Ferrocenyl-contained dendritic-like antioxidants with dihydropyrazole and pyrazole as the core: Investigations into the role of ferrocenyl group and structure-activity relationship on scavenging radical and protecting DNA

Article abstract of DOI:10.1016/j.tet.2013.08.053

Eight ferrocenyl and three corresponding phenyl dendritic-like antioxidants with dihydropyrazole or pyrazole as the core were synthesized and their antioxidant abilities to scavenge 2,2′-azinobis(3-ethylbenzothiazoline-6- sulfonate) cationic radical (ABTS⁺) and to protect DNA against 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation were evaluated. It was found that the antioxidant abilities of almost all of the ferrocenyl dendritic-like antioxidants are greater than those of the corresponding phenyl dendritic-like antioxidants remarkably. Moreover, the structure-activity relationships of all these dendritic-like antioxidants were investigated in detail. The quantitative contributions of ferrocenyl group, hydroxyl group, and dihydro-structure in the heterocyclic core to the values of n, n_app and n+n_app in scavenging ABTS⁺ and the values of n in protecting DNA against AAPH-induced oxidation were also calculated.

Full text of DOI:10.1016/j.tet.2013.08.053

Tetrahedron 69 (2013) 9898e9905
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Ferrocenyl-contained dendritic-like antioxidants with
dihydropyrazole and pyrazole as the core: investigations into the
role of ferrocenyl group and structureeactivity relationship on
scavenging radical and protecting DNA
*
Pei-Ze Li, Zai-Qun Liu
Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road, Changchun 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2013
Received in revised form 1 August 2013
Accepted 20 August 2013
Available online 27 August 2013
Eight ferrocenyl and three corresponding phenyl dendritic-like antioxidants with dihydropyrazole or
pyrazole as the core were synthesized and their antioxidant abilities to scavenge 2,20-azinobis(3-
ꢀ
ethylbenzothiazoline-6-sulfonate) cationic radical (ABTSþ ) and to protect DNA against 2,20-azobis(2-
amidinopropane hydrochloride) (AAPH)-induced oxidation were evaluated. It was found that the anti-
oxidant abilities of almost all of the ferrocenyl dendritic-like antioxidants are greater than those of the
corresponding phenyl dendritic-like antioxidants remarkably. Moreover, the structureeactivity re-
lationships of all these dendritic-like antioxidants were investigated in detail. The quantitative contri-
butions of ferrocenyl group, hydroxyl group, and dihydro-structure in the heterocyclic core to the values
Keywords:
Dendritic-like antioxidant
Ferrocene
Free radical
Oxidation of DNA
ꢀ
of n, napp and nþnapp in scavenging ABTSþ and the values of n in protecting DNA against AAPH-induced
oxidation were also calculated.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
structures with pyrazole as the core were investigated freshly.12
Nevertheless, these pyrazoles do not possess rigid dendritic struc-
It is proved that the oxidation of lipids, DNA, membranes, and
proteins caused by reactive oxygen species (ROS) incorporating free
radicals in vivo is associated with a variety of chronic health
problems, such as cancers, atherosclerosis,1 neurodegenerative
processes like Alzheimer’s and Parkinson’s diseases.2 Free radicals
are single-electron species produced in metabolism3 or absorbed
from environment,4 which can attack many kinds of cells and tis-
sues.5 Therefore, supplementation with antioxidants becomes
a therapeutic strategy for some diseases6e8 as the antioxidants can
provide hydrogen atoms or electrons to neutralize the single elec-
tron.9 For this reason, exploring effective antioxidants is of impor-
tance in medicine and chemistry. Dendrimers are widely used in
drug design because of their special affinities toward DNA, mem-
branes, and other biological species.10 In recent years, some anti-
oxidants with analogous structures called dendritic antioxidant
have also been synthesized and their abilities to protect DNA
against 2,20-azobis(2-amidinopropane hydrochloride) (AAPH)- and
Cu2þ-induced oxidation were found greater than those of some
polyphenols.11 As a promising sort of dendritic antioxidants,
tures, which is suitable to be called dendritic-like antioxidants and
their antioxidant abilities are still lower than that of our
expectation.
