Article pubs.acs.org/JAFC
Re-evaluation of the 2,2-Diphenyl-1-picrylhydrazyl Free Radical (DPPH) Assay for Antioxidant Activity J. Xie and K. M. Schaich* Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: Kinetics and stoichiometry of reactions between the 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical and 25 antioxidant compounds with different structure, molecular weight, number of −OH groups, and redox potential were investigated by recording the loss of DPPH• absorbance at 515 nm continuously for 10 min. A series of antioxidant concentrations was tested to determine linear response ranges and reaction saturation points. The primary feature distinguishing antioxidant activityrate of initial reaction ( 1) act increasingly by hydrogen atom transfer, have reaction impeded by steric hindrance, or both. Effects of Antioxidant Concentration on Reactivity with DPPH. Results of this study revealed significant variation among concentration−response patterns (Figure 3), k2 and linear response range (Table 2), and saturation concentration (Figure 3) from the different reactivity groups. For Group 1 (compounds with instantaneous reaction), k2 values were the highest, and increase of initial reaction rates (Ri) was close to linear with antioxidant concentrations up to about 250 μM (final reaction concentration), then the reaction slowed. The one exception to this pattern was ascorbic acid, which was very reactive at the lowest concentrations, and small increases in concentration made dramatic differences in reaction rates, reflected in k2 orders of magnitude larger than those of other antioxidants. This high reactivity is probably due to a combination of better accessibility to DPPH and low redox potential. At the same time, ascorbic acid reaction with DPPH also saturated rapidly with concentration (see linear range, Table 2) due to its propensity for secondary reactions at higher concentrations, and this was not reflected in k2. The k2 values dropped as reactivity slowed progressively in Groups 1 through 4 (Table 2), indicating that increasing antioxidant concentrations had less effect on response. Indeed, for many compounds in Groups 2 and 3, reaction rates increased linearly with antioxidant only at the very low concentrations (100 and 10−50 μM, respectively), above which the response curves flattened, exhibiting more
RESULTS AND DISCUSSION Preliminary studies in this laboratory recording DPPH loss continuously revealed a marked difference in the shapes of reaction curves for different antioxidants. Some antioxidants showed an immediate very fast reaction that was complete within seconds, while others showed a combination of fast and slower stages, and some showed only slow sustained reactions. To further investigate the reasons for these differences, determine best protocols for standardization of the DPPH antioxidant assays, and identify what useful chemical information can be derived from full reaction curves, reactions of 28 phenolic and nonphenolic compounds were studied in detail. Visual patterns of reaction curves were used for initial categorization, then initial reaction rates, antioxidant concentration dependence and linear reaction ranges, reaction stoichiometry, and degree of early reaction completion were calculated and compared with standard EC50 values, number of phenolic −OH (or other H donating) groups, and antioxidant redox potentials to learn more about factors controlling antioxidant reactions with DPPH. Table 2 compiles these chemical properties and calculated reaction characteristics of tested antioxidants. Kinetic Reaction Patterns and Initial Reaction Rates. Response curves of antioxidant reactions with DPPH revealed at least five distinctly different groups of reactivity (Figure 2) based on their reaction curve patterns and initial rates (Ri) at 100 μM (where antioxidant and DPPH concentrations are nearly equal, 0.1 mM AOX/0.0917 mM DPPH). (1) Extremely fast: instantaneous initial absorbance drop, Ri >20.0 nmol DPPH/s, the reaction is complete in less than 10 s, and ΔAf/ ΔAi = 1; e.g., n-propyl gallate, pyrogallol, and ascorbic acid. (2) Fast: rapid initial absorbance drop, then the reaction continues more slowly, Ri = 4.0−10.0 nmol DPPH/s, and ΔAf/ΔAi = 1− 1.76; e.g., 3-methylcatechol, quercetin, catechol, α-tocopherol, caffeic acid, epicatechin, and gallic acid. (3) Medium: absorbance drop slow and continual over the reaction period, Ri = 0.5−4.0 nmol DPPH/s, and ΔAf/ΔAi = 1.6−8.6; e.g., protocatechuic acid, rosmarinic acid, hydroquinone, Trolox, ferulic acid, and chlorogenic acid. (4) Slow: absorbance drop very slow and almost linear over time, Ri = 0.0−0.5 nmol 4255
