Formation of dehydrodiisoeugenol and dehydrodieugenol from the reaction of isoeugenol and eugenol with DPPH radical and their role in the radical scavenging activity

Article abstract of DOI:10.1016/j.foodchem.2009.04.115

The aim of this work was to investigate the products of the reactions between isoeugenol and eugenol with the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and their role in the radical scavenging mechanism. The reaction of isoeugenol and eugenol with the DPPH radical produced, as evidenced by GC-MS and HPLC-MS, a complex mixture of dimeric species in which dehydrodiisoeugenol and its adducts with methanol (reaction solvent) and dehydrodieugenol were the main reaction products, respectively. The antioxidant activity of dehydrodiisoeugenol, determined by the DPPH method, resulted lower than that of isoeugenol considering both the parameters Effective Concentration (EC₅₀) and Antiradical Efficiency (AE). In particular, due to its very slow kinetic behaviour (T_EC50 = 201 min), the possible contribution of dehydrodiisoeugenol to the DPPH radical scavenging activity of isoeugenol (T_EC50 = 3.1 min) was practically negligible. On the contrary, dehydrodieugenol had an antioxidant activity higher than that of eugenol and its lower T_EC50 (85 min with respect to 126 min) made it possible to contribute to the DPPH radical scavenging activity of eugenol.

Full text of DOI:10.1016/j.foodchem.2009.04.115

Food Chemistry 118 (2010) 256–265
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Food Chemistry
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Formation of dehydrodiisoeugenol and dehydrodieugenol from the reaction
of isoeugenol and eugenol with DPPH radical and their role in the radical
scavenging activity
b
a
b
Renzo Bortolomeazzi a, , Giancarlo Verardo , Anna Liessi , Alessandro Callea
*
a Department of Food Science, University of Udine, via Sondrio 2a, 33100 Udine, Italy
b Department of Chemical Sciences and Technologies, University of Udine, via Cotonificio 108, 33100 Udine, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this work was to investigate the products of the reactions between isoeugenol and eugenol
with the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and their role in the radical scavenging
mechanism. The reaction of isoeugenol and eugenol with the DPPH radical produced, as evidenced by
GC–MS and HPLC–MS, a complex mixture of dimeric species in which dehydrodiisoeugenol and its
adducts with methanol (reaction solvent) and dehydrodieugenol were the main reaction products,
respectively. The antioxidant activity of dehydrodiisoeugenol, determined by the DPPH method, resulted
lower than that of isoeugenol considering both the parameters Effective Concentration (EC50) and Anti-
Received 10 February 2009
Received in revised form 9 April 2009
Accepted 28 April 2009
Keywords:
Radical scavenging activity
DPPH radical
Isoeugenol
radical Efficiency (AE). In particular, due to its very slow kinetic behaviour (TEC ¼ 201 min), the possible
50
contribution of dehydrodiisoeugenol to the DPPH radical scavenging activity of isoeugenol
(TEC ¼ 3:1 min) was practically negligible. On the contrary, dehydrodieugenol had an antioxidant activ-
Eugenol
50
ity higher than that of eugenol and its lower TEC (85 min with respect to 126 min) made it possible to
Dehydrodiisoeugenol
Dehydrodieugenol
Oxidative dimers
GC–MS
50
contribute to the DPPH radical scavenging activity of eugenol.
Ó 2009 Elsevier Ltd. All rights reserved.
HPLC–MS
1. Introduction
(EC50) that represent the [antioxidant]/[DPPH] concentration ratio,
which decreases the initial DPPH concentration by 50%, the stoichi-
Several analytical methods are routinely used to evaluate the
antioxidant capacity of a large variety of compounds both in pure
form and in complex mixture like herbs, spices, fruits and seeds ex-
tracts. Among them, the oxygen radical absorbance capacity
(ORAC) assay, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as-
say, the trolox equivalent antioxidant capacity (TEAC) assay and
the ferric reducing antioxidant power (FRAP) assay are probably
the most widely used. The ORAC method is based on the scaveng-
ing of peroxyl radicals generated by an azo compound, and fluores-
cein is used as target molecule. The decrease in fluorescence in the
absence and in the presence of an antioxidant is monitored. The as-
say is carried out in phosphate buffer at pH 7.4, and the results are
expressed as trolox equivalents (Cao, Alessio, & Cutler, 1993; Ou,
Hampsch-Woodill, & Prior, 2001). The DPPH method is based on
the scavenging of the stable DPPH radical by the antioxidant. The
decrease in absorbance of the radical is monitored at 515 nm, in
methyl alcohol, until the reaction reaches a steady state. The re-
sults are expressed by the parameters Effective Concentration
ometry value (n) defined as the DPPH moles scavenged by one
mole of antioxidant and the Antiradical Efficiency defined as
AE ¼ 1=ðEC50 ꢀ TEC Þ, where TEC represents the time needed to
50
50
reach the steady state for EC50 (Brand-Williams, Cuvelier, & Berset,
1995; Sanchez-Moreno, Larrauri, & Saura-Calixto, 1998). With a
similar approach, the Trolox Equivalent Antioxidant Capacity
(TEAC) method is based on the ability of a compound to scavenge
the 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical
(ABTSÅꢁ) with respect to trolox C. The decrease in absorbance of
the blue-green radical is monitored at 652 nm for 6–10 min. The
assay is carried out in ethanol or in phosphate-buffered saline
(PBS) at pH 7.4 and the results are expressed as trolox equivalents
(Re et al., 1999). On the contrary, the FRAP method does not use
free radical scavenging but is based on the reducing ability of the
antioxidant versus the ferric-tripyridyltriazine complex. The in-
crease in absorbance due to the formation of the coloured reduced
ferrous complex is monitored and the results are expressed as trol-
ox equivalents (Benzie & Strain, 1996). This brief description is en-
ough to show the different approach and the different conditions of
reaction time, reaction medium, temperature and treatment of the
results used by these methods and the consequent difficulty to
* Corresponding author. Tel.: +39 0432558147; fax: +39 0432558130.
E-mail address: renzo.bortolomeazzi@uniud.it (R. Bortolomeazzi).