Ferrocene, as an effective phenyl bioisostere, is becoming an
applicable platform for drug design by virtue of its redox proper-
ties, high liphophilicity and three-dimensional metallocene unit,
which may lead to some changes in selectivity toward biological
targets compared with a phenyl or alkyl group.13e15 A mass of drugs
have possessed higher activities by introducing ferrocene moiety
into molecules, such as antitumor drugs,16 steroidal drugs,17 anti-
tubercular drugs,18 etc. The same strategy was also used to develop
antioxidants and the results were satisfactory.19e21
Therefore, such useful strategy of introducing ferrocene moiety
into molecules was also used in this work to design eight ferrocenyl
and three corresponding phenyl dendritic-like antioxidants with
dihydropyrazole or pyrazole as the core. The aim of this work is
trying to improve the antioxidant abilities of dendritic-like antiox-
idants with pyrazole as the core aforementioned in literature12 and
investigate the structureeactivity relationship, quantitative contri-
butions of ferrocenyl group, dihydro-structure in the heterocyclic
core, and hydroxyl group to the antioxidant activities of such species
of dendritic-like antioxidants in detail. The synthetic route and the
names of these dendritic-like antioxidants are shown in Scheme 1.
* Corresponding author. Tel.: þ86 431 88499174; fax: þ86 431 88499159; e-mail
address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
0040-4020/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.08.053

P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
9899
Ar2
Ar2
4
5
4
5
a
b
O
3
1
3
1
N
N
Ar1
Ar2
Ar1
Ar1
N
N
2
2
Chalcone
DH
AR
Chalcone DH AR
Ar1
Ar2
Reaction Conditions:
a) phNHNH2,
CH3COOH-EtOH-
H2O, reflux.
b) The datas of 1AR
to 3AR are used
directly accroding
to literatue12, so
they were not
1DH 1AR
2DH 2AR
1
2
3
HO
3DH 3AR
4DH 4AR
HO
4
5
6
7
Fe
synthesized in this
work; MnO2,
5DH 5AR
6DH 6AR
7DH 7AR
Fe
toluene, reflux for
4AR and 5AR;
patial spontaneous
oxidation during
the reaction for
6AR and 7AR.
Fe
HO
Fe
HO
Scheme 1. The synthetic route and the structures of the dendritic-like antioxidants with dihydropyrazole and pyrazole as the core.
Their antioxidant abilities were evaluated via scavenging 2,20-
azinobis(3-ethylbenzothiazoline-6-sulfonate)
cationic
radical
ꢀ
(ABTSþ ), which is an oxidant-type radical commonly used to test
the redox capacities of antioxidants,22 and protecting DNA against
AAPH-induced oxidation, which is commonly used to mimic the
oxidation of DNA caused by peroxyl radicals (ROOꢀ) in vivo.23 The
ꢀ
structures of ABTSþ and AAPH are shown in Scheme 2.
O
O
S
O
N
S
O
O
S
N
S
O
N
N
ꢀ
Fig. 1. The decrease in the absorbance of ABTSþ (734 nm) in the presence of 10.0
mM
dihydropyrazole and pyrazole derivatives.
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)
ꢀ
The decay of the absorbance of ABTSþ in the presence of an
antioxidant was studied by means of chemical kinetics expressed
by two equations eventually.25 The difference between the con-
NH
N
NH2
NH
2HCl
H2N
N
ꢀ
ꢀ
centration of ABTSþ at the beginning ([ABTSþ ]0) and the end
ꢀ
([ABTSþ
]
N) of the reaction is divided by the concentration of the
2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)
ꢀ
antioxidant to express the number of ABTSþ (n) trapped by the
ꢀ
Scheme 2. Structures of AAPH and ABTSþ used in this work.
antioxidant in the primary period, as shown in Eq. 1.
