the reaction mixture also affect the absorbance of DPPH (Ozcelik et al., 2003). The accuracy range of spectrophotometric measurements falls within an absorbance of 0.221–0.698, which is equal to a transmittance of 20–60% (Ayre, 1949). DPPH concentration corresponding to this range is 25–70 μm
2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH•) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) O2N NO2 N• N NO2 O2N NO2 N N H NO2 RH (antioxidant) R´
(Sharma and Bhatt, 2009). The absorbance of DPPH without any addition was stable over 30 min, and the suitable solvent for the DPPH assay was methanol or buffered methanol for the antioxidant assay of non-polar and polar compounds/extracts. DPPH radical scavenging activity is influenced by the polarity of the reaction medium, the chemical structure of the rad- ical scavenger and the pH of the reaction mixture (Ozcelik et al., 2003; Saito et al., 2004; Shizuka and Kawata, 2005). The EC50 values of three standard antioxidants, ascorbic acid, BHT and propyl gallate, in methanol and buffered methanol was reported by Sharma and Bhatt (2009). The rad- ical scavenging profiles of ascorbic acid and propyl gallate were similar in methanol and buffered methanol as solvents. However, unlike ascorbic acid and propyl gallate, the DPPH radical scavenging activity of BHT was markedly high in buffered methanol as compared to methanol alone. The difference in the IC50 values of BHT in methanol and buffered methanol could be due to one or more factors, as described above.
Longhi et al. (2011) reported the ARP of five standard antioxidants in the following order: BHA (2.11), vitamin C (3.70), a-tocopherol (3.85), quercetin (10.95) and gallic acid (12.72).
The DPPH scavenging activity of phenolics was correlated positively with the number of hydroxyl groups (Sroka and Cisowski, 2003). This observation explains the relative EC50 values of BHT and propyl gallate. Propyl gallate with three hydroxyl groups has lower IC50 values as com- pared to BHT with one hydroxyl group. The radical scavenging reaction of ascorbic acid with DPPH was instantaneous. On the other hand, the radical scavenging reaction of BHT with DPPH was slow and absorbance continued to decrease for a period of 90 min of observation. The reaction of DPPH with propyl gallate was also quite fast, but slower as compared to that with ascorbic acid. For the sake of uniformity, a time interval of 30 min was taken for ascorbic acid, BHT and propyl gallate radical scavenging cap- acity measurements. However, it is important to carry out a time course of radical scavenging activity while using DPPH radicals for the assay of antioxidant assay.
The DPPH method is very rapid, simple, sensitive and reproducible, and does not require special instrumentation. Very mild experimental conditions are required for this assay. DPPH assay is not discriminative with respect to radical species, but gives a general idea of the radical quenching ability.
Table 8.1. EC50 values of ascorbic acid, BHT and propyl gallate using DPPH antioxidant assay. (From Sharma and Bhatt, 2009.)
Standard IC50 (μm)
Ascorbic acid 11.8,a 11.5b
BHT 60,a 9.7b
Propyl gallate 4.4,a 4.7b
8.3.2 2-2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) has high water solubility and chemical stability. It is a peroxidase substrate and generates a metastable radical with a characteristic absorption spectrum and high molar absorptivity at 414 nm when oxidized in the presence of hydrogen peroxide (Arnao et al., 1996). However, there are secondary absorption maxima in the wavelength regions of 645, 734 and 815 nm. ABTS assay is based on the generation of a blue/green ABTS
∙
+ that canbe reduced by antioxidants in the reaction medium on a timescale dependent on the antioxidant activity. This ABTS radical cation decol- onization assay is applicable to both lipophilic and hydrophilic antioxi- dants, including flavonoids, hydroxycinnamates, carotenoids and plasma antioxidants. Generation of the ABTS (2,2′-azinobis(3-ethylbenzothiazo- line-6-sulfonic acid) radical cation forms the basis of this spectrophoto- metric antioxidant assay. The preformed radical mono cation of ABTS
∙
+is generated by the oxidation of ABTS with potassium persulfate and is reduced in the presence of hydrogen donating antioxidants (Fig. 8.2). The addition of antioxidants to the preformed radical cation reduces it to ABTS to an extent and on a timescale depending on the antioxidant activity, the concentration of the antioxidants and the duration of the re- action. The extent of decolonization as the percentage inhibition of the ABTS
∙
+ radical cation is determined as a function of concentration andHO3S S S N N N N SO3H CH2CH3 CH2CH3 ABTS HO3S S S N N N N SO3H CH2CH3 CH2CH3 ABTS +K2S2O8 (potassium persulfate) + . + ·
time, and is calculated relative to the reactivity of trolox as standard and under the same conditions. ABTS is dissolved in water to a 7-mM con- centration. ABTS radical cation (ABTS
∙
+) is produced by reacting ABTSsolution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use.
