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The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties
of plant constituents for scavenging free radicals. The method is based on the spectrophotometric
measurement of the DPPH concentration change resulting from the reaction with an antioxidant.
Several protocols have been followed for this assay using different conditions such as different reaction
times, solvents, pH and different compounds used as antioxidant standards. This review shows to what
extent the mentioned parameters have the influence on the presented results.
Оригинальное название
Application of free radical diphenylpicrylhydrazyl
(DPPH) to estimate the antioxidant capacity of food
samples
The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties
of plant constituents for scavenging free radicals. The method is based on the spectrophotometric
measurement of the DPPH concentration change resulting from the reaction with an antioxidant.
Several protocols have been followed for this assay using different conditions such as different reaction
times, solvents, pH and different compounds used as antioxidant standards. This review shows to what
extent the mentioned parameters have the influence on the presented results.
The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties
of plant constituents for scavenging free radicals. The method is based on the spectrophotometric
measurement of the DPPH concentration change resulting from the reaction with an antioxidant.
Several protocols have been followed for this assay using different conditions such as different reaction
times, solvents, pH and different compounds used as antioxidant standards. This review shows to what
extent the mentioned parameters have the influence on the presented results.
Application of free radical diphenylpicrylhydrazyl
(DPPH) to estimate the antioxidant capacity of food
samples Krystyna Pyrzynska * and Anna Pekal The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties of plant constituents for scavenging free radicals. The method is based on the spectrophotometric measurement of the DPPH concentration change resulting from the reaction with an antioxidant. Several protocols have been followed for this assay using dierent conditions such as dierent reaction times, solvents, pH and dierent compounds used as antioxidant standards. This review shows to what extent the mentioned parameters have the inuence on the presented results. Introduction There is more recent evidence that free radicals induce oxidative damage to biomolecules. This damage has been implicated in ageing and in several human pathologies and other diseases. 1 Several studies indicate that the active dietary constituents of fresh fruits, vegetables and beverages prevent these free radical- induced diseases and protect foodstus against oxidative deterioration. 2 These protective eects have been attributed to the antioxidant species, such as polyphenolic compounds, carotenoids, vitamins C and E, which scavenge free radicals. 3,4 Interest in natural sources of antioxidants for use in the food, beverage and cosmetic industries has resulted in a large body of research in recent years. Antioxidant capacity is widely used as a parameter to char- acterize dierent substances and food samples with the ability of scavenging or neutralizing free radicals. This capacity is related to the presence of compounds capable of protecting a biological system against harmful oxidation. There are several methods for the evaluation of antioxidant eciency of pure compounds and plant extracts such as ORAC (oxygen radical absorbance capacity), FRAP (ferric reducing antioxidant power), CUPRAC (cupric reducing antioxidant capacity), the 2,2- azinobis(3-ethyl-benzothiazoline-6-sulphonate) radical cation (ABTS + _) assay and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH_) assay. 512 The most common assays are those involving chromogen compounds of radical nature, which in the presence Krystyna Pyrzynska received her PhD in Chemistry from the University of Warsaw, Poland. Now she is working in the Laboratory of Flow Analysis and Chromatography, University of Warsaw. Her research interest is focused on the application of solid sorbents for preconcentra- tion and speciation purposes of some metal ions in environ- mental samples as well as chro- matographic analysis of phenolic compounds and anti- oxidant properties of food samples. Anna Pe kal received her Mas- ter's degree in analytical chem- istry from the University of Warsaw. She is currently a PhD candidate in the Department of Chemistry at the University of Warsaw. Her research interest includes analytical methods for the evaluation of antioxidant capacity of food samples. University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland. E-mail: kryspyrz@chem.uw.edu.pl; Fax: +48 22 8223532 Cite this: Anal. Methods, 2013, 5, 4288 Received 7th March 2013 Accepted 11th June 2013 DOI: 10.1039/c3ay40367j www.rsc.org/methods 4288 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013 Analytical Methods MINIREVIEW P u b l i s h e d