0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.04.115

R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
257
compare the results obtained for the same compounds. Foti and
Ruberto (2001) observed a dependence of the antioxidant activity
of some flavonoids and phenolic antioxidants on the hydrogen
bond acceptor activity of the solvents. The effect of solvent and
food components on the antioxidant activity obtained by applying
the ORAC, DPPH, TEAC and FRAP methods to the mixture of cate-
chin and gallic acid has been recently outlined by Perez-Jimenez
and Saura-Calixto (2006). Also the oxidation processes involved
are different: hydrogen atom transfer (HAT) in the case of the
ORAC assay and single electron transfer (SET) in the other methods
(Foti, Daquino, & Geraci, 2004; Huang, Ou, & Prior, 2005). These
problems are even more enhanced by the possible occurrence of
side reactions involving chemical species other than the antioxi-
dant under testing. Studying the reaction mechanism of the DPPH
radical with BHT, eugenol and isoeugenol, Brand-Williams et al.
(1995) and Bondet, Brand-Williams, and Berset (1997) evidenced
an inverse relationship between stoichiometry and reaction rates
suggesting the intervention of a more complex reaction mecha-
nism, eventually involving dimeric species, for the compounds
with a slow kinetic behaviour such as eugenol. Recently, Ordoudi,
Tsimidou, Vafiadis, and Bakalbassis (2006), studying the DPPH rad-
ical scavenging activity of guaiacol acid derivatives, reported the
difficult to interpret the stoichiometry values of some compounds.
Eklund et al. (2005) reported the formation of dimers in the reac-
tion of lignans with DPPH radical. The EC50 of compounds with
long reaction time can therefore reflect a radical scavenging capac-
ity comprehensive not only of the contribution of the original anti-
oxidant but also of other chemical species. Similar considerations
were more recently outlined by Arts, Dallinga, Voss, Haenen, and
Bast (2003) and Arts, Haenen, Voss, and Bast (2004) about the TEAC
assay. In the reaction between ABTSÅꢁ and the flavonoid chrysin,
these authors reported the formation of a product with an antiox-
idant capacity higher than that of the parent compound. Hotta,
Sakamoto, Nagano, Osakai, and Tsujino (2001) and Hotta, Nagano,
Ueda, Tsujino, Koyama and Osakai (2002), studying the antioxidant
capacity of natural compounds by column flow electrolysis, ob-
served an increase of exchanged electrons by decreasing the flow
and they attributed this effect to the oxidation of compounds
formed by chemical reactions following the first oxidation process.
The aim of this work was to investigate the products formed by
reaction of isoeugenol (1) and eugenol (2) with the DPPH radical
under the same conditions used in the DPPH assay. The structural
identification of these compounds, as well as the determination of
their radical scavenging activity, may provide an insight into the
antioxidant mechanism involved.
calibration was performed with indium, temperature calibration
was done with n-hexane, distilled water and indium. Samples were
placed into 40-
lL aluminium pans that were hermetically sealed
and scanned under 10 mL minꢁ1 dry nitrogen flow to at least
10 °C above the endset of melting at a rate of 1 °C minꢁ1. The
reported values were the average of two onset Tm.
2.3. 1H and 13C NMR spectra
1H and 13C NMR spectra were recorded on a Bruker AC-F 200
spectrometer at 200 and 50 MHz, respectively, using CDCl3 as sol-
vent at room temperature. NMR chemical shifts are reported as d
values from TMS.
2.4. Gas chromatography–mass spectrometry (GC–MS)
A Shimadzu gas chromatograph coupled to a quadrupole mass
spectrometer QP-2010 (Shimadzu Corporation, Kyoto, Japan) was
used. The column was a 30 m ꢀ 0.25 mm i.d., 0.25
lm film thick-
ness, fused silica Equity 5 (Supelco, Sigma–Aldrich, Milan, Italy).
The initial oven temperature was 60 °C for 2 min, then pro-
grammed to 280 °C at 15 °C minꢁ1. The injection was in splitless
mode (2 min) with helium as carrier gas at a flow rate of
1 mL minꢁ1. The injector, transfer line and ion source temperatures
were, respectively 280, 280 and 200 °C. The mass spectrometer
operated in electron impact ionisation mode at 70 eV.
2.5. High performance liquid chromatography–mass spectrometry
(HPLC–MS)
A Finnigan LXQ linear trap (Thermo Electron Corporation) was
used for ESI-MSn analysis in the positive and negative ion modes.
The general conditions were capillary temperature 275 °C, sheath
and auxiliary gas flows 25 and 5 (arbitrary units), respectively,
source and capillary voltage in the positive polarity 4.0 kV and
2.0 V and 4.7 kV and ꢁ50.0 V in the negative polarity, respectively.
Collision-induced dissociation (CID) multiple MS spectra (MSn
experiments) were acquired using helium as the collision damping
gas in the ion trap at a pressure of 1 mTorr. Direct infusion of the
standard solutions of pure dehydrodiisoeugenol (3) and dehydro-
dieugenol (4) and intact reaction mixtures diluted with MeOH or
MeCN were performed with the aid of a syringe pump, using a flow
rate of 5
same instrument coupled with a Finnigan Surveyor LC Pump Plus
equipped with a Waters Spherisorb 5 m ODS2 analytical column
l
L minꢁ1. The LC-ESI-MS analyses were obtained with the
l
(4.6 ꢀ 250 mm) at 30 °C. Elution was carried out at a flow rate of
0.8 mL minꢁ1 using an Accurate post-column flow splitter with a
split ratio of 1:4 and MeCN/H2O (65/35, v/v) as mobile phase.
2. Materials and methods
2.1. Materials and reagents
2.6. DPPH radical scavenging method
All solvents were of analytical grade or LC–MS grade. 2,2-Diphe-
nyl-1-picrylhydrazyl (DPPH) radical, eugenol (1), isoeugenol
(2; mixture of E and Z isomers), N,O-bis(trimethylsilyl)trifluoroac-
etamide (BSTFA) and horseradish peroxidase (HRP, type II) were
purchased from Sigma–Aldrich (Milan, Italy). Silica gel 60, 70–
230 mesh ASTM and the thin-layer chromatography (TLC) silica
gel plates, 0.25 mm thickness were provided by Merck (Darmstadt,
Germany).