For the study of phenolic compounds and food extracts, the ABTS
∙
+ so-lution is diluted with ethanol, and for plasma antioxidants with 5 mM phos- phate buffered saline (PBS) pH 7.4, to an absorbance of 0.70 ± 0.02 at 734 nm and equilibrated at 30°C. After the addition of 1.0 ml of diluted ABTS
∙
+solution (A734 = 0.70 ± 0.02) to 10 μl of antioxidant compounds or trolox
standards (final concentration = 0–15 μM) in ethanol or PBS, the absorbance reading was recorded at 30°C exactly 1 min after initial mixing and up to 6 min. Appropriate solvent blanks are run in each assay. All determinations are car- ried out at least three times and in triplicate. The reduction of the absorbance is calculated according to the following equation:
Percentage inhibition = (Abst= 0 – Abst = t)/Abst = 0 × 100,
where Abst= 0 = absorbance at 0 min; Abst= t = absorbance after t min. The percentage inhibition of absorbance at 734 nm is calculated and plotted as a function of concentration of antioxidants and trolox for the standard and the reference data (Re et al., 1999). The reduction of the absorbance is plotted against the amount of sample to draw a regression line. The ratio between the sample and trolox’s slope of regression line is calculated and expressed as the TEAC. Results are expressed as TEAC (micromole trolox equivalents per milligram of dry extract). Higher TEAC values correspond to higher antioxidant activity.
Both DPPH and ABTS are convenient in their application and large number of samples can be screened in a short time; nevertheless, they are limited as they use non-physiological radicals. The results of DPPH and ABTS assays differ for plant extracts as the stiochiometry of reactions be- tween the antioxidant compounds in the extract and ABTS
∙
+ and DPPH∙
differ. Also, the electron reduction potential of ABTS
∙
+ and DPPH∙
is dif-ferent for different compounds (Amensour et al., 2010).
Relative antioxidant capacity (RACI) was defined as an integrated approach to compare the antioxidant capacity of different foods or food components measured with two or more chemical assays (Sun and Tanumihardjo, 2007; Sun et al., 2009).
8.3.3 Phosphomolybdenum method
The phosphomolybdenum method is a quantitative method and total antioxi- dant activity is expressed as the number of equivalents of ascorbic acid. Spectrophotometric measurement of antioxidant capacity by the phos- phomolybdenum method is based on the reduction of molybdenum (VI) to molybdenum (V) by the analyte sample (Prieto et al., 1999). Sample solution
is mixed with a reagent solution comprising sulfuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM) and incubated in a boiling water bath for 90 min. Absorbance of green coloured phosphate/ molybdenum (V) compounds with absorption maxima at 695 nm is meas- ured. For samples of unknown composition, water-soluble antioxidant capacity is expressed as the equivalent of ascorbic acid, μmole g–1 of ex-
tract (Gupta and Prakash, 2009).
8.3.4 Reducing power assay
Reducing power has been used as one of the antioxidant capability indi- cators of medicinal herbs (Duh and Yen, 1997). Reducing properties are generally associated with the presence of reductones, which are believed to break radical chains by the donation of a hydrogen atom, indicating that antioxidative properties are concomitant with the development of reducing power (Gordon, 1990). The reducing power may accord with the overall antioxidant activity. Tanaka et al. (1988) noted that the anti- oxidative effect increased exponentially as a function of the development of the reducing power, indicating that the antioxidative properties were concomitant with the development of the reducing power.
In this assay, the Fe3+/ferricyanide complex is reduced to the ferrous
form (Fe2+) by antioxidants. The ferrous ion formed is monitored by meas-
uring the formation of Perl’s Prussian blue at 700 nm (Oyaizu, 1986). The higher the absorbance, the stronger the reducing power (Guo et al., 2001). Reducing power is increased by increasing the sample concentration.