ꢀ
ꢀ
½ABTSþ ꢁ0 ꢂ ½ABTSþ ꢁN
½antioxidantꢁ0
n ¼
(1)
2. Results
ꢀ
2.1. Radical-scavenging ability
If the oxidized product of the antioxidant can also trap ABTSþ
,
ꢀ
the reaction between the antioxidant and ABTSþ will undergo the
secondary stage. Thus, the decay of the concentration of ABTSþꢀ can
be expressed by the Eq. 2, which shows the relationship between
The radical-scavenging ability is commonly regarded as the
basic property of an antioxidant24 and evaluated by trapping
ꢀ
ꢀ
ꢀ
ABTSþ in this work. As shown in Fig. 1, the absorbance of ABTSþ
[ABTSþ ] and the corresponding reaction time (t). In this equation,
decreases in the presence of dendritic-like antioxidants.
a¼napp[antioxidant]0 and b¼1/(kapp[antioxidant]0).

9900
P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
Table 2
ꢀ
ꢀ
ab
b þ t
½ABTSþ ꢁ ¼ ½ABTSþ ꢁN
þ
(2)
The contributions of ferrocenyl group to the incremental values of n, napp, and
nþnapp in scavenging ABTSþꢀ radical and n in protecting DNA against AAPH-induced
oxidation
Apparently, if the values of a and b are ciphered out, the values
of napp and kapp will be obtained easily. Therefore, in order to cal-
culate the values of a and b practically, the Eq. 2 should be changed
to Eq. 3, in which the intercept (1/a) and the slope (1/ab) can be
obtained by linear regression analysis in Origin 6.0 Professional
software.
a
b
Antioxidant
D
n
D
napp
D
(nþnapp
)
DnDNA
4DH
5DH
6DH
7DH
4ARc
5AR
6AR
7AR
1.42ꢃ0.07
3.10ꢃ0.16
0.83ꢃ0.04
3.03ꢃ0.15
2.25ꢃ0.11
d
6.13ꢃ0.31
2.22ꢃ0.11
0.88ꢃ0.04
14.72ꢃ0.74
d
ꢂ0.56ꢃ0.03
2.46ꢃ0.12
1.04ꢃ0.05
1.19ꢃ0.06
3.00ꢃ0.15
2.67ꢃ0.13
ꢂ0.66ꢃ0.03
1.50ꢃ0.08
0.57ꢃ0.03
0.08ꢃ0.00
0.61ꢃ0.03
1.72ꢃ0.09
ꢂ1.22ꢃ0.06
3.96ꢃ0.20
1.61ꢃ0.08
1.27ꢃ0.06
3.61ꢃ0.18
4.39ꢃ0.22
d
1
1
a
1
ab
0.80ꢃ0.04
7.40ꢃ0.37
¼
þ
t
(3)
ꢀ
ꢀ
½ABTSþ ꢁ ꢂ ½ABTSþ ꢁN
a
ꢀ
The values of
D
n,
D
napp, and
D
(nþnapp) are calculated by the n, napp, and nþnapp
napp refers to the apparent number of ABTSþ trapped by the oxi-
dized products of the antioxidant, while kapp is the apparent rate
constant in this period but it is not our interest because it can’t act
as an index of antioxidant ability. Therefore, nþnapp is the total
values of ferrocenyl dihydropyrazole and pyrazole derivatives minus those of the
corresponding phenyl dihydropyrazole and pyrazole derivatives in scavenging
ꢀ
ABTSþ radical.
b
The values of DnDNA are calculated by the n values of ferrocenyl dihydropyrazole
ꢀ
number of ABTSþ trapped by the antioxidant. However, it should
and pyrazole derivatives minus those of the corresponding phenyl dihydropyrazole
and pyrazole derivatives in protecting DNA.
be noted that the secondary period can’t be completely separated
c
The data of 1AR, 2AR, and 3AR used in calculating
D
n,
D
napp
,
D
(nþnapp), and
nDNA of 4ARe7AR are gotten from the literature.12
from the primary one because the oxidized products generated
D
ꢀ
from the primary period may react with ABTSþ immediately.