The radical scavenging activity of the compounds was deter-
mined following the method used by Bortolomeazzi, Sebastianutto,
Toniolo, and Pizzariello (2007). DPPH was dissolved in methanol at
a final concentration of about 6 ꢀ 10ꢁ5 M. The exact concentration
of DPPH was calculated from
a
calibration curve,
e
= 11870 Mꢁ1 cmꢁ1 at 515 nm. Different aliquots of a methanolic
solution of the phenolic compounds were added to 2450 L of
DPPH solution and the volume was adjusted to a final value of
2500 L with methanol. Five different concentrations were used
l
l
2.2. Melting point determination
for each assay. The decrease in the DPPH radical was followed at
515 nm until the reaction reached a steady state by using a Varian
Cary 1E spectrophotometer equipped with a thermally controlled
multicell block set at 25 °C. The concentrations of DPPH at the stea-
dy state, corrected for the natural disappearance of the DPPH rad-
ical, were plotted as a function of the molar concentration ratio
Melting point determination was performed by differential
scanning calorimetry (DSC) (Mettler TA 4000 system equipped
with TC15 TA Controller, DSC 30 measuring cell and Star software
version 8.10, Mettler, Greinfensee, Switzerland). Heat flow

258
R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
[AH]/[DPPH] to determine the Effective Concentration (EC50). The
The GC–MS analysis of the crystallised product evidenced the
presence of four compounds with practically the same mass spec-
tra and M+ at m/z 326. The relative retention times (RRTs) to the
most abundant compound were 0.96, 0.97, 1, 1.01 and the corre-
sponding percent chromatographic areas, calculated on the basis
of the m/z 326 ion signal, were 2.1, 0.1, 95.7 and 2.1%, respectively.
The HPLC–MS analysis of the same product evidenced the presence
of three compounds with m/z 327 [M + H]+ and the same fragmen-
tation patterns till MS3. Sarkanen and Wallis (1973) reported the
formation of (E)-1-[(2RS,3SR)-2,3-dihydro-2-(4-hydroxy-3-meth-
oxy phenyl)-7-methoxy-3-methyl-1-benzofuran-5-yl]-1-propene
[(E)-( )-trans-dehydrodiisoeugenol] (3a) and (Z)-1-[(2RS,3SR)-2,3-
dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methyl-1-
ben zofuran-5-yl]-1-propene [(Z)-( )-trans-dehydrodiisoeugenol]
(3b) by peroxidase catalysed oxidation of (E)- and (Z)-isoeugenol,
respectively. Due to the presence in isoeugenol (1) standard of both
the (E)- and (Z)-isomers in the proportion of about 93% and 7%,
respectively, the formation of both the dehydrodimers 3a and 3b
would be expected. On this basis the structure of (E)-( )-trans-
dehydrodiisoeugenol (3a) has been assigned to the most abundant
compound and the structure of (Z)-( )-trans-dehydrodiisoeugenol
(3b) to one of the other three compounds. Regarding the other
two, the following structures (E)-1-[(2RS,3RS)-2,3-dihydro-2-
(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methyl-1-ben zofu-
ran-5-yl]-1-propene [(E)-( )-cis-dehydrodiisoeugenol] (3c) and
(Z)-1-[(2RS,3RS)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-
time needed to reach the steady state for EC50 (TEC ) and the Anti-
50
radical Efficiency AE ¼ 1=ðEC50 ꢀ TEC Þ were also calculated.
50
2.7. Reaction of isoeugenol-DPPH radical
The conditions used to analyse the reaction products between
isoeugenol (1) and DPPH were similar to those used in the DPPH
assay. To 2450
lL of a methanolic DPPH solution were added
40 L of methanol and 10 lL of an isoeugenol (1) methanolic solu-
l
tion. The final concentration was about 6 ꢀ 10ꢁ5 M and 4.4 ꢀ 10ꢁ5
M for DPPH and isoeugenol (1), respectively. After 5 min, the reac-
tion mixture was reduced to dryness by a rotavapour at 40 °C, dis-
solved with 200
lL of acetonitrile and analysed by GC–MS. To the
acetonitrile solution was then added 100
lL of BSTFA and was let
to react for 1 h at room temperature before GC–MS analysis. The
reaction was carried out also by using acetonitrile as solvent both
for DPPH and isoeugenol (1) and analysed after 5 and 30 min.
2.8. Reaction of eugenol-DPPH radical
Similar conditions were used also for the analysis of the reac-
tion products between DPPH radical and eugenol (2), final concen-
tration was about 6 ꢀ 10ꢁ5 M and 4.7 ꢀ 10ꢁ5 M, respectively. The
reaction was stopped after 10 min using methanol and after
30 min in the case of acetonitrile as solvents.
methoxy-3-methyl-1-benzofuran-5-yl]-1-propene
[(Z)-( )-cis-
2.9. Synthesis of dehydrodiisoeugenol (3)
dehydrodiisoeugenol] (3d) have been hypothesised (Scheme 1).
The 1H and 13C NMR spectra were in agreement with the structure
of (E)-( )-trans-dehydrodiisoeugenol (3a) (Barbosa-Filho, Leitão
da-Cunha, & Sobral da Silva, 1998; Juhász et al., 2000; Shiba
et al., 2000) which purity was about 95.7%. The structure-number-
ing system used in the 1H and 13C NMR data does not follow the
actual compound names but is based on the numbering of the
monomer so as to maintain consistency.
Dehydrodiisoeugenol (3) was prepared by oxidative coupling of
isoeugenol (1) using the peroxidase-H2O2 system, following the
method reported by Nascimento, Lopes, Davin and Lewis (2000)
with minor modifications. To a solution of isoeugenol (1) (1.01 g,
6.2 mmol) in 30 mL of MeOH were added 275 mL of citrate–phos-
phate buffer (20 mM, pH 3) and horseradish peroxidase (8.5 mg,
1500 U) in 10 mL of buffer. 0.31 mL of H2O2 (3.05 mmol) was then
added dropwise over 10 min and under magnetic stirring. The
reaction mixture was stirred for an additional 10 min and then
transferred in a separatory funnel and extracted with 100 mL of
ethylacetate. The organic phase was washed with water, dried over
anhydrous Na2SO4 and reduced to dryness. The crude product was
loaded into a silica chromatography column (20 cm length ꢀ 2 cm
i.d.) and eluted with n-hexane/ethylacetate mixture from an initial
8.5/1.5 (v/v) ratio. The chromatographic separation was monitored
by TLC. The fractions containing the product (Rf 0.41, n-hexane/
ethylacetate, 8/2, v/v) were combined, reduced to dryness
(268 mg, 27%) and crystallised from MeOH/H2O. m.p. 132.5 °C
[lit. 128–130 °C (Shiba, Xiao, Miyakoshi, & Chen, 2000); 129–
132 °C (Juhász, Kürti, & Antus, 2000)]; calcd. for C20H22O4: C,
73.60; H, 6.79; found C, 73.56; H, 6.71. EIMS m/z (rel. int.): m/z
326 (M+, 100), 311 (M+ꢁCH3, 16), (1 ꢀ TMS) m/z 398 (M+, 100),
383 (M+ꢁCH3, 14), 368 (M+ꢁ2 ꢀ CH3, 10). ESI (+)-MS (infusion,
MeOH), m/z: 327 [M + H]+ (MS3 transitions 327 > 203 > 171), 349
[M + Na]+, 675 [2 M + Na]+. ESI (ꢁ)-MS (infusion, MeOH), m/z:
325 [MꢁH]ꢁ (MS5 transitions 325 > 310 > 295 > 277 > 249).