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pronounced saturation with each group. There was no range of linear response in Group 4. That none of the antioxidants exhibited a perfectly linear response range and all exhibited some saturation demonstrate that factors other than antioxidant concentration also have important influences on reactivity. This poses a severe problem for quantitation of activity, especially in mixed extracts, since quantitative assays require that (1) the response is continually linear with reactant concentration or the assay must be conducted in a concentration range with linear response and that (2) analytes have comparable concentration dependence, as reflected in the second order rate constant (k2), or a range of concentrations must be tested for each material. The variability of k2 within all groups warrants additional comment. As more antioxidant is added to the system, crowding of antioxidant molecules around the DPPH increases. This has two possible consequences, depending on antioxidant structure: (1) forcing reactive phenolic −OH groups into closer proximity to the DPPH radical site, thus facilitating reaction and (2) creating random molecular packing that impedes access to the DPPH radical. For compounds with lower k2 values (e.g., hydroquinone, Trolox, and ferulic acid in Group 3), reaction curves were consistent with increased packing interference at higher antioxidant concentrations, while the curves of protocatechuic acid, rosmarinic acid, and chlorogenic acid showed greater reaction rate increase with added antioxidant (higher k2), consistent with forced DPPH access by the increased packing. Stoichiometry of Antioxidant Reactions. At 10 μM antioxidant concentrations (DPPH/AOX ∼10, conditions where multiple DPPH molecules should be sufficiently close to each antioxidant molecule that diffusion limitations on reaction are minimal), n-propyl gallate, tocopherol, and catechol were the only compounds to donate the expected two electrons per phenolic−OH group (Table 2). Ascorbic acid reacted one electron per OH group and other monophenols donated slightly less than one electron, while stoichiometry with compounds having more complex structure (epicatechin, rosmarinic acid, and quercetin) dropped still further. At antioxidant concentrations (100 μM) comparable to those of DPPH, stoichiometry dropped dramatically to fractions of an electron per OH group for every compound except tocopherol. Plots of stoichiometry over the entire antioxidant concentration range confirmed that stoichiometry was not constant but decreased with antioxidant concentration (Figure 4). The greatest stoichiometry change with concentration occurred at the lowest concentrations of the most reactive phenolic compounds. Differences in stoichiometry were obscured at high antioxidant concentrations, showing once again that reaction conditions markedly affected the picture given for both relative and absolute reactivity. Such nonconstant stoichiometry creates distinct problems for use of the DPPH assay in quantitating and comparing the antioxidant action of extracts with unknown composition and concentration. As with initial rate, stoichiometry showed negligible correlation to the number of phenolic −OH groups per antioxidant molecule or to redox potential (Table 3). There was also little correlation between stoichiometry and initial reaction rate (0.52 and 0.14 for 10 and 100 μM antioxidant, respectively), so evaluations of antioxidant activity based on stoichiometric measures alone will miss differences between antioxidants at high concentrations and misrepresent the actual reactivity of many antioxidants at low concentrations.
Figure 4. Inverse relationship between stoichiometry and antioxidant concentration for representative antioxidants from each reactivity group.