Meanwhile, the fresh antioxidant molecules still undergo the pri-
ꢀ
mary reaction with ABTSþ . Therefore, the reaction between the
2.2. Antioxidant effect on AAPH-induced oxidation of DNA
ꢀ
antioxidant and ABTSþ is artificially divided into two periods in
this chemical kinetic model. All of the dendritic-like antioxidants
The guanine bases in DNA can be oxidized by radicals generated
from the decomposition of AAPH.26 In this process, the oxidation of
linear DNA generates more than 20 carbonyl species, which can
react with TBA to form colorful TBARS (lmax¼535 nm).27 Thus, the
oxidation process can be examined by detecting TBARS using
ultravioletevisible spectroscopy. As shown in Fig. 2A, in the blank
experiment, the increase of the absorbance of TBARS indicates that
more carbonyl species will generate as the reaction period in-
creases. The slope of the line can be regarded as the formation rate
of the carbonyl species during the oxidation of DNA. However, if
addition of an antioxidant can bend the increase line of TBARS at
the beginning of the reaction period, that is, to say the formation of
TBARS is lagged for a period, it will imply that the antioxidant
added is able to inhibit the generation of carbonyl species from the
oxidation of DNA.
ꢀ
reacting with ABTSþ were treated with this method in this work.
Moreover, in order to investigate the structureeactivity relation-
ship of all these dendritic-like antioxidants and quantitative con-
tributions of ferrocenyl group, dihydro-structure in the heterocyclic
core, and hydroxyl group to the values of n, napp, and nþnapp in
detail, nDH,n, nDH,app, nDH,t, nOH,n, nOH,app, nOH,t, Dn, Dnapp and
D
(nþnapp) were defined in addition as the contribution of dihydro-
structure, hydroxyl group, and ferrocenyl group, respectively. The
values of nDH,n, nDH,app, and nDH,t can be calculated by the n, napp
,
and nþnapp values of dihydropyrazole derivatives minus those of
the corresponding pyrazole derivatives, respectively, while the
values of nOH,n, nOH,app, and nOH,t can be calculated by the n, napp
,
and nþnapp values of hydroxylpyrazole derivatives minus those of
the corresponding non-hydroxylpyrazole derivatives, respectively.
The values of
D
n,
D
napp, and
D
(nþnapp) can be calculated by the n,
For the antioxidants, which possess such inhibited effect as
some of the dendritic-like antioxidants with dihydropyrazole and
pyrazole as the core used in this work, the time corresponding to
the cross point of two tangents (short dot lines) can be regarded as
the inhibition period (tinh), as shown in Fig. 2A. Furthermore, Fig. 3
napp, and nþnapp values of ferrocenyl dihydropyrazole and pyrazole
derivatives minus those of the corresponding phenyl dihydropyr-
azole and pyrazole derivatives. All these parameters are listed in
Tables 1 and 2.
Table 1
ꢀ
Characteristic parameters of dendritic antioxidants with dihydropyrazole and pyrazole as the core reacting with ABTSþ
na
napp
nþnapp
nDH,n
nDH,app
nDH,t
nOH,n
nOH,app
nOH,t
b
c
Antioxidant
1DH
2DH
3DH
4DH
5DH
6DH
7DH
1AR
2AR
3ARd
4AR
5AR
6AR
7AR
0.62ꢃ0.03
3.85ꢃ0.19
1.73ꢃ0.09
2.04ꢃ0.10
3.72ꢃ0.19
3.29ꢃ0.16
4.19ꢃ0.21
0.25ꢃ0.01
