Observations on the Estimation of Scavenging Activity of Phenolic Compounds Using Rapid 1,1-Diphenyl-2-picrylhydrazyl (DPPH•) Tests

Nikolaos Nenadis and Maria Tsimidou*
Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

ABSTRACT: Rapid 1,1-diphenyl-2-picrylhydrazyl (DPPH•) tests are often applied to classify the scavenging activity of phenolic compounds (AH). Published analytical protocols differ in more than one experimental condition, and results for the relative order or magnitude of activity are often contradictory. In this work, parameters such as duration of test, [AH]/[DPPH•] molar ratio, and solvent effects were examined and discussed. The test duration and the value of the [AH]/[DPPH•] ratio did not influence the order of activity among tested antioxidants. Ethanol, commonly used as solvent in such tests, was compared with acetonitrile and tert-butyl alcohol. Solvent properties such as the ability to form hydrogen bonds with the AH seem to influence the level of the relative activity (%RSA). Higher %RSA values were observed in ethanol. The activity of the most polar compounds was affected the most, and in some cases (caffeic, dihydrocaffeic, and rosmarinic acids) the order of activity was changed owing to different kinetics. Standardization of the analytical protocol should include a 20-min reaction period and a molar ratio that permits attainment of a 60–80% RSA value for the most potent antioxidant. Solvent choice is critical for classifying activity. Safe classification can be based only on results from kinetic studies. Paper no. J10184 in JAOCS 79, 1191–1195 (December 2002). KEY WORDS: DPPH• test, phenolic antioxidants, radical scavenging activity, solvent effect.

free radicals from certain substrates or the antioxidant activity of compounds carrying –SH, –OH, and –NH groups (8–10). DPPH• does not dimerize, i.e., it remains in its monomeric form in solutions, exhibits a stable absorbance over a wide pH range, and resists oxidation. In addition, the reaction conditions are mild and, as reported, the results provide basic information on the reactivity of compounds with regard to their structure (5–7,10). All these characteristics explain the increasing popularity of applying rapid DPPH• tests to foods either for screening or to highlight the mechanism of reaction with the AH. An overview of the literature reveals that the analytical protocols of these tests differ in more than one experimental condition. The latter may be the reason why the results concerning the relative order or the magnitude of the radical scavenging activity are often contradictory (11,12). The aim of this work was to examine parameters of the rapid DPPH• tests that may influence the scavenging activity of phenolic antioxidants. A wide range of phenolic compounds was used to support the observations and justify the proposals for the standardization of the analytical protocol. MATERIALS AND METHODS Standards, reagents, and solvents. Caffeic acid and 6-hydroxy2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) were from Riedel-de Haën (Seelze, Germany). Dihydrocaffeic acid, o-coumaric acid, p-coumaric acid, sinapic acid, chlorogenic acid, tyrosol, BHA, BHT, TBHQ, and DPPH• were from Sigma Chemical Co. (St. Louis, MO). Ferulic acid and isoferulic acid were from Aldrich, Chemical Co. (Steinheim, Germany); m-coumaric acid was from Fluka Chemie (Buchs, Switzerland). Oleuropein and rosmarinic acid were from Röth (Karlsruhe, Germany). Hydroxytyrosol was a generous gift of Dr. G. Blekas. α-Tocopherol was from Merck (Darmstadt, Germany). Absolute ethanol and acetonitrile of HPLC grade were from Riedel-de Haën. tert-Butylalcochol was from Fluka Chemie. Apparatus. A U-2000 Hitachi spectrophotometer (Tokyo, Japan) was used for the measurement of the reduction of DPPH• absorbance at 516 nm. Estimation of radical scavenging activity (%RSA) by the DPPH• test. The %RSA activity of phenols was based on the method of Pekkarinen et al. (11). The decrease of the absorption at 516 nm of the DPPH• solution after addition of the AH