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View Article Online View Journal | View Issue of an antioxidant disappear. This reaction can be easily followed by common spectrophotometric detection. The DPPH assay is one of the most popular and frequently employed methods to test the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate the antioxidant capacity of foods. The DPPH_ radical is a long-lived organic nitrogen radical with a deep purple color. It is commercially available and does not have to be generated before assay as in other scavenging assays. When a solution of DPPH_ radical is mixed with an antioxidant/reducing compound, its color turns from purple to yellow of the corre- sponding hydrazine (Fig. 1). The reducing ability of antioxi- dants towards DPPH_ can be evaluated by monitoring the decrease of its absorbance at 515528 nm as the formed cor- responding hydrazine DPPH 2 yields a yellow solution 13 or by electron spin resonance. 14 Originally, it was believed that the DPPH assay involved a hydrogen transfer reaction, but Foti et al. 15 have suggested a dierent mechanism. The initial elec- tron transfer occurs very quickly and the subsequent hydrogen transfer occurs more slowly and this depends on the hydrogen- bond accepting solvent used such as methanol and ethanol. This paper presents anoverviewof the publications regarding the use of the DPPH method. Various research groups have used dierent conditions for this assay (dierent concentrations of DPPH_ radical, incubation times and reaction solvents). The sample pHandthe presence of metal ions inthe studiedextracts, as natural components of plants, are very oen ignored. Stability of DPPH_ reagent The DPPH_ reagent is soluble only in organic solvents. Tsimo- giannis and Oreopoulou 16 found that the reactivity of DPPH_ is much more enhanced in the presence of methanol than in ethyl acetate. The stability of DPPH_ solution is inuenced by the solvent used for its preparation. The absorbance of DPPH_ in methanol and acetone decreased by 20% and 35%, respectively, for 120 min at 25
C under light, while in the dark it did not change signicantly for 150 min. 17 Deng et al. 17 reported that the eect of temperature on the concentration of DPPH_ is more notable than that of time. The decrease in absorbance by 6% within 90 min was observed as the temperature changed from 4
C to 25
C. The authors concluded that it is better to keep the reagent solution for a short period of time at room temperature than in a refrigerator. On the other side, some authors conducted the DPPH assay at elevated temperatures, such as 37
C (ref. 19) or even 50
C. 20 Chemical conditions for measurement The experiments have been performed under dierent chemical conditions. The excess of DPPH_reagent should be used in order to exhaust the H-donating capacity of polyphenols. However, in accordance with Beer's law and the normal practice in spec- trophotometry, the initial DPPH_ concentration in a cuvette should be chosen such to give absorbance values less than 1.0 (which corresponds to the light intensity being reduced no more than ten-fold while passing through the sample). This implies that the nal concentration of the DPPH_ solution in the cuvette is in the range of 2570 mM. 21 Some workers used the DPPH_ concentration higher than 200 mM. 19,22,23 For most of the compounds which exhibit antioxidant properties, their reaction with DPPH_ is biphasic, with a fast decay in absorbance in the rst few minutes, followed by a slower step in which degradation products are involved, until equilibrium is reached. As can be seen from Fig. 2, the rate of reaction varies widely among dierent compounds. 24 The DPPH assay is most frequently simplied by measuring the DPPH_ concentration at the beginning of the reaction and aer a certain incubation time. In the original assay the reaction time of 30 min was recommended. 25 However, a shorter time in the range of 316 min has been used, 2629 while the use of 24 h incubation time has also been reported in some papers. 30,31 This is one of the reasons for the diversity of the results published for similar samples which consequently limits the comparison of data between studies. Fig. 1 DPPH_ radical's chemical structure and its reaction with a scavenger indicated by AH. Fig. 2 The kinetic curves of the scavenged DPPH_ by some phenolic compounds at the concentration of 0.1 mM (ref. 24) (reproduced with permission). This journal is The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 42884295 | 4289 Minireview Analytical Methods P u b l i s h e d