0.28ꢃ0.01
0.25ꢃ0.01
1.29ꢃ0.06
1.44ꢃ0.07
3.28ꢃ0.16
2.92ꢃ0.15
0.58ꢃ0.03
1.32ꢃ0.07
1.82ꢃ0.09
1.05ꢃ0.05
3.25ꢃ0.16
0.66ꢃ0.03
3.32ꢃ0.17
0.22ꢃ0.01
0.12ꢃ0.01
0.16ꢃ0.01
0.79ꢃ0.04
0.30ꢃ0.02
0.73ꢃ0.04
1.88ꢃ0.09
1.20ꢃ0.06
5.17ꢃ0.26
3.55ꢃ0.18
3.09ꢃ0.15
6.97ꢃ0.35
3.95ꢃ0.20
7.51ꢃ0.38
0.47ꢃ0.02
0.40ꢃ0.02
0.41ꢃ0.02
2.08ꢃ0.10
1.74ꢃ0.09
4.01ꢃ0.20
4.80ꢃ0.24
0.37ꢃ0.02
3.57ꢃ0.18
1.48ꢃ0.07
0.75ꢃ0.04
2.28ꢃ0.11
0.01ꢃ0.00
1.27ꢃ0.06
d
0.36ꢃ0.02
0.73ꢃ0.04
d
d
d
1.20ꢃ0.06
4.77ꢃ0.24
3.23ꢃ0.16
1.11ꢃ0.06
d
0.74ꢃ0.04
1.24ꢃ0.06
d
3.97ꢃ0.20
2.35ꢃ0.12
d
1.66ꢃ0.08
3.14ꢃ0.16
0.26ꢃ0.01
1.01ꢃ0.05
2.95ꢃ0.15
5.23ꢃ0.26
d
d
d
ꢂ0.07ꢃ0.00
ꢂ0.06ꢃ0.00
1.25ꢃ0.06
0.47ꢃ0.02
d
ꢂ0.39ꢃ0.02
0.07ꢃ0.00
d
0.86ꢃ0.08
0.54ꢃ0.03
d
1.44ꢃ0.07
2.71ꢃ0.14
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
0.03ꢃ0.00
0.00ꢃ0.00
d
ꢂ0.10ꢃ0.01
ꢂ0.06ꢃ0.00
d
ꢂ0.07ꢃ0.01
ꢂ0.06ꢃ0.00
d
d
d
d
d
d
d
d
1.99ꢃ0.10
1.48ꢃ0.07
ꢂ0.06ꢃ0.00
1.58ꢃ0.08
1.93ꢃ0.10
3.06ꢃ0.15
d
a
The values of n are calculated by Eq. 1.
The values of napp are calculated by Eq. 3.
b
c
The values of nDH,n, nDH,app, and nDH,t are calculated by the n, napp, and nþnapp values of dihydropyrazole derivatives minus those of the corresponding pyrazole derivatives,
respectively, while the values of nOH,n, nOH,app, and nOH,t are calculated by the n, napp, and nþnapp values of hydroxylpyrazole derivatives minus those of the corresponding non-
hydroxylpyrazole derivatives, respectively.
d
All of the values of n, napp, and nþnapp of 1AR, 2AR, and 3AR are gotten from the literature.12

P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
9901
A
C
B
Fig. 2. (A) The range of the absorbance of TBARS in the mixture of DNA (2.0 mg/ml) and 40 mM AAPH with various concentrations of dihydropyrazole and pyrazole derivatives
added, and (B) the incremental values of the absorbance of TBARS with 4AR and 5AR (300
concentrations of 4DH added.
mM) added, and (C) the incremental values of the absorbance of TBARS with various
illustrates that tinh also changes with the concentrations of anti-
oxidant employed. The quantitative equations between tinh and the
concentrations of these dendritic-like antioxidants can be obtained
by linear regression analysis and listed in Table 3.
Chemical kinetics demonstrates that tinh generated by an anti-
oxidant (AH) is linearly related to its concentration as expressed as
equation tinh¼(n/Ri) [AH].28 Ri is the initiation rate of a radical-
induced reaction. The stoichiometric factor (n) implies the number
of radicals trapped by one molecule of the antioxidant, and can be
used as a quantitative index to express the antioxidant ability. This
equation has been applied to treat the protective effects of some
antioxidants on AAPH-induced oxidation of DNA.12,19,29 Ri is as-
sumed to be equal to the rate of the radical generated from the
decomposition of AAPH (Rg¼(1.4ꢃ0.2)ꢄ10ꢂ6 [AAPH] s
)
ꢂ1 30 because
AAPH and DNA are both dissolved in PBS0, and
Ri¼Rg¼1.4ꢄ10ꢂ6ꢄ40 mM sꢂ1¼3.36
m
M minꢂ1 when 40 mM AAPH
is employed.29 The n values of these dendritic-like antioxidants
Fig. 3. The relationships between tinh and the concentrations of dihydropyrazole and
pyrazole derivatives in AAPH-induced oxidation of DNA.