JAOCS, Vol. 79, no. 12 (2002)

The main mechanism of action of phenolic antioxidants (AH) is considered to be the scavenging of free radicals by hydrogen-atom donation, although other mechanisms may be involved (1). A methodology to estimate the scavenging of free radicals by the phenolic antioxidants that is rapid, inexpensive, easily applicable to a large number of samples (standard compounds or extracts), accurate, and robust is needed. Much effort has been devoted to the development of procedures based on reaction of AH with different radical species of biological significance such as O2•−, OH•, NO•, or lipid peroxyl radicals LOO• (2–4). Tests using the reduction of the 1,1-diphenyl-2picrylhydrazyl radical (DPPH•) in the presence of phenolic compounds are also quite popular among food scientists (5–7). These tests are based on the decrease of the absorbance of the radical solution according to the reaction DPPH•(purple) + HA → DPPH-H(yellowish) + A•. This radical has been used for many decades to study the mechanism of hydrogen-atom donation to
*To whom correspondence should be addressed. E-mail: tsimidou@chem.auth.gr Copyright © 2002 by AOCS Press


B 47.5 ± 0. that is.2 ± 0.B 9.5b.A 53.4 ± 0. no.A 34.5 ± 0.3 ± 0.A 16.3c.05 confidence level). An aliquot (2960 µL) of 0.5c.7d.4 ± 0.8c.1e.1f.1g.8a.1 mM DPPH• solution in ethanol or in acetonitrile. the higher the antioxidant activity. (11).A 37.8 ± 0.5. The radical scavenging activity of the AH was determined using 0.A 73.A 92. This difference in %RSA is due to the substituents in the aromatic ring that affect the reactivity of the compounds toward the sta- TABLE 1 Effect of Reaction Period and Relative Concentration of the AH on the Ability of Phenolic Antioxidants to Scavenge the DPPH• Radical [AH]/[DPPH•] (mol/mol) 0.A 56.A 25.4b.A 0.2d.A 17.1 ± 2.A 2.A 26.5e.A 39.A 2.1-diphenyl-2picrylhydrazyl. Different uppercase letters as superscripts indicate significantly different values within the same row at P ≤ 0.3 ± 0.A 41.A 2.B 5.25 Reaction period (min) 20 a 0.3 ± 0.0 ± 1. The results are expressed as %RSA = [Abs516nm(t = 0) − Abs516nm (t = t′) × 100/Abs516nm (t = 0)].1f.1e.9e.4 ± 2.1f.4f.A 0. followed by the multiple Duncan test (P ≤ 0.6i.3 ± 0. Statistical comparisons of the mean values for each experiment were performed by one-way ANOVA.B 22.A 14.6 ± 0.A 90.6c.A 46.2d.3 ± 0.A 58.1 ± 0. The experimental procedure was also performed using AH solutions of 1.B 21.1c.6 ± 0.1g.5 ± 0.2 ± 0.1e.5b.A 37.4e.6f.A 94.B 88.9 ± 0.7h.0 ± 0.4 ± 2.3f.2 ± 0.A 3.A 41.A 3.4 ± 0.5 ± 0.A 41.A 93.4 ± 0.5 ± 0.2h.A 33.7e. TSIMIDOU was measured in a glass cuvette (1 cm long).A 2.8 ± 1.7.A 1.1g.6 ± 0. Absorbance values were corrected for radical decay using blank solutions.A 1.8i.9d.5a. 6-hydroxy-2.1d.3 49.7 ± 0.2 ± 0.1f.9 ± 0.A 72.3d.5 10 20 50.A 0.B 4.A 54.2d.3 ± 0.7 ± 0.4a.A 39.A a.A 52.13 AH 10 20 10 (%) RSA Dihydrocaffeic acid Rosmarinic acid Caffeic acid Chlorogenic acid Sinapic acid Ferulic acid Isoferulic acid o-Coumaric acid m-Coumaric acid p-Coumaric acid trans-Cinnamic acid Hydroxytyrosol Oleuropein Tyrosol α-Tocopherol Trolox TBHQ BHA BHT a 0. phenolic compound.7 ± 2.4 ± 1.A 0. Graphs were constructed showing the percentage of residual DPPH• vs.2 82.A 70.3j.6 ± 0.A 88.A 32.B 76.7 ± 0.9 ± 0.5 ± 0.A 2.A 60.4e.6i.A 2. 