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View Article Online Magalh~aes et al. 32 proposed a kinetic matching approach, where the oxidation kinetic behavior of a standard mixture (caeic acid, (+)-catechin, hesperetin, morin and ()-epigallo- catechin gallate) was compared to that attained for red wines, selected as a model for a complex food matrix. This comparison involved the calculation of antioxidant capacity values and, when a kinetic matching standard is found, these values are constant along the reaction time. The second part of this strategy consisted of converting the calculated antioxidant capacity value (expressed as equivalents of the kinetic matching standard) into an antioxidant capacity value expressed as equivalents of trolox by taking into account the number of electrons transferred by each compound. There was no statis- tical dierence between the results obtained by the kinetic matching approach (aer <10 min of reaction) and those obtained under end point conditions (aer 120 min). The sample throughput increased from <18 (end point measure- ments) to >108 per hour using the proposed kinetic approach. Expression of results The results of the DPPH assay have been presented in many ways. The majority of studies expressed the results in terms of the reduction percentage of the DPPH_ solution, referred to also as the percent of inhibition or quenching, and calculated as I% [(A 0 A t )/A 0 ] 100, where A 0 and A t are the absorbance values in the absence and presence of an antioxidant. In some papers the results are presented in the form of the percentage of residual DPPH_ calculated in the following way: DPPH_ res
[(DPPH_) t /(DPPH_) 0 ] 100, where DPPH_ 0 and DPPH_ t are the concentrations at the initial and steady state, respectively, obtained from a calibration curve. The antioxidant concentration necessary to decrease the initial DPPH_ concentration by 50% inhibition (named as inhi- bition concentration IC 50 or eciency concentration EC 50 ) is also oen used for the comparison of the antioxidant capacities of dierent compounds or extracts of natural samples. A lower value of IC 50 indicates higher antioxidant activity. The IC 50 value is calculated using the graph obtained by plotting the inhibition percentage against the extract or compound concentration. However, the values of IC 50 parameter were expressed in dierent units: in grams of antioxidant per kg of DPPH_, 16 the molar or the mass ratio of antioxidant to DPPH_, 3337 as mmoles of an antioxidant 21,3840 or in a concentration unit as mg mL 1 . 41 Moreover, dierent values of IC 50 could be found in the literature for the same compounds. This parameter highly depends on the reaction time and the initial DPPH_ concentra- tion as shown in Table 1 for gallic and ascorbic acids as examples. The time needed to reach the steady state with IC 50 concentration dened as TIC 50 was calculated graphically by plotting the time at the steady state against the concentration of each antioxidant compound. 44 However, this may lead to a large variation of TIC 50 values as there is no linear correlation between the time at the steady state and the concentration of antioxidants and as a consequence dierent TIC 50 values would be obtained. Moreover, the absolute values of IC 50 and TIC 50 are highly dependent on the units of the antioxidant concentration (mg L 1 or mgL 1 ). Sanchez-Moreno et al. 44 further proposed another parameter to express the antioxidant activity called antiradical eciency (AE) where AE 1/EC 50 TIC 50 . The larger the AE the more ecient is the antioxidant. A concep- tually similar parameter for the evaluation of the radical scav- enging eciency (RSE) was suggested by De Beer et al. 41 RSE was calculated as the ratio of the starting rate of DPPH_decay to IC 50 . Recently, Scherer and Godoy 43 proposed a new antioxidant activity index (AAI) calculated as the ratio of the nal concen- tration of DPPH_ (in mg mL 1 ) to IC 50 (in mg mL 1 ). AAI takes into consideration the mass of DPPH_and the mass of the tested compounds in the reaction resulting in a constant for each antioxidant, independent of the concentration of DPPH_ and sample used. However, the concentration of DPPH_ used in this study produced absorbance values between 2 and 3, which fell outside the range and accuracy of spectral measures. The change of DPPH_ absorbance could be compared to the change induced by a reference compound. Several compounds have been used as a standard antioxidant in the performed experiments including ascorbic acid, 25,4547 a-tocopherol, 48,49 gallic acid, 45 butylhydroxytoluene (BHT) 46,50 and trolox. 27,31,5153 Trolox, a water soluble analogue of vitamin E, does not have any physiological signicance and its choice as the standard for antioxidant activity is arbitrary. However, the expression of the results as trolox equivalents (mmol of trolox necessary to provide the same antioxidant activity as a gram of the sample) helps for comparison of the published data. Eect of solvent The antioxidant properties of extracts, estimated by the DPPH method, obtained from the food samples using dierent solvents (methanol, ethanol, acetone or their mixtures with water in dierent proportions as well as ethyl acetate or chlo- roform) are discussed in many papers. 