EC50. As expected, antioxidant concentrations necessary to reduce DPPH by 50% appeared in reverse order to reactivity (Table 2, EC50 column). Less than 20 μM final concentrations were required by very fast reacting compounds (Group 1), and this value increased progressively in Groups 2 and 3. Compounds in Group 4 were unable to reduce DPPH by 50%. There was considerable variability within reactivity groups, which probably accounts for the lack of correlations between EC50 and Mw or redox potentials, and only a weak negative relationship to the number of phenolic −OH groups (−0.52), initial reaction rate (−0.36), and stoichiometry (−0.40) (Table 3). Thus, as with stoichiometry, the use of EC50 alone will give a distorted and inaccurate measure of actual antioxidant reaction with DPPH when comparing antioxidants from different structural classes. Molecular Properties Controlling Antioxidant Reactivity with DPPH. Although previous studies have associated antioxidant activity within structural classes of phenolic compounds with redox potential41 and number of phenolic −OH groups,9 low Pearson correlation coefficients indicated that these factors could not explain the differences in reactivity between structural classes in this study (Table 3). Thus, alternate explanations must be sought. Steric accessibility must be recognized as a key problem in reaction with DPPH since the radical site is protected inside a reaction cage formed by the two phenyl rings orthogonal to each other, and the picryl ring angled about 30° with its two nitro groups oriented above and below the radical site14,15 (Figure 1). Most phenolic compounds in Groups 2−4 have more complex ring adducts or multiple rings that can impede the access of phenolic −OH groups to the hindered radical site of DPPH and reduce observed reactivity. Comparing ΔDPPHf/ ΔDPPHi provides support for steric accessibility to the DPPH radical site as a major controller of reactivity (last column, Table 2). Small monophenols with only −OH ring adducts showed a ΔDPPHff/ΔDPPHi ratio of ∼1, indicating that the reaction was complete within seconds. This ratio increased with the number and complexity of ring adducts, reflecting a slowing of reaction as molecules had to rotate to orient reactive groups toward the DPPH radical site and as bulky ring adducts or multiple rings interfered with free diffusion to DPPH. The pronounced saturation of DPPH reaction rates with increasing antioxidant concentration shown in slower reacting Groups 2− 4 (Figure 3) as well as inhibition of reactions in mixtures of 4256
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Table 3. Pearson Coefficients for Relationships of Antioxidant Properties to Reactivity with DPPH rates mol wt. phenol −OH E7 (V)
stoichiometry
ΔAi
ΔAF
Ri/s
k2
10 μM
100 μM
EC50
−0.06 0.44 −0.48
0.11 0.63 −0.44
−0.16 0.30 −0.34
−0.13 0.31 −0.09
−0.04 0.57 −0.23
0.43 0.22 −0.17
0.02 −0.52 0.13
Table 4. Comparison of Solvent Effects on DPPH Quenching Rate (Ri) and Total DPPH Consumption after 10 min of Reaction by Selected Antioxidants (100 μM Final Concentration) Solvent antioxidants
methanol
pyrogallol catechol hydroquinone resorcinol glutathione
21.02 6.18 1.26 0.13 0.18
± ± ± ± ±
1.24 0.44 0.03 0.01 0.01
pyrogallol catechol hydroquinone resorcinol glutathione
0.25 0.24 0.17 0.01 0.02
± ± ± ± ±
0.02 0.01 0.01 0.00 0.00
50% aq. methanol Initial Rate, Ri (nmol DPPH/s) 30.56 ± 1.32 11.92 ± 0.54 6.84 ± 0.46 0.35 ± 0.02 1.30 ± 0.09 Total DPPH Consumption (μmol) 0.24 ± 0.02 0.24 ± 0.02 0.23 ± 0.02 0.07 ± 0.00 0.14 ± 0.01
ethanol
acetone
1.56 ± 0.05 0.47 ± 0.02 0.48 ± 0.03 no reaction 0.10 ± 0.01
0.30 ± 0.01 0.18 ± 0.01 0.12 ± 0.02 no reaction no reaction
0.21 ± 0.01 0.20 ± 0.01 0.13 ± 0.01 no reaction 0.005 ± 0.000
0.015 ± 0.000 0.003 ± 0.000 0.044 ± 0.001 no reaction no reaction
methanol, ethanol, acetone, and 50% aqueous methanol, solvents most often in assays of antioxidant activity. For the phenolic compounds, reactions were fastest in methanol and slowed dramatically in ethanol or acetone (Table 4). A number of papers have documented that hydrogen bonding solvents generally decrease hydrogen atom transfers because they complex with both DPPH4,14 and phenolic compounds,16,19,31 thus blocking reaction. Consistent with these observations, the relative rates for solvents in this study are in the reverse order of hydrogen bond accepting ability of the three solvents, i.e., hydrogen bond parameters β2H are 0.41, 0.44, and 0.50 for methanol, ethanol, and acetone, respectively.42 Litwinienko and Ingold have shown that HAT cannot occur from hydrogen bonded complexes between solvent and hydrogen atom donor, XH···S, so the most simplistic explanation for the solvent effects is differences in the strength of hydrogen bonding of the solvent to phenolic −OH groups and/or to DPPH that interferes with the release of H atoms.17,18,43 Slowing of reactions in ethanol may also be attributed to increased viscosity slowing the diffusion of antioxidants to the DPPH radicals or to increased steric interference with phenol access to the DPPH radical site. Acetone is particularly effective in inhibiting hydrogen transfer from vicinal diphenols because it hydrogen bonds very strongly to both phenolic −OH groups, completely blocking both electron and hydrogen transfer.16 In addition, DPPH reacts with phenolic compounds via rapid sequential proton loss electron transfer (SPLET) in ionizing solvents and by slower hydrogen atom transfer (HAT) in nonionizing solvents or in the presence of acid.18 Both methanol and ethanol partially ionize, allowing both electron and hydrogen transfer, but acetone is nonionizing. Thus, DPPH radical quenching is minimal in acetone because both electron and hydrogen atom transfers are strongly inhibited. Diluting methanol to 50% with water greatly increased the reaction rate by about 50% for pyrogallol, which is a strong electron transfer compound, but considerably more for