79.A 3.A Mean value of three measurements ± SD.2 ± 0.0d.4f.4 ± 0.1g.5f.6 ± 0.2 ± 0.1 ± 0.4a. respectively). JAOCS.1 ± 2.2g.1a. namely.A 6.6 ± 0.7 ± 0.A 69.1g.4f. DPPH•. From these graphs the percentage of DPPH• remaining at the steady state was determined.A 2.A 2.B 52.0 ± 0.1192 N.6 ± 0. and 20-min reaction periods.3j.4a.A 53.2 ± 1.A 63.1 ± 0.A 56.6d.A 30.1f.A 3.3 ± 0.A 49.6 ± 1.5.2b.2 ± 0. the reaction time needed to reach the steady state for EC50 (TEC50) and antiradical efficiency.2 ± 0.3f. Vol.B 88. AH.4 ± 1.2a. the order of DPPH• scavenging by the compounds was not affected by the reaction period.1h.4 ± 0.8k.5 ± 0.7 ± 0.3 ± 0.5 ± 1.6c. time.4l.05.13.6k.5 ± 0.2 ± 1.A 81. All tests were performed in triplicate at 25°C.6 ± 0.6 ± 0.A 28.6 ± 0.1f.A 82.3f. The lower the EC50 is.9e.5b.1h.5k.6d.1h.6 ± 0.A 54.B 47.A 3. %RSA values are presented for 10-min.3e.9 ± 2.7 mM (mole AH/mole DPPH• = 0.A 52.2 ± 1.5 ± 1. 12 (2002) .A 0.5 ± 0.3 ± 0.7e.A 4. According to these results. %RSA.A 3.A 57.A 85. the amount of antioxidant necessary to de- crease the initial [DPPH•] by 50%.9e.4e. as proposed by Pekkarinen et al.A 3.A 3.A 2.8d.5g. Reduction of DPPH• was followed by monitoring the decrease in absorbance at 516 nm until the reaction reached a plateau.A 31.1c.A 93.5f. 1.A 3. AE = 1/EC50 × TEC50 were also calculated (6).5 ± 0.A 2.B 8.1b.A 79. Kinetic studies were undertaken in certain cases.4 ± 1.1f.0 ± 0.1 mM ethanolic DPPH• solution was mixed with 40 µL of a 1 mM AH solution so that the relative concentration of AH vs.4 ± 0.A 63.5 ± 0. The %RSA values within each column indicated the different activity of each compound toward the stable radical.2 ± 0.85 mM solution of the AH was also carried out in acetonitrile or tert-butylalcohol. Determination of the %RSA using the 1.6 ± 0.3 ± 0.6 ± 0.A 89.5 ± 0.2e.8c.8 ± 0.A 94.5 ± 0.7 ± 0. the stable radical (mole AH/mole DPPH•) in the cuvette was 0.6 ± 1. Moreover.4f.0 ± 0. percent relative activity.A 5.8d.5 ± 0.5 ± 0.7 ± 1.A 41.9 ± 0.A 41.A 41.5 ± 1.9 ± 0. The results of the effect of the test duration and of the relative concentration of the antioxidant on the ability of the compounds to scavenge the DPPH• are presented in Table 1.8 ± 0.5g.1h.A 3.2 ± 0. The absorption was monitored at the start and at 10 and 20 min.1j.A 4.A a.4j.2e. and the values were transferred onto another graph showing the percentage of residual stable radical at the steady state as a function of the molar ratio of antioxidant to DPPH•.3g.3f.2f.1d.7i.2 56. Different lowercase letters as superscripts indicate significantly different values within each column.8 ± 0.85 and 3.A 2. hydroxycinnamic acid derivatives and secoiridoids as well as α-tocopherol and a group of synthetic antioxidants often used as a reference in autoxidation studies.1g.3 ± 0.0d.9 ± 0.0 ± 0.0 ± 1.0 ± 0.3 ± 0.9 ± 0.A 4. Statistical analysis.A 39.2 ± 2.4g.3 ± 1.B 92.A 0.6f.A 4.2h.2i.9f.5 ± 0. The latter was used to determine the efficient concentration (EC50).A 36.A 2.A 2.A a.5f.B 83.4 ± 0.A 13.A 86. Trolox.5g.5 ± 0.0 ± 0.1f.A 25.25 and 0.3 ± 0.8j.5e. RESULTS AND DISCUSSION In the present study a large number of phenolic compounds were examined.1a.A 89.7h. NENADIS AND M.8-tetramethyl-chroman-2-carboxylic acid.5c.A 3.1 ± 1.0 ± 0.7 ± 0.1d.