30,5456,65 The antioxidant properties of such extracts are largely related to dierences in their quantitative and qualitative composition resulting from dierent extraction ability of the used solvents. Antioxidant compounds in food may be water soluble, fat soluble, insoluble Table 1 IC 50 values for gallic and ascorbic acids from various reported studies IC 50 (mM) [DPPH_] 0 a (mM) Time (min) Ref. Gallic acid 0.74 29.3 40 18 1.48 58.5 40 18 4.15 50 30 29 4.2 50 30 37 5.1 63 20 42 6.88 78 90 43 17.1 195 90 43 Ascorbic acid 1.35 29.3 40 18 2.74 58.5 40 18 10.2 50 30 37 50 250 30 23 55.9 85.7 30 38 629 100 10 39 a [DPPH_] 0 the nal concentration in the cuvette. 4290 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013 Analytical Methods Minireview P u b l i s h e d
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View Article Online or bound to cell wall. Certain antioxidants require polar solvents such as methanol or ethanol, while ethyl acetate or chloroform are used to extract lipophilic antioxidants. Hence, extraction eciency is an important factor in the evaluation of antioxidant capacity. It was observed that the addition of water to alcohol increases the eciency of extraction, until it reaches the optimum. 57,58 There is no solvent that would be entirely satisfactory for extraction of all the antioxidants present in food, particularly those associated with complex carbohydrates and proteins. Consequently, there is a considerable amount of bioactive compounds with antioxidant properties in the extraction residues, which are ignored in most chemical and biological studies. Perez-Jimenez et al. 55 proposed at least two extraction cycles from plant foods performed with aqueous organic solvents with dierent polarities in order to extract antioxidant compounds with dierent chemical structures. Although higher extract yields were obtained by the reux extraction technique, in general higher amounts of total phenolic contents and better antioxidant activity were found in the extract prepared from some medicinal plants using a shaker. It has been reported that thermal extraction conditions might result in the loss of natural antioxidants and the eect of degradation of avonoids depends on the extraction conditions and their chemical structure. 59 The high number of hydroxyl groups promotes degradation of avonoids, whereas sugar moiety and methoxyl groups protect them. The smallest decomposition was observed by the heated reux extraction procedure within 30 min in water bath and by microwave assisted extraction (160 W during 1 min). 59 On the other hand, Dutra et al. 60 reported that among dierent extraction tech- niques (reux, maceration, ultrasound, heating plate) extrac- tion performed under reux using ethanolwater (70 : 30, v/v) oered the highest polyphenols level from plant material. Dawidowicz et al. 61 reported that the type and the amount of solvent used for antioxidant dissolution signicantly inuenced the dierence in the amount of unreacted DPPH_. The presence of ethyl acetate or dioxane in the system decreases the kinetics of the reaction between DPPH_ and BHT in relation to its kinetics in the system containing only methanolic solutions. This eect was linear for the ethyl acetate volume in the examined range of 501000 mL, while the increase of the dioxane volume caused almost constant suppression of the DPPH_BHT reaction kinetics. According to Musialik and Lit- winienko, 62 aprotic solvents (such as ethyl acetate or dioxane) suppress the kinetics of DPPH_antioxidant reaction. On the other side, small volumes (up to 440 mL in the total volume of 3 mL) of chloroformic solution of BHT accelerated the reaction kinetics, whereas large volumes (up to 1000 mL) decelerated the reaction rate. While the latter observed a decrease of the reac- tion rate, which is unstable (chloroform is also an aprotic solvent), the eect at small volumes of this solvent is intriguing. The authors explained that it might be connected with the structural changes of bulk methanol caused by the addition of chloroform to the measuring system. The increase in water content in the mixture of methanol generally leads to the acceleration of the DPPH_antioxidant reaction kinetics in comparison to the kinetics of the system without water and that dierence was greater in the case of small water content (up to 30 mL with 910 mL of methanol). 61 Stasko et al. 33 studied the limits for water as a component of the mixed waterethanol solvent for the DPPH radical assay. They concluded that 50% (v/v) aqueousethanol solution is a suitable choice for lipophilic and hydrophilic antioxidants and the reaction rate increases considerably with the increasing water ratios. However, at the water content over 60% (v/v), the anti- oxidant capacity decreases, since a part of DPPH_coagulates and it is not easily accessible in the reaction with antioxidants. The evaluation of the antioxidant capacity of the mixtures obtained aer mixing equal volumes of catechin and gallic acid standard solutions (at a concentration of 1 mol L 1 each) in dierent solvents (water, methanolwater (30 : 70 v/v), methanolwater (50 : 50 v/v), methanol and acetonewater (50 : 50 v/v)) showed a weak inuence on the DPPH assay. 