2.10. Synthesis of dehydrodieugenol (4)
Dehydrodieugenol (4) was prepared by oxidative coupling of
eugenol (2) using potassium ferricyanide as oxidising agent follow-
ing the method reported by De Farias Dias (1988). To a solution of
eugenol (2) (1.0 g, 6.1 mmol) in an acetone/H2O mixture (2:1, v/v,
30 mL) 18 mL of aqueous NH4OH (28%) was added and the mixture
was stirred for 10 min. To the mixture a saturated aqueous solution
of K3Fe(CN)6 (2.0 g, 6.1 mmol) was then added dropwise over a
period of about 5 h and then 18 mL of aqueous NH4OH (28%) was
added. The mixture was stirred for an additional 16 h at room tem-
perature and then neutralised with HCl 10%. There was the forma-
tion of a solid precipitate which was filtered, washed with water
and dried (720 mg, 72%). The crude product was crystallised from
ethanol after decolourisation with activated charcoal. m.p.
105.8 °C, [lit. 105–106 °C, (De Farias Dias, 1988)]; calcd. for
C20H22O4: C, 73.60; H, 6.79; found C, 73.10; H, 6.84. EIMS m/z
(rel. int.): m/z 326 (M+, 100), 297 (M+ꢁHCO, 34), 285 (M+ꢁCH2-
CHCH2, 6), 284 [M+ꢁ(CH2CHCH2 + H), 7], 267 [M+ꢁ(CH2CHCH2 +
H2O), 6], 253 [M+ꢁ (CH2CHCH2 + H + CH3O), 22], 244
(M+ꢁ2 ꢀ CH2CHCH2, 11), 221 [M+ꢁ(CH2CHCH2 + H + CH3O +
CH3OH), 9]; (2 ꢀ TMS) m/z 470 (M+, 100), 455 (M+ꢁCH3, 46), 440
(M+ꢁ2 ꢀ CH3, 36). ESI (+)-MS (infusion, MeOH), m/z: 327
[M + H]+, 349 [M + Na]+ (MS3 transitions 349 > 308 > 277), 675
[2 M + Na]+. ESI (ꢁ)-MS (infusion, MeOH), m/z: 325 [MꢁH]ꢁ (MS5
transitions 325 > 310 > 295 > 254 > 226). 1H NMR (CDCl3/TMS): d
3.36 (d, 4H, J = 6.7 Hz, CH2-7, CH2-70), 3.89 (s, 6H, 2 ꢀ OMe),
5.01–5.18 (m, 4H, CH2-9, CH2-90), 5.87–6.09 (m, 2H, CH-8, CH-80),
6.05 (br s, 2H, OH), 6.72 (d, 2H, J = 1.9, CH-2, CH-2’), 6.75 (d, 2H,
1H NMR (CDCl3/TMS): d 1.37 (d, 3H, J = 6.8 Hz,
3H, J = 6.4, 1.5 Hz, CH3-9), 3.37–3.52 (m, 1H,
MeO-3), 3.88 (s, 3H, MeO-3), 5.09 (d, 1H, J = 9.5 Hz,
5.67 (br s, 1H, OH), 6.10 (dq, 1H, J = 15.6, 6.5 Hz, CH-8), 6.36 (dd,
1H, J = 15.6, 1.5 Hz, CH-7), 6.76 (br s, 1H, CH-6), 6.78 (br s, 1H,
CH-2), 6.87–6.93 (m, 2H, CH-5, CH-6), 6.97 (br s, 1H, CH-2).
13C NMR (CDCl3): d 17.5 (
C-9), 18.3 ( C-9), 45.5 ( C-8), 55.8
MeO-3), 55.9 ( MeO-3), 93.7 ( C-7), 108.8 ( C-2), 109.1 ( C-2),
113.2 ( C-6), 114.0 ( C-5), 119.9 ( C-6), 123.4 ( C-8), 130.8 ( C-
7), 132.0 ( C-1), 133.2 ( C-5), 144.1 ( C-3), 145.7
C-3) (Scheme 1).
A
CH3-9), 1.86 (dd,
CH-8), 3.86 (s, 3H,
CH-7),
B
A
A
B
A
B
B
B
B
A
A
A
A
B
A
(A
B
A
A
B
B
A
A
B
B
B
C-1), 132.1 (
A
B
B
13
(A
C-4), 146.5 (
B
C-4), 146.6 (
A
J = 1.9, CH-6, CH-60). C NMR (CDCl3): d 39.9 (C-7, C-70), 56.0

R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
259
.
O
OH
O
O
O
4
MeO
MeO
MeO
MeO
MeO
3
5
6
.
+DPPH·
-DPPH-H
2
8
.
1
7
.
9
1
B
O
O
OMe
A
OMe
OMe
O
RO
RO
OMe
OMe
3a, 3c
3b, 3d
O
3, R = H (C8-C5; m/z = 326; ESI(+): m/z 327 [M+H]+, 349 [M+Na]+
and 675 [2M+Na]+; ESI(-): m/z 325 [M-H]-)
3-TMS, R = TMS (m/z = 398)
OMe
MeO
O
O
MeO
RO
OR
OMe
OMe
7
5, R = H (C8-C8; m/z = 326)
5-TMS, R = TMS (m/z = 470)
O
OR
OR
O
OMe
OMe
MeO
MeO
6, R = H (C5-C5; m/z = 326)
6-TMS, R = TMS (m/z = 470)
Scheme 1.