2 ± 0. by performing a rapid test this piece of information cannot be revealed and results may not be directly used in structure–activity relationship studies.3 ± 1. Unexpectedly.2g 57.2a.A 41.7 ± 0. Simple phenols without an aromatic ring substituent (coumaric acids.A 30. The behavior of dihydrocaffeic acid differed in the three solvent systems. For example.1b.8c. in line with previous reports (17). optimization of the [AH]/ [DPPH•] ratio is important with regard to the potency of the antioxidants under examination.3a.7d.A 2.A 22.5g.0 ± 0. the esterified form of hydroxytyrosol with elenolic acid.B 6.7h. and the results were compared with those in acetonitrile and in tert-butyl alcohol.B 37. This ability is well described by the β2H constant (16). The behavior of α-tocophJAOCS.B 0 0 0 0 31.5 ± 0. caffeic acid) were the most active. The results of the effect of the reaction environment on the %RSA are presented in Table 2. The β2H value is the same for ethanol and acetonitrile (0.A 3.7g. however. In some cases. whereas in acetonitrile it was less active than caffeic and rosmarinic acids.A 76. the relative order in the antioxidant activity seemed to be in line with the rules of structure–activity relationship. respectively) when acetonitrile or tert-butyl alcohol was used as a solvent.7 ± 0.8d.A a. the effect of the number of the phenolic hydroxyl groups on the activity of the compounds was more obvious (rosmarinic acid was 1.6 times more active than caffeic and dihydrocaffeic acids.A 19.0 ± 1. the reaction with certain compounds is completed within a few minutes.A 45. BHA. %RSA values were higher in ethanol and.7 ± 1. However. The environment of the reaction is expected to affect the hydrogen-atom transfer from AH to the free radical (13).B 40. consequently. was less active than hydroxytyrosol. in tert-butyl alcohol its activity was equal to that of caffeic acid.5h. as did hydroxytyrosol and oleuropein. this parameter seems not to be taken into consideration although in most applications ethanol or methanol is used as the solvent. most likely as a result of higher levels of AH molecules available to scavenge the same amount of the stable radical.5 ± 0.A 60.2e. and rosmarinic acids had similar %RSA values. tyrosol. dihydrocaffeic.5 ± 0.8e. rosmarinic acid.B 46.2d.5 ± 0.OBSERVATIONS ON RAPID DPPH• TESTS 1193 ble radical and.7 ± 1. The choice of solvent for preparing the radical solution could be important in structure–activity studies.6h.9 ± 1.7). This is expected when the maximal %RSA values range from 60 to 80%. no.5 88. Caffeic acid remained more active than its esterified form with quinic acid (chlorogenic acid). but the relative differences varied from solvent to solvent. These solvents have been used previously to study the kinetics of the phenolic hydrogen-atom transfer from α-tocopherol and phenol to DPPH• (13–15).2 and 1. The rate of this transfer was found to be affected by the ability of the solvent to form intermolecular hydrogen bonds with the AH (13–15).7 ± 0.3 ± 0.B 26.B 2.1a.3 ± 0. The order of reactivity of secoiridoids was the same in all three solvents. since rosmarinic acid was found to be as active as dihydrocaffeic acid in ethanol. may provide better insight into the real “potency” of each tested compound (5. Vol.6f 