54 The greatest signicant dierence was between water (IC 50 0.067) and acetonewater mixture and pure methanol (IC 50 0.083 for both systems). Noipa et al. 29 have developed a modied DPPH assay using surfactant aggregates or micelles, which do not require an organic solvent. Among three types of surfactants studied such as nonionic (Triton X-100), anionic (sodium dodecyl sulphate, SDS) andcationic (cetyltrimethylammoniumbromide, CTAB), in the last medium gallic acid and protocatechuic acid (used as the model compounds) showed higher percent of inhibition than TritonX-100 andSDS. The optimummicelle systemwas found to be 2 mMCTABin0.1 Macetate buer. The reactionof DPPH_with gallic acid observed in the CTAB solution was faster than other micelle systems and reached the steady state absorbance within 1 min. The rate constant inthis systemwas about 30 times higher than in methanol. It was assumed that the direct abstraction of phenol H-atom by DPPH_ occurred inside the micelle and the electrostatic interactions allowedthe radical toremaininside the hydrophobic core of the CTAB micelle. This allowed the use of shorter analysis time and to evaluate the antioxidant capacity from aqueous extracted plant samples. pH of sample In the original Blois paper 12 regarding the DPPH method, it was suggested that the system should be maintained at the pH range of 5.06.5 by using acetate buers. However, these conditions were abandoned in the later current practice, as there is a great uncertainty in the meaning of pH values for methanol or ethanol used mainly as reaction media. Organic acids alone (such as acetic, malonic and citric acids) exhibited no scavenging activity towards DPPH_, but when added to the solution of ascorbic acid its action enhanced towards DPPH_. 35 Thus, the antioxidant action of ascorbic acid could be enhanced by some components that are not directly involved in the free radical scavenging activity. The greatest eect was observed in the presence of citric acid in relation to water. This medium had the lowest pH value of 3.28. Sharma and Bhat 21 examined the DPPH radical scavenging activity of ascorbic acid, BHT and propyl gallate using the reagent prepared in methanol and in buered methanol, This journal is The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 42884295 | 4291 Minireview Analytical Methods P u b l i s h e d
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View Article Online which was prepared by mixing 0.1 Macetate buer (pH 5.5) with methanol (2 : 3, v/v). They found that the radical scavenging proles of ascorbic acid and propyl gallate were comparable in both reagent media, while for BHT it was markedly higher in buermethanol solution in comparison to that in methanol alone (Fig. 3). Dawidowicz et al. 61 reported that the increase of hydrogen ion concentration in phosphoric buermethanol solution (in the pH range of 1.64.1) slowed down the kinetic reaction between BHT and DPPH_ in relation to pure solvent. The application of buer with pH 5.5 gave the similar eect to the case when pure water was applied, leading to the accelera- tion of this reaction kinetics. Generally, the increase of hydrogenionconcentrationleads to the decrease of the reaction rate of chromogen radical scav- enging, whereas under basic conditions proton dissociation of polyphenolics would enhance the reducing capacity of compounds. Following, 62,63 the reaction between phenolic anti- oxidants and DPPH radical occurs by a combination of Proton Coupled-Electron Transfer (PC-ET) and Sequential Proton Loss Electron Transfer (SPLET) mechanisms. The rst is slower and dominates innon-polar solvents of lowdielectric constant andof low basicity, whereas the second one is faster and is character- istic for solvents of high dielectric constant and of high basicity supporting antioxidant ionization. The SPLET mechanism is favored in methanol, which possesses high dielectric constant and high ability to solvate phenolic anions. In non-ionizing solvents, the SPLET mechanism can be ever eliminated. It was found that the kind of the used buer system also aected the measurements. 64 Phosphate buer showed a decrease in DPPH_ radical scavenging activity at increasing concentration (0.050.3 mM), while the acetate buer concen- tration did not cause a signicant dierence. Therefore, careful monitoring of these aspects is essential for the successful use of the DPPH assay and the comparison of the antioxidant activity of food samples tested under varying conditions is not appropriate. The presence of metal ions Metal ions are natural components of plants and their content is inuenced by the type of plant, the soil composition and local environment. Their concentration in the obtained extracts of food samples depends on the metal, plant type as well as extraction conditions. As transition metal ions play a vital role in the initiation of free radical processes (via the Fenton reaction), metal chelation by polyphenolic compounds is widely considered as another mechanism of their antioxidant activity. 