(2 ꢀ OMe), 110.6 (C-2, C-20), 115.6 (C-9, C-90), 123.0 (C-6, C-60),
124.4 (C-5, C-50), 131.8 (C-1, C-10), 137.6 (C-8, C-80), 140.8 (C-4,
C-40), 147.2 (C-3, C-30). The 1H and 13C NMR spectra were in agree-
ment with the data reported in literature (De Farias Dias, 1988).
9 arising from the addition of methanol to the C8–O–C4 quinone
methide intermediate 8 (Scheme 2).
The formation of two diastereomeric forms of 9 was previously
reported by Miller (1972) among the dimeric compounds obtained
by 2,4,6-tri-tert-butylphenoxyl radical oxidation of (E)- or (Z)-iso-
eugenol in benzene/methanol solution. Also Chioccara et al.
(1993) reported the formation of a mixture of erythro and threo
diastereomers 9 by addition of methanol to a quinone methide
intermediate formed by C8-O-C4 coupling of two phenoxyl radicals
during the reaction of (E)-isoeugenol with hydrogen peroxide and
HRP in aqueous buffer and methanol from 10% to 90%. The same
authors observed an increase in the yields of these compounds
with increasing methanol content. The addition of methanol to a
quinone methide structure formed by reaction of lignans and DPPH
radical was recently reported by Eklund et al. (2005). To confirm
these structures, the reaction mixture was acetylated (Ac2O, pyri-
dine) and analysed by GC–MS. The analysis evidenced the presence
of four compounds with m/z 400 (M+) and fragment ions at m/z
237, 236, 209, 195, 194, 167 and 164. The Mr and the fragmenta-
tion patterns were in agreement with those reported by Chioccara
et al. (1993) for the acetate derivatives of the methanol adducts of
the C8–O–C4-coupled dimers 9.
3. Results and discussion
3.1. Reaction products of isoeugenol (1)
3.1.1. GC–MS analysis
The GC–MS chromatogram of the reaction mixture of isoeuge-
nol (1) and DPPH radical in methanol evidenced the presence of
two groups of compounds characterised by the Mr 326 and 358,
respectively. The first group was composed of six compounds with
M+ at m/z 326 (Scheme 1), four of which had the same retention
times of those present in the GC–MS chromatogram of the synthes-
ised dehydrodiisoeugenol (3). In particular, the compound present
in the highest amount (83.0%) had the same RT and mass spectrum
of (E)-( )-trans-dehydrodiisoeugenol (3a). The other two com-
pounds with m/z 326 accounted for only 2.0% of the total chro-
matographic area.
The other group was composed of three compounds character-
ised by the same mass spectrum with M+ at m/z 358. Compounds
with Mr 358, which corresponded to an increase of 32 u with
respect to the Mr 326, can be considered as the methanol adducts
The GC–MS chromatogram of the TMS derivatives of the reac-
tion products of isoeugenol (1) and DPPH radical in methanol is
shown in Fig. 1A. Some compounds were detectable only after

260
R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
MeO
O
O
+MeOH
OMe
OMe
MeO
MeO
OR
O
9, R = H (C8-O-C4; m/z 358; ESI(+): m/z 381 [M+Na]+
and 739 [2M + Na]+; ESI(-): m/z 357 [M-H]-)
9-TMS, R = TMS (m/z 430)
8
+H2O
HO
O
OMe
MeO
OH
13 (C8-O-C4: ESI(+): m/z 367 [M+Na]+ and 711 [2M+Na]+)
Scheme 2.
TMS derivatisation probably due to a better chromatographic
behaviour of the TMS derivatives and, in particular, of the com-
pounds present in very low amount. The GC–MS analysis evi-
denced the presence of four compounds with M+ at m/z 398
corresponding to 326 + TMS, four compounds with M+ at m/z 430
corresponding to m/z 358 + TMS, three compounds with M+ at
m/z 470 corresponding to 326 + 2 ꢀ TMS and three compounds
with M+ at m/z 502 corresponding to 358 + 2 ꢀ TMS. The mixture
of products resulting from the reaction between DPPH radical
and 1 was very complex, also due to the presence of (E)- and
(Z)-isoeugenol, and can be rationalised by considering basically
two groups of compounds characterised by Mr 326 and 358,
respectively. The compounds with Mr 326 (Scheme 1) are dimeric
form of 1 which, after silylation, produced two separate classes
of isomers. The first class had originally one phenolic hydroxy
group and the Mr of 398 corresponds to a mono-TMS derivative.
Among the dimers with one phenolic hydroxy group, the most
abundant was that corresponding to (E)-( )-trans-dehydrodiisoeu-
genol (3a) arising from a C8–C5 coupling process (Scheme 1).
The second class had two phenolic hydroxy groups and the Mr of
470 corresponded to a bis-TMS derivative. The dimers with two phe-
nolic hydroxy groups 5 and 6 were present at a very low amount
compared to 3a and all the other compounds and can be originated
from the C8–C8 and C5–C5 coupling processes, respectively, as
hypothesised in Scheme 1. Sarkanen and Wallis (1973) reported
the formation of compounds arising from the addition of water to
a bisquinone methide system, such as 7 shown in Scheme 1, formed
by a C8–C8 couplingprocess in the case of the enzyme-catalysed oxi-
dation of both (E)- and (Z)-isoeugenol. Although the same authors
did not report the formation of the C8–C8-coupled dimer 5 (Scheme
1), its formation in very low amount by aromatisation of bisquinone
methide 7, before the addition of water, cannot be excluded. Regard-
ing the C5–C5-coupled dehydrodimer 6 (Scheme 1), Sarkanen and
Wallis(1973)did notreport itsformationamongtheoxidation prod-
ucts of both (E)- and (Z)-isoeugenol. On the other hand, Ralph, Qui-
deau, Grabber and Hatfield (1994) and Hatfield, Ralph and Grabber
(1999) reported both the presence of C8–C8 and low amounts of
the C5–C5 dehydrodimers of ferulic acid in plant cell wall extracts.
Like the compounds with Mr 326, also the compounds with Mr
358 produced, after silylation, two classes of isomers. The first class,
which comprehended the methanol adducts of the C8–O–C4-cou-
pled dimers 9, had originally one phenolic hydroxy group and the
Mr of 430 (326 + 32 + 72) corresponded to the mono-TMS deriva-
tives 9-TMS (Scheme 2).