3.6 ± 0.2 ± 0.7 ± 0.3 ± 0.2 ± 0.B 23. caffeic.1e.6f.2h.3f 2.2c. Compounds with more than one hydroxyl group (dihydrocaffeic acid.2 ± 0. Oleuropein.5b. For abbreviations see Table 1. was inactive.9 ± 1. BHT).2c. Monophenols with no substituents in the aromatic ring (coumaric acids) were inactive in all solvents.A 53.9 ± 0.g.A 56.8 ± 0.B 1.8 ± 0.1f.3f 54.2b. 12 (2002) . Different uppercase letters as superscripts indicate significantly different values within the same row at P ≤ 0. Therefore.5 ± 1.A 58.6 ± 0. Relative differences were of similar size in all solvents. the %RSA values were significantly lower and differences among AH were evident. ferulic) or alkyl substituents (e.0 ± 0.8d.5i.6i. butyl alcohol.7 ± 0. the kinetics of the reaction.9e. Thus.B 59.49).9d.0 ± 0. the relative order of activity in the compounds changed.B 60.A 3. However. the activity of the AH in acetonitrile was similar to that in tert-butyl alcohol.A 8. Kinetic studies using DPPH•.6c.5 ± 0. As known. these molecules gave rather low %RSA values (≤50%). although time-consuming.A 52.5g. At a high [AH]/[DPPH•] ratio.4 ± 0.4a.6 ± 0.A 6. [AH]/[DPPH•] = 0. no or slight differences could be found among structurally similar AH.1d.8f.2d.B 65. More critical was the relative concentration of the antioxidant ([AH]/[DPPH•]) ratio that affected the level of the %RSA. In the presence of acetonitrile and tert- TABLE 2 Solvent Effect on the Ability of Phenolic Antioxidants to Scavenge the DPPH• Radicala Solvent AH Ethanol Acetonitrile (%) RSA Dihydrocaffeic acid Rosmarinic acid Caffeic acid Chlorogenic acid Sinapic acid Ferulic acid Isoferulic acid o-Coumaric acid m-Coumaric acid p-Coumaric acid trans-Cinnamic acid Hydroxytyrosol Oleuropein Tyrosol α-Tocopherol Trolox TBHQ BHA BHT a b tert-Butylalcohol b 93.A 52.B 0 51.4 ± 1. 79.9 ± 0. The relative order of reactivity was retained among methoxy-substituted phenols.4a. Sinapic was more active than ferulic and isoferulic acids. it was the most active of all the derivatives.B 0 0 1.5a.3 ± 1. It is therefore implied that the activity of an AH would be similar in the first two solvents and lower in the third. This affected the level of the differences observed among the activities of the AH.05. Different lowercase letters as superscripts indicate significantly different values within each column. in general.B 71.1 ± 0. In the case of cinnamic acid derivatives.B 14.0d.4f. The %RSA value for most of the compounds increased concomitantly with the value of the [AH]/[DPPH•] ratio.9 ± 2.44) and somewhat higher in tert-butyl alcohol (0. whereas slow kinetics have been reported for a great number of antioxidants owing to a more complex mechanism (5).B 0 49.A 1.25. At the lowest [AH]/[DPPH•] ratio.6 ± 0.1 ± 2. In our study the scavenging of DPPH• was carried out in ethanol.6 ± 1.8 ± 0.B 24. although the relative difference depended heavily on the solvent.A 45. the presence of a second catechol moiety did not infer higher reactivity as expected.B 46. whereas the monophenol. followed by monophenols with methoxy (sinapic.4j.7i.4d.B 27.5 ± 0. In ethanol.7g.A Reaction period 20 min. tyrosol) were almost inactive. Mean value of three measurements ± SD..B 1.7h.5 ± 0.B 0 32.8 ± 1.B 8.0 ± 0.6 ± 0.

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