65 For steric reasons the complexes usually include no more than two avonoid mole- cules. In addition, some avonoids exhibit the ability to reduce Fe(III) and Cu(II) ions. 6567 Several experimental data indicate that these complexes are more eective free radical scavengers than the corresponding free avonoids. 6870 The naringinCu complex exhibits a much stronger DPPH_ scavenging than nar- ingin alone and is higher than vitamin C (Fig. 4). 69 Dawidowicz et al. 61 studied the dierence between BHT DPPH_ reaction rates in the systems with Fe(III) and Cu(II) and Fig. 3 Scavengingof DPPH_radical by (A) ascorbic acid(1 mM) and(B) BHT (1 mM) prepared in methanol or buered methanol 21 (reproduced with permission). Fig. 4 Inhibition of DPPH by rutin, vitamin C, naringin and its complex with copper. All compounds at 10 mM concentration 69 (reproduced with permission). 4292 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013 Analytical Methods Minireview P u b l i s h e d
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View Article Online without these metal ions. The increase of metal concentration (up to 1 mg mL 1 and 20 mg mL 1 for Cu and Fe, respectively) caused an almost linear deceleration of the reaction kinetics. Cu(II) suppressed BHTDPPH_ reaction kinetics more than Fe(III). The authors concluded that the observed changes in the reaction kinetics can be attributed to the formation of metal complexes with the components of the measuring system. However in their next paper, 71 it was reported that the dierence between the percent of remaining DPPH_with and without Cu(II) or Fe(III) aer 60 min of the BHTDPPH_ reaction is almost the same. In both studies, the experiments were conducted in non- buered solutions. As metal ions can behave as acids in protic solvents (protons are released in the solution from the OH groups due to complexation reaction), the presented data interpretation becomes spectaculative. Espinoza et al. 72 evaluated the change in radical scavenging activity against DPPH_ when copper or iron compounds were added to four dierent types of wines. For Cabernet Sauvignon 2004 red wine, the addition of 0.6 mg L 1 and 1.2 mg L 1 copper resulted in 22% and 46%, respectively, DPPH_ peak area increase with respect to native wine. In the same way, the addition of 3 mg L 1 and 6 mg L 1 of iron produced 46% and 69% peak area increase with respect to native wine. These results show that the formation of redox inactive complexes between polyphenols of wine and studied metal ions resulted in reduction of their antioxidant activity. The authors mentioned that these interactions require a certain time to be established as in the other case it produced erratic and inconsistent results in all the cases, however, this interval time was not specied. As certain inorganic salts occurring in extracts of plants aect the DPPH_ radical scavenging activity, Al-Dabbas et al. 64 have proposed the desalting method using cationic or anionic exchange resins. The anionic euent of the crude water extract of Varthemia iphionoides plant, commonly used in folk medi- cine, exhibited signicantly higher antioxidant properties. This may be related to the actual DPPH_ radical scavenging activity of antioxidants present in this extract as all the inorganic matter was removed. The reported dierences in the antioxidant properties of the extracts obtained from the same plant material (growing for example under dierent conditions) can be caused not only by the dierent contents of polyphenolic compounds but also by dierences in the kinds of metal ions and their concentration in the nal extract. The results of the DPPH assay for samples polluted with metal ions can be dierent in relation to the samples free of these metal ions. Thus, this can be a source of erroneous conclusions in comparison to dierent samples. Comparison with other assays There are several dierent methods to assess the relative anti- oxidant capacity of dierent food samples. The ORAC method utilizes uorescence detection, while CUPRAC, FolinCiocalteu (FC), FRAP, DPPH, and ABTS are applied with spectrophoto- metric measurements. Electrochemical detection has also been used for the analysis of the anodic current waveform in cyclic voltammetry and for the potentiometric measurement of redox potential. Spectrophotometric methods are still the most widely used because the reagents used are easy to get, results are given relatively quick and the experiments are convenient. The relative antioxidant capacity of many compounds present in food samples varies widely according to dierent testing assays, which dier from each other in terms of substrates, reaction conditions and quantitation methods. The FC method, very popular and widely used for the determination of the so-called total polyphenol content, is based on a non- specic phenol oxidation reaction by the two strong inorganic oxidants (phosphotungstic and phosphomolibdic acids). This assay is conducted in alkaline medium and gives dierent responses to dierent phenolic compounds, depending on their chemical structures. 