(x1,000,000)
TIC
4.0
3.0
2.0
1.0
430
A
398
502
398
430
502
470
398
502
470
430
398
470
430
19.0
19.5
20.0
20.5
21.0
min
(x1,000,000)
TIC
470
B
5.0
4.0
3.0
2.0
1.0
P
470
500
500
470
500
398
17.0
18.0
19.0
20.0
21.0 min
Fig. 1. GC–MS chromatograms of the dimer regions of the silylated reaction
mixtures of isoeugenol (1) and eugenol (2) with DPPH radical in methanol. The
number above the peaks represents the m/z ratio of the molecular ion of the
corresponding compound. (A) isoeugenol – DPPH radical (for m/z 398 and 470 refer
to Scheme 1, for m/z 430 refer to Scheme 2, for m/z 502 refer to Scheme 3); (B)
eugenol – DPPH radical (for m/z 470 and 398 refer to Schemes 4 and 5, for m/z 500
refer to Scheme 6); P = phthalate contaminant.
The second class, present in very low amount, had two phenolic
hydroxy groups and the Mr of 502 (326 + 32 + 2 ꢀ 72) corre-
sponded to a bis-TMS derivative. The addition of methanol can take
place only to the quinone methide intermediate 7 formed by the
C8–C8 coupling process yielding 10 (Scheme 3). This consideration

R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
261
OMe
O
OR
MeO
O
MeO
RO
+MeOH
OMe
OMe
7
10, R = H (C8-C8; m/z 358; ESI(+): m/z 381 [M+Na]+
and 739 [2M + Na]+; ESI(-): m/z 357 [M-H]-)
10-TMS, R = TMS (m/z 502)
+H2O
OH
MeO
HO
OMe
OH
OH
MeO
HO
+
O
OMe
11 (C8-C8: ESI(+): m/z 367 [M+Na]+
12 (C8-C8: ESI(+): m/z 367 [M+Na]+
and 711 [2M+Na]+)
and 711 [2M+Na]+)
Scheme 3.
suggested that the dimers with two hydroxy groups were probably
of the C8–C8 type (Scheme 1). The necessity of the presence of
methanol to produce the adducts was demonstrated by carrying
out the reaction in acetonitrile. In this case neither compounds
with Mr 358, due to methanol addition, nor compounds with Mr
430 and 502 after silylation were observed.
obtained by GC–MS. The reactions carried out in acetonitrile were
visibly slower than those carried out in methanol.
Neither termination products by coupling of 1 and DPPH
radicals nor compounds arising by the abstraction of two
hydrogen atoms from 1 were detected under our experimental
conditions.
Besides the dimers, other peaks corresponding to unreacted iso-
eugenol (1) and DPPH radical thermodegradation products were
present in the GC–MS chromatogram, in particular diphenylamine
and 2,4,6-trinitroaniline, which were the most abundant com-
pounds in the reaction mixture as identified on the basis of the
mass spectrum and comparison with the NIST library.
From the data obtained it can be concluded that the main prod-
ucts formed by reaction of isoeugenol (1) and DPPH radical in
methanol were the (E)-( )-trans-dehydrodiisoeugenol (3a) and
the methanol adducts 9.
3.2. Reaction products of eugenol (2)
3.1.2. HPLC–MS analysis
3.2.1. GC–MS analysis
Due to the possible risk of artifacts formation in the hot injection
port of the gas chromatograph, the reaction mixture between
isoeugenol (1) and DPPH radical was analysed also by HPLC–MS.
The HPLC–MS analysis evidenced the presence of three compounds
with ESI (+)-MS at m/z 327 [M + H]+, 349 [M + Na]+ and 675
[2 M + Na]+ andESI(ꢁ)-MSat m/z325[M ꢁ H]ꢁ withthesamereten-
tion timesand fragmentationpatternsof thesynthesised dehydrodi-
isoeugenol (3). Moreover, three compounds with ESI (+)-MS at m/z
381 [326 + 32 + Na]+ and 739 [2 ꢀ (356 + 32) + Na]+ and ESI (ꢁ)-
MS at m/z 357 [326 + 32 ꢁ H]ꢁ which corresponded to the methanol
adducts 9 and/or 10 were present (Schemes 2 and 3). These results
confirmed the previous findings obtained by GC–MS apart from
the number of isomers detected which can depend by the different
resolving powers of the two chromatographic techniques. HPLC–
MS analysis showed six other compounds with m/z at 367
[326 + 18 + Na]+ and 711 [2 ꢀ (326 + 18) + Na]+ which probably cor-
responded to the addition of water to the quinone methides 7 and 8
with the formation of 11 and 12 (Scheme 3) and 13 (Scheme 2),
respectively.
The formation of 11 (Sarkanen and Wallis, 1973) and 13 (Sarka-
nen and Wallis, 1973; Shiba et al., 2000) was previously reported in
the case of the enzyme-catalysed oxidation of 1 moreover, like the
addition of methanol leading to 10, the addition of water to 7 could
afford 12 (Scheme 3). The formation of water adducts could be due
to the dilution of the methanolic reaction mixture with water to a
final concentration of about 35% like the mobile phase prior to
HPLC–MS analysis.
The GC–MS chromatogram of the TMS derivatives of the reac-
tion products of eugenol (2) and DPPH radical in methanol is re-
ported in Fig. 1B. The GC–MS analysis evidenced the presence of
three peaks with Mr 470, one peak with Mr 398 and three peaks
with Mr 500. The compounds with Mr 470 (326 + 2 ꢀ TMS) corre-
sponded to the bis-TMS derivatives of eugenol dimers and the
most abundant had the same retention time and mass spectrum
of the TMS derivative of the dehydrodieugenol (4) synthesised, de-
rived from a C5–C5 coupling process (Scheme 4). The other two
compounds, which accounted for only 0.2% of the chromatographic
area, could arise from the addition of eugenol-5-radical 14 at the 7
and 9 position of the quinone methide intermediate 15 affording
16 and 17, respectively, as hypothesised in Scheme 5.
The compound with Mr 398 (326 + TMS) could be interpreted as
mono-TMS derivative of eugenol dimer with only one hydroxy
phenolic group, a C5–O–C4 coupling process could be the mecha-
nism leading to compound 18 (Scheme 5). The formation of a
compound formed by a C5–O–C4 coupling process was reported
by Sy and Brown (1998) in the case of the oxidative coupling of
4-allylphenol.