phenolic compounds (manuscript in preparation) are similarly consistent with steric interference imposed by molecules blocking access to the radical site, rather than accumulation of DPPH2 that reportedly inhibits the reaction.4 These results suggest that steric hindrance may be the most important factor limiting the reaction of phenolic compounds with DPPH and thus raise questions about the validity of the DPPH assay for rating the activity of antioxidants with different structures. Computer analysis of molecular docking of antioxidants with DPPH should clarify the role of steric accessibility in this assay. Inductive effects of ring substituents on reactivity with DPPH must also be considered. Slower reacting phenolic compounds tended to have more electron-withdrawing ring substituents (COOH, CC, and COOR) or multiple rings which deactivate phenolic −OH groups to ionization or H atom transfer. However, there was little direct correlation between reaction rates and estimated Hammett sigma constants, measures of how strongly ring substituents at meta and para positions donate or withdraw electrons from reactive groups (data not shown). Thus, other factors have a stronger influence than inductive effects on DPPH reaction rates. Finally, DPPH is a mixed mode reagent, so the balance between electron and hydrogen atom transfer for individual compounds and in different reaction environments must contribute to the control of reaction rates with DPPH. As shown above, compounds capable of electron transfer react more rapidly with DPPH, while hydrogen atom donors react more slowly. Some insights into the relationship between the dominant mechanism and reaction rate are provided by the solvent effects on reactions discussed below. Effects of Solvent on Antioxidant Reactivity with DPPH. To determine the optimum solvent for standardization and to explore whether these solvent effects could be further used to advantage to investigate reaction processes, we reacted DPPH with glutathione (a strong H donor) and phenols (pyrogallol, catechol, hydroquinone, and resorcinol) having closely related structure from each of the reactivity groups in 4257
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Recommendations for the Use of DPPH Reactions in the Evaluation of the Radical Quenching Activity of Antioxidants. The DPPH reaction has been used extensively to compare and rank the antioxidant effectiveness of a wide range of natural extracts in now thousands of studies. Nonetheless, results reported here raise serious questions about its validity for quantitating and comparing the activity of antioxidants with different sizes and structures, and especially for ranking their relative reactivity due to the following significant limitations of the DPPH reaction: (1) Absorbance measurements only after long reaction periods are inadequate for quantitating antioxidant reaction with DPPH because they miss important initial reactions that provide critical distinction between the reactivity of antioxidants with different structures and do not determine reaction rates. Final stoichiometry determined from total absorbance drops reflects the numbers of hydrogen atom donating groups almost exclusively and thus overestimates the activity of polyphenols and underestimates activity of monophenols. These conventional analyses also ignore antioxidant concentration effects and reaction saturation. (2) Full and accurate evaluation of antioxidant reactivity rather than capacity requires recording DPPH loss continuously over the full reaction time, with calculation of reaction kinetics from early response (immediate to 30 s). However, accurate measurements of the initial very rapid reaction rates requires fast kinetic techniques such as continuous or stopped flow mixing because the initial electron transfer from antioxidant to DPPH is too fast to quantitate using manual mixing and conventional spectrophotometers. (3) Accurate information cannot be gained from single antioxidant concentrations, particularly in extracts not normalized to the concentration of phenolic compounds. Rather, reactions must be run over a range of antioxidant concentrations from dilute (diffusion controlled) to approximately equal to DPPH. Concentrations greater than DPPH as conventionally used to determine second order rate constants are not valid due to reaction saturation and interference with steric accessibility to DPPH. Determining reaction response as a function of antioxidant concentration clearly identifies valid linear response ranges and saturation