Oxidant
Fe(S-CN)2 Fe(S-CN)3
Ferrous thiocyanate Fe3+ thiocyanate
8.3.5 Ferric reducing antioxidant power
Ferric reducing antioxidant power (FRAP), also, known as ferrous ion chelating ability assay, is different from other assays as no free radicals are involved, but the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) is
monitored. Ferrous ion, commonly found in food systems, is well known as an effective pro-oxidant due to its high reactivity. Ferrous ion partici- pates in the direct or indirect initiation of lipid oxidation (Wettasinghe and Shahidi, 2002).
FRAP assay is carried out to determine the capability of a substance to bind with the oxidation catalytic ferrous ion. The FRAP method is based on the reduction of a colourless ferroin analogue, Fe3+ complex of tripyrid-
yltriazine Fe (TPTZ)3+, to the intensely blue coloured ferrous (Fe2+) com-
plex, Fe (TPTZ)2+, by antioxidants in acidic media (at low pH). A complex
Ferrozine can form complexes with Fe2+ quantitatively. The complex
formation is disrupted in the presence of other chelating agents, with the result that the colour of the complex decreases.
A measure of the rate of colour reduction allows estimation of the che- lating activity of the coexisting chelator (Yamaguchi, 1980). The higher the ferrous ion chelating ability of the test sample gives the lower ab- sorbance. The percentage inhibition of the formation of the Fe2+/ferrozine
complex is calculated using the formula:
Scavenging effect (%) = [(Acontrol – Asample)/Acontrol] × 100,
where Acontrol = absorbance of the Fe2+/ferrozine complex and A
sample =
absorbance of the test compound.
The percentage inhibition of absorbance is plotted as a function of concentration of ethylenediamine tetra acetic acid (EDTA) for the standard reference data. The results are expressed in terms of EDTA equivalents (micromole EDTA equivalents per gram of dry extract).
The FRAP assay is quick and simple to perform to measure the anti- oxidant capacity not only of pure compounds but also of fruit, wines and animal tissues (Wojdyło et al., 2007). The reference antioxidant for this assay must be water soluble, such ascorbic acid, uric acid or trolox. This method is used mostly in conjugation with other assays. However, the reducing capacity does not necessarily reflect the antioxidant activity (Katalinic et al., 2006; Wong et al., 2006).
8.3.6 Oxygen radical absorbance capacity
Oxygen radical absorbance capacity (ORAC) has been found to be the most relevant method for biological samples. ORAC measures the absorb- ance capacity of peroxyl radicals. The ORAC method is the only one so far that combines the total inhibition time and the percentage of free radical damage by the antioxidant into a single quantity (Zulueta et al., 2009). The method is based on the detection of chemical change in a fluorescent molecule caused by a free radical attack. It measures the antioxidant scav- enging activity against peroxyl radicals generated by thermal decompos- ition of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) (Fig. 8.3). AAPH is used as a peroxyl radical generator, fluorescein (FL), as a fluor- escent probe and trolox as a standard. The loss of fluorescence of FL is an indication of the extent of damage caused from its reaction with the per- oxyl radicals. The ORAC assay is carried out on a multi-label counter with fluorescence filters. Initially developed by Cao et al. (1993), the method consists of measuring the decrease in the fluorescence of a protein as a result of the loss of its conformation when it suffers oxidative damage caused by a source of peroxyl radicals (ROO.). The method measures the
ability of the antioxidants in the sample to protect protein from oxidative damage. The protective effect of an antioxidant is measured by assessing the area under the fluorescence decay curve, and antioxidant activity is
expressed in micromole trolox equivalents per gram of dry weight of food or biological samples (Kratchanova et al., 2010). ORAC is extremely sen- sitive. The samples must be diluted appropriately before analysis to avoid interference.