The compounds with Mr 500 (326 ꢁ 2 + 32 + 2 ꢀ TMS), present
in very low amount, could have structures 19 and 20, correspond-
ing to the bis-TMS derivatives of the addition products of methanol
to the quinone methide 21, as hypothesised in Scheme 6. As in the
case of isoeugenol (1), the necessity of the presence of methanol to
produce these adducts was demonstrated by carrying out the reac-
tion in acetonitrile, in this case no compounds with Mr 500 were
observed.
The HPLC–MS analysis of the reaction mixture carried out in
acetonitrile and diluted with water, before analysis, evidenced
the formation of the dimers of 1 and the water adducts, but not
the formation of the methanol adducts, confirming the results
Unreacted eugenol (2) and DPPH radical thermodegradation
products were also present in the GC–MS analysis of the reaction
mixture.

262
R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
.
O
O
OH
4
O
MeO
MeO
MeO
MeO
3
.
5
+DPPH·
-DPPH-H
2
8
6
.
1
7
9
2
OR
OR
O
O
4'
4
MeO
OMe
MeO
OMe
3
5
5'
3'
2'
6'
6
2
8
1
7
1'
7'
8'
9
9'
4, R = H (C5-C5; m/z = 326; ESI(+): m/z 327 [M+H]+,
349 [M+Na]+ and 675 [2M+Na]+; ESI(-): m/z 325 [M-H]-)
4-TMS, R = TMS (m/z = 470)
Scheme 4.
3.2.2. HPLC–MS analysis
radical by extending the delocalisation and lowers the Bond Dissoci-
The HPLC–MS analysis of the reaction mixture of eugenol (2)
and DPPH radical evidenced the presence of a compound with
ESI (+)-MS at m/z 327 [M + H]+, 349 [M + Na]+ and 675
[2 M + Na]+ and ESI (ꢁ)-MS at m/z 325 [M ꢁ H]ꢁ corresponding,
as retention time and mass spectrum, to the synthesised dehydro-
dieugenol (4). Moreover, two compounds with m/z 379
[326 ꢁ 2 + 32 + Na]+ and 355 [326 ꢁ 2 + 32 ꢁ H]ꢁ which could cor-
respond to 19 and 20 (Scheme 6) were present. These results con-
firmed the previous findings obtained by GC–MS.
As in the case of isoeugenol (1), the reactions carried out in ace-
tonitrile were visibly slower than those carried out in methanol,
and neither termination products by coupling of 2 and DPPH rad-
icals nor compounds arising by the abstraction of two hydrogen
atoms from 2 were detected under our experimental conditions.
ation Enthalpy (BDE) of the phenolic O–H bond (Barclay, Xi, & Norris,
1997; Wrigth, 2002; Wrigth, Johnson, & Di Labio, 2001). This struc-
turewas lostintheformationof 3 andtheeventuallystabilisingelec-
tron-donating (+I) effect of the aliphatic part of new heterocyclic
moiety was moreover impaired by the ꢁI effect of the oxygen linked
to the AC7 in 3 (Scheme 1).
The radical scavenging activity of the methanol adducts 9 was
not determined, due to the lack of the pure compounds; however,
on the basis of their structure it was sensible to hypothesise an
antioxidant activity similar to that of 3.
Sanchez-Moreno et al. (1998) outlined the presence of com-
pounds with comparable EC50 but with very different kinetic
behaviour. These authors introduced the parameter AE that takes
into account both the radical scavenging activity (EC50) and the ki-
netic behaviour (TEC ). On the basis of the AE values, isoeugenol (1)
50
3.3. DPPH radical scavenging activity of dehydrodiisoeugenol (3) and
dehydrodieugenol (4)
resulted about 90 times more active than its corresponding dimer
dehydrodiisoeugenol (3) (Table 1).
Dehydrodieugenol (4) had an EC50 value lower than that of
eugenol (2) and one mole can scavenge about 2.7 mol of DPPH,
The DPPH radical scavenging activity of 3 and 4 was determined
in order to evaluate their possible contribution to the activity of
the corresponding parent compounds, isoeugenol (1) and eugenol
(2), respectively. Only these two dimeric species have been consid-
ered from the point of view of the antioxidant activity being the
most abundant among the products originated from the reactions
whereas 2 scavenged 2 mol of DPPH radical. Also the TEC value
50
was lower and, on the basis of the AE parameter, the antioxidant
activity of 4 resulted about twice that of 2 (Table 1). The higher
activity of 4 with respect to 2 can be explained by the presence
of two phenolic OH groups and an ortho benzene ring which
allowed an extensive conjugation stabilising the phenoxyl radical
of 4.
of 1 and 2 with the DPPH radical. The results expressed as EC50
,
stoichiometry (n), TEC and AE are reported in Table 1 together
50
with the radical scavenging activity of 1 and 2 previously deter-
mined by Bortolomeazzi et al. (2007) under similar conditions of
analysis.
The EC50 of dehydrodiisoeugenol (3) was slightly higher than that
of isoeugenol (1) and one mole of both compounds roughly scav-
enged one mole of DPPH, with a stoichiometry value of 0.7 and 0.9,
respectively, corresponding to the number of phenolic OH present
in the molecular structure. On the contrary, their kinetic behaviour
The TEC values of the compounds analysed deserve some more
50
comments. The TEC of isoeugenol (1) was very low in comparison
50
to those of dehydrodiisoeugenol (3) and eugenol (2) attributing
this effect to the lower BDE of the phenolic OH bond in 1 as a con-
sequence of the extended conjugation. A similar consideration has
been applied also to 4 although, in this case, the substituent is
attached to the ortho position, relative to the two phenolic OH.
Wrigth et al. (2001) reported that a vinyl group in the ortho posi-
tion relative to phenol showed a stabilising electronic effect only
slightly lower than in the para position. On this basis, the kinetic
behaviour of dehydrodieugenol (4) could be expected to be more
was completely different with
3 acting much more slowly
(TEC ¼ 201 min) than 1 (TEC ¼ 3:1 min). The very rapid kinetic of
50
50
1 has been explained by the presence, at the para position relative
to phenol, of a propenyl group with the double bond conjugated to
the benzene ring. This allows a better stabilisation of the phenoxyl
rapid with a TEC more similar to that of isoeugenol (1). The slow
50
reaction between 4 and DPPH radical can, however, be explained

R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
263
.