levels, and differentiates reactivity patterns. (4) Allowing reactions to run to completion over many minutes to hours is warranted for the quantitation of phenols, as described in early applications of DPPH,2 and reflects potential antioxidant capacity but is irrelevant for determining the important initial rates of antioxidant reaction with DPPH. Analyses of immediate (0−2 min) rather than long reactions should be stressed when determining antioxidant reactivity. (5) Steric accessibility to the DPPH radical site appears to control the reaction more than specific identifiable chemical properties of the antioxidants. Initial reaction rates are most rapid with the simplest phenolic compounds and decrease with the addition of bulky ring adducts and multiple rings. These differences are largely obscured and may even be reversed when reactions are monitored only after the long periods allowed for reaching equilibrium. Furthermore, compounds known to have strong antioxidant activity, e.g., glutathione and BHT,11 show only slow reactivity with DPPH at least in part due to strongly steric limitations. Because structure rather than chemistry appears to dominate the reaction rate, it is our scientific judgment that reaction with DPPH should not be used to compare or infer the potential antioxidant activity of antioxidant mixtures, prepared or in
antioxidants that acted preferentially by hydrogen atom transfer. Glutathione, a strong hydrogen donor, increased more than seven times in rate. Water is a known proton transfer medium; it supports ionization of solutes, has lower hydrogen bonding strength (β2H = 0.3842) than the organic solvents tested, and stabilizes the polar transition states necessary for hydrogen atom abstraction.41 All of these actions markedly facilitate hydrogen atom transfer. Solvent effects extended variably to reaction stoichiometry. Fifty percent methanol did not alter the reactivity of pyrogallol and catechol (strong electron transfer antioxidants) relative to methanol but increased the total reaction of hydroquinone by 50% and of resorcinol and glutathione by 7 times. In terms of DPPH assay standardization, these results indicate that of the solvents studied here, methanol is the solvent of choice. That it selects for antioxidants acting predominantly or exclusively by SET does present the disadvantage that strong electron transfer agents appear to be the most active antioxidants17 while known physiological antioxidants such as glutathione and uric acid that act almost exclusively by HAT are tremendously underrated. Nevertheless, running DPPH assays in 50% aqueous methanol in parallel with neat methanol offers opportunities to distinguish compounds that act by HAT, elucidate dominant reaction mechanisms of individual antioxidants and of mixed composition extracts, and provide important insights into behaviors that may be expected in multiphase systems such as cells and foods where lipid phases would favor electron transfers and aqueous phases would favor hydrogen atom transfers. Effects of pH on Kinetics and Reaction. Antioxidants from the solvent tests were further evaluated in 50% aqueous methanol in which the water portion was varied in pH (5, 7, and 9) by the addition of high purity HCl or NaOH. Initial rates for known electron transfer agents pyrogallol and catechol increased notably with pH, while hydrogen atom donors glutathione and resorcinol showed negligible change (Figure 5).
Figure 5. Effect of pH on initial rates of antioxidant reactions with DPPH. [AOX] = 100 μM (final); solvent = 50% aqueous methanol. pH effects on final stoichiometry in the same reactions were negligible.
Hydroquinone, which acts by both electron and hydrogen transfers, exhibited intermediate rate increases. Thus, pH effects on rates of antioxidant reaction with DPPH in 50% methanol can be advantageously combined with solvent effects to distinguish between electron and hydrogen atom transfers as dominant reaction mechanisms. Differences were only seen with reaction rates; essentially, no change occurred with reaction stoichiometry as pH was varied (data not shown). 4258
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extracts, where different structural classes are present and may not even be known. Ascorbic acid contents, in particular, will enhance reactivity disproportionately, so its concentrations in extracts must be determined to accurately interpret results. A potential alternative application of the DPPH reaction may be distinction of electron transfer versus hydrogen atom transfer as dominant reaction mechanisms of individual antioxidant compounds. Even here, though, qualitative information about reaction mechanisms of mixtures and extracts can be derived from reaction response patterns and kinetic solvent effects, but rates of general radical reactivity for mixtures cannot be quantified accurately due to limitations in steric accessibility to the DPPH radical site.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 848-932-5454. Fax: 732-932-6776. E-mail: schaich@ aesop.rutgers.edu. Notes
The authors declare no competing financial interest.
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REFERENCES
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