8.3.7 b-Carotene–linoleate method
Real food systems consist of multiple phases in which lipid and water co- exist with some emulsifier; therefore, an antioxidant assay using a hetero- geneous system such as oil in water emulsion is also required. Linoleic acid and linoleic acid emulsion systems represent homogeneous and heteroge- neous systems (Osawa and Namiki, 1981). A linoleic acid system can be correlated with a homogeneous systems or bulk oil phase system. Also, a linoleic acid emulsion system can be simulated with a biological lipid system, or with food or fat emulsion. In b-carotene–linoleic acid bleaching and linoleic acid emulsion system–thiocyanate methods, inhibition of per- oxidation is taken as the index of activity. In a b-carotene–linoleate model of an antioxidant assay system, the mechanism of bleaching of b-carotene is a free radical-mediated phenomenon resulting from the hydroperox- ides formed from linoleic acid. b-carotene in this model system under- goes rapid discoloration in the absence of an antioxidant because of the coupled oxidation of b-carotene and linoleic acid generating free radicals. The linoleic acid free radical formed on the abstraction of a hydrogen atom from one of its diallylic methylene groups attacks the highly unsaturated b-carotene molecules and as they lose their double bond, b-carotene loses its chromophore and characteristic orange colour, which can be moni- tored spectrophotometrically. The presence of antioxidants can hinder the extent of b-carotene bleaching by neutralizing the linoleate free radical and
H N N N H N+ H CL– AAPH N+• O•O • N N N + 2N C. N+• H H Cl+ H Cl+ Stable product (loss of fluorescence) Fluorescent probe H N+• 2 N O–O• Antioxidants Stable products H Cl+
other free radicals formed in the system (Jayaprakash et al., 2001). In an antioxidant assay system using a linoleic acid–thiocyanate system, lino- leic hydroperoxides are generated because of the oxidation of the linoleic acid, which further decomposes to many secondary oxidation products (Hua-Ming et al., 1996). The oxidized products react with ferrous sulfate to form ferric sulphate, then to ferric thiocyanate of a blood-red colour. In the presence of antioxidants, the oxidation of linoleic acid is slowed; therefore, colour development due to the formation of thiocyanate will be slow.
The results are expressed in percentage basis of preventing bleaching of b-carotene. The antioxidant activity of the extracts is calculated in terms of b-carotene bleaching using the following formula (Abdille et al., 2005):
Antioxidant activity = 100 [1 – (A0 – At)/(A0 0 – A0t)],
where A0 and A0
0 are the absorbance values measured at zero time of the
incubation for the test sample and control, respectively.
At and A0
t are the absorbance values measured at zero time of the in-
cubation for the test sample and control, respectively, after incubation for 120 min. The method is sensitive due to the strong absorption of b-carotene, but is slower than the DPPH method. This assay has poor reproducibility because of variations in the b-carotene bleaching reaction. It is not spe- cific as it is subject to interference from oxidizing and reducing agents in the extracts, and also, linoleic acid is not representative of typical food lipids (Spigno and De Faveri, 2007).
8.3.8 Thiobarbituric reactive substance (TBARS) assay
Thiobarbituric reactive substance (TBARS) assay is one of most popular as- says for studies related to lipid peroxidation and it is used widely to evaluate the antioxidant activities of various natural products. Thiobarbituric acid re- acts with many different carbonyl compounds formed from lipid peroxida- tion. Their TBA adducts absorb the same UV wavelength absorbed by the malonyl-thiobarbituric acid adduct.
Various concentrations of testing samples are added to an aqueous solution containing tris buffer (pH 7.4), potassium chloride (1 M), sodium dodecyl sulfate (SDS) (1%) and cod liver (this can be any kind of lipid such as linoleic acid, arachidonic acid or w-3 fatty acids), ferrous chloride FeCl2 and hydrogen peroxide (H2O2, 0.5 μM) in a non-transparent vial. The sample is incubated at 37o c for 18 h, with shaking. After the incuba- tion, oxidation is terminated by adding BHT (4% in ethanol) solution and TBA reagent (0.67% TBA, trichloroacetic acid (TCA) 1%, SDS, 5N HCl) is added to the sample. The sample is heated at 80°C for 1 h and then cooled in an ice bath for 10 min. A blank is prepared following the same procedure without a test sample. In the thiobarbituric acid assay, malonaldehyde TBA-MA adduct product formed is measured using a spectrophotometer
at 532 nm. A known antioxidant such as BHT, vitamin A or vitamin C is used as a positive control in the assay (Moon and Shibamoto, 2009).
8.3.9 Superoxide dismutase (SOD) assay
The superoxide anion scavenging activity of plant extracts is determined by the WST (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium monosodium salt) reduction method using the SOD assay kit-WST. Here, O2
∙
- reduces WST to produce the yellow formazan measuredspectrophotometrically. Antioxidants inhibit the formation of yellow WST (Dudonné et al., 2009).
8.3.10 Electron spin resonance (ESR) spectroscopy
Electron spin resonance (ESR) spectroscopy determines the presence of unpaired electrons of oxygen and is commonly used for free radical evalu- ation. Superoxide anion (O2
∙
-) scavenging capacities are measured by ESRassay (Tabart et al., 2009). It has been applied to some foods to measure free radical production and to establish their antioxidant capacities (Noda
et al., 1997).