OH
O
O
O
O
MeO
MeO
MeO
MeO
MeO
.
+DPPH·
-DPPH-H
+DPPH·
-DPPH-
.
15
2
13
14
OR
.
.
O
O
MeO
MeO
MeO
+DPPH-H
-DPPH·
14 + 15
OR
O
OH
OMe
OMe
OMe
16, R = H (C5-C7; m/z 326)
16-TMS, R = TMS (m/z = 470)
.
.
O
OR
O
MeO
MeO
MeO
+DPPH-H
-DPPH·
OR
O
OH
OMe
OMe
OMe
17, R = H (C5-C9; m/z 326)
17-TMS, R = TMS (m/z = 470)
OR
OMe
MeO
O
13 + 14
18, R = H (C5-O-C4; m/z 326)
18-TMS, R = TMS (m/z = 398)
Scheme 5.
by the intervention of steric hindrance which can interfere with
the planar conformation of 4 necessary to achieve optimal stabili-
sation of the phenoxyl radical by resonance as hypothesised by
Barclay et al. (1997) in the case of 2,20-dihydroxy-3,30-dime-
thoxy-5,50-dimethoxymethylbiphenyl.
hydrogen atom to DPPH radical, dimerisation by coupling of two
antioxidant radicals and coupling of antioxidant and DPPH radi-
cals. In particular, for 2, these authors suggested a dimerisation
mechanism, while in the case of 1 none of these reactions would
take place.
Brand-Williams et al. (1995) and Bondet et al. (1997) reported
that in the case of compounds reacting very quickly, the number
of DPPH molecules reduced equals the number of hydroxyl groups
present in the antioxidant molecule (n = 1). On the contrary, in the
case of slow reacting compounds the stoichiometric value is, in
general, larger than one (n > 1). This aspect was evidenced by the
behaviour of isoeugenol (1) and eugenol (2) which had a kinetic
fast (0.5 min) and slow (120 min) and n = 1.1 and 1.9, respectively.
These authors explained the finding of stoichiometric values larger
than one by hypothesising three reaction pathways after the initial
formation of the antioxidant radical, such as donation of a second
The results obtained in our work evidenced that, contrary to the
hypothesis of Brand-Williams et al. (1995) and Bondet et al.
(1997), the phenoxyl radical of isoeugenol (1) underwent exten-
sive dimerisation and, moreover, the solvent methanol was in-
volved in the reaction mechanism. The 0.9 stoichiometric value
of 1 (Table 1) can, however, be explained by the very low kinetic
behaviour of dehydrodiisoeugenol (3) with respect to 1. When
the DPPH method was applied to 1, the time of the analysis was
too short for the formed 3, and eventually the methanol adducts
9, to reveal their contribution to the total radical scavenging activ-
ity of 1.

264
R. Bortolomeazzi et al. / Food Chemistry 118 (2010) 256–265
.
O
OH
OH
OH
O
OH
MeO
OMe
+DPPH·
MeO
OMe
MeO
OMe
.
-DPPH-H
4
+DPPH·
-DPPH-H
OR
OR
OR
OR
O
OH
MeO
OMe
MeO
MeO
OMe
MeO
+MeOH
OMe
+
OMe
20, R = H (C5-C5; m/z 356)
20-TMS, R = TMS (m/z 500)
19, R = H (C5-C5; m/z 356)
19-TMS, R = TMS (m/z 500)
21
Scheme 6.
Barbosa-Filho, J. M., Leitão da-Cunha, E. V., & Sobral da Silva, M. (1998). Complete
assignment of the 1H and 13C NMR spectra of some lignoids from lauraceae.
Magnetic Resonance in Chemistry, 36, 929–935.
Table 1
Effective Concentration (EC50), stoichiometry (n), TEC and antiradical efficiency (AE)
50
Barclay, L. R. C., Xi, F., & Norris, J. Q. (1997). Antioxidant properties of phenolic lignin
model compounds. Journal of Wood Chemistry and Technology, 17, 73–90.
Benzie, I. F. F., & Strain, J. J. (1996). The Ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power”: The FRAP assay. Analytical Biochemistry, 239,
70–76.
Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and mechanisms of
antioxidant activity using the DPPH free radical method. Lebensmittel-
Wissenschaft und-Technologie, 30, 609–615.
obtained with the DPPH assay. (The values are the mean of three determina-
tions standard deviation).
a
c
Compound
EC50
nb
TEC
50
AE ꢀ 103d
Isoeugenol (1)
Dehydrodiisoeugenol (3)
Eugenol (2)
0.54e 0.04
0.74 0.02
0.26e 0.01
0.184 0.002
0.93 0.07
0.67 0.02
1.94 0.07
2.72 0.03
3.12 0.06
592 45e
6.7 0.3
31 2e
201
126
85
3
8
1
Dehydrodieugenol (4)
64
1
Bortolomeazzi, R., Sebastianutto, N., Toniolo, R.,
& Pizzariello, A. (2007).
Comparative evaluation of the antioxidant capacity of smoke flavouring
phenols by crocin bleaching inhibition, DPPH radical scavenging and
oxidation potential. Food Chemistry, 100, 1481–1489.
a
Mol AH/mol DPPH.
Mol DPPH/mol AH.
Min.
b
c
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method
to evaluate antioxidant activity. Lebensmittel-Wissenschaft und-Technologie, 28,
25–30.
d
Mol DPPH/(mol AH ꢀ t); AH, phenolic antioxidant.
e
Bortolomeazzi et al. (2007).
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In the case of eugenol (2), the formation of dehydrodieugenol
(4), as the main reaction product, confirmed the hypothesis of
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the dimerisation of 2. The 1.9 stoichiometry value of 2 (Table 1)
can then be explained by the formation of the dimer and its reac-
tion with DPPH radical. In fact, considering the TEC of 2 and 4, the
50
time window of the reaction between DPPH radical and 2 can over-
lap with that of the reaction between DPPH radical and 4 which
can then contribute to the radical scavenging activity of eugenol
(2).
The formation of a complex mixture of dimeric compounds
originated by different coupling processes and finding that also
the solvent, methanol, and eventually water can be involved, evi-
denced that the reaction mechanism can be more complex than
previously hypothesised. The possible contribution of the reaction
products to the radical scavenging activity of a compound limits
the use of the DPPH method to evaluate structure–activity rela-
tionship and the possibility to correlate the results with the antiox-
idant activity obtained by other methods.
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