11 Moreover, the FC reagent could simul- taneously oxidize several nonphenolic organic compounds as well as some inorganic substances to give an elevated apparent phenolic content, thus it can be used for the measurement of the total reducing capacity of samples. The CUPRAC method is based on the reduction of Cu(II) to Cu(I) at neutral pH by anti- oxidants present in a sample utilizing the copper(II)neo- cuproine reagent as the chromogenic oxidant. Slow reacting antioxidants needed an increased temperature incubation to complete their oxidation. 73 The FRAP method utilizes the reduction of the ferric tripyridyltriazine complex at low pH to its ferrous form which has an intense blue colour (l max 593 nm). The ABTS + _radical cation during its reaction with an antioxidant at pH 7.4 is converted to its colorless neutral form and the decrease in absorbance is monitored. This assay is oen referred to as the trolox equivalent antioxidant capacity (TEAC) assay. Thus, there is no single, widely acceptable assay appli- cable to a reasonable variety of compounds in food matrices. It is essential to use a minimum of two methods depending on the expected antioxidant potential and/or on the origin of the substance. Usually linear correlations between the results obtained by dierent assays have been checked. However, dierent results have beenreportedevenfor the similar kindof foodsamples. For example the DPPH assay showed similar trends for studied tea infusions to the FC and CUPRAC methods, 73 but contrasting results were reported for green tea preparation. 74 The total quantities of polyphenols in tea samples as well as the percentage of the individual compounds variedwiththe varieties andthe change of tea plant living conditions. Besides, it couldbe also the result of the synergies or antagonisms of tea infusion components in a given assay. 75 It should be remembered that all the used assays are intended for the estimation of the anti- oxidative potential of food. As for the antioxidative actionof food constituents in real biological systems, this will depend also on their availability and food antioxidant metabolism. Conclusions The DPPH assay is considered as an easy and useful spectro- photometric method with regards to screening or measuring the antioxidant activity of model compounds and extracts of food samples. It can be used for solid and liquid samples. The DPPH_ radical is stable, commercially available, and does not This journal is The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 42884295 | 4293 Minireview Analytical Methods P u b l i s h e d
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View Article Online have to be generated before assay like ABTS + _. It is not specic to any particular antioxidant component, but applies to the overall antioxidant capacity of the sample. The steric accessibility of the DPPH_ radical is a major determinant of the reaction, since small molecules that have better access to the radical site have relatively higher antioxidant capacity. On the other hand, many large antioxidant compounds that react quickly with peroxyl radicals may react slowly or may even be inert in this assay. The inexistence of DPPH_ or similar radicals in biological systems is also a shortcoming. Spectrophotometric measurements can be aected by compounds, such as carotenoids, which can absorb at the wavelength of determination. 76 The interactions between the polyphenols and the food constituents have also been found. 35,54 Most of them produced no eect by themselves but altered the original polyphenol IC 50 value. The glucidic compounds such as glucose, galacturonic acid and pectin reduced the IC 50 , indicating a greater antioxidant capacity. Cysteine is an amino acid that mostly reduced the IC 50 and simultaneously increased the AE values. 54 The main problem is the compatibility of the obtained values between several laboratories due to dierent reaction times, solvents, pH and dierent antioxidant standard compounds, which are frequently applied. This paper shows the complexity of the problem. The DPPH assay should be validated and standardized with a large body of comparable data available in the literature. It was postulated to express the H-donating capacity as the amount of DPPH_, which may be scavenged by the tested sample (the stoichiometric coecient for individual antioxidants or the mass of sample). 77 However, it should be taken into consideration that the parameter increases with the incubation time. 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