were calculated by the equation mentioned above and the results

9902
P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
Table 3
The equations of tinhw[dendritic antioxidants with dihydropyrazole and pyrazole as the core] and n, nDH, and nOH of dendritic antioxidants with dihydropyrazole and pyrazole
as the core in protecting DNA against AAPH-induced oxidation
tinh(min)¼(n/Ri)a [AH(
d
m
M)]þconstantb
n
nDH
nOH
c
Antioxidant
1DH
2DH
3DH
4DH
5DH
6DH
7DH
1AR
2AR
3ARd
4AR
5AR
6AR
7AR
d
0.00ꢃ0.00
d
tinh¼0.70 (ꢃ0.04) [2DH]ꢂ49.4 (ꢃ2.5)
2.35ꢃ0.12
d
0.64ꢃ0.03
2.35ꢃ0.12
0.00ꢃ0.00
d
d
ꢂ1.81ꢃ0.09
d
d
0.00ꢃ0.00
tinh¼0.66 (ꢃ0.03) [5DH]ꢂ28.0 (ꢃ1.4)
tinh¼0.69 (ꢃ0.03) [6DH]þ76.2 (ꢃ3.8)
tinh¼4.40 (ꢃ0.22) [7DH]ꢂ41.6 (ꢃ2.1)
d
2.22ꢃ0.11
3.23ꢃ0.16
14.72ꢃ0.74
d
2.22ꢃ0.11
d
0.72ꢃ0.04
3.23ꢃ0.16
12.50ꢃ0.63
d
5.51ꢃ0.28
d
d
d
d
d
d
d
tinh¼0.51 (ꢃ0.03) [2AR]þ1.4 (ꢃ0.1)
tinh¼0.54 (ꢃ0.03) [3AR]þ9.4 (ꢃ0.3)
d
1.71ꢃ0.09
1.81ꢃ0.09
d
1.71ꢃ0.09
1.81ꢃ0.09
d
d
d
d
tinh¼0.75 (ꢃ0.04) [6AR]ꢂ17.2 (ꢃ0.9)
2.51ꢃ0.13
2.51ꢃ0.13
9.21ꢃ0.46
tinh¼2.74 (ꢃ0.14) [7AR]þ59.2 (ꢃ3.0)
9.21ꢃ0.46
a
Ri¼Rg¼1.4ꢄ10ꢂ6 [AAPH] sꢂ1¼3.36
m
M minꢂ1 when [AAPH]¼40 mM in protecting DNA, thus, n¼coefficientꢄ3.36
m .
M minꢂ1
b
c
The constant was generated from the linear regression analysis.
The values of nDH are calculated by the n values of dihydropyrazole derivatives minus those of the corresponding pyrazole derivatives, while the values of nOH are cal-
culated by the n values of hydroxylpyrazole derivatives minus those of the corresponding non-hydroxylpyrazole derivatives.
d
The n values of 1AR, 2AR, and 3AR are gotten from the literature.12
are also listed in Table 2. Moreover, in order to investigate the
structureeactivity relationship of these dendritic-like antioxidants
and quantitative contributions of ferrocenyl group, dihydro-
structure in the heterocyclic core, and hydroxyl group to the
replacement of phenyl by ferrocenyl can improve the antioxidant
abilities of this kind of dendritic-like antioxidants remarkably, ex-
cept the same change in 6DH exhibiting a negative effect. Such
positive result may be due to the higher electron cloud density of
p65 in ferrocenyl group than that of p66 in phenyl group. Another
reason may be the ferrocenyl ring in these dihydropyrazole and
pyrazole derivatives can form a conjugation system with the
dihydropyrazole or pyrazole ring more completely than phenyl due
to its special three-dimensional metallocene unit. Thus, the anti-
oxidant radicals generated by reacting with ABTS radical may be
stabilized by the whole conjugation system. Nevertheless, such
promotive effect of ferrocenyl group does not operate in-
dependently. Compared 6AR and 7AR with 4AR or compared 4DH
and 5DH with 5AR, it can be found that only hydroxyl group or
dihydro-structure one exists with ferrocenyl group together in the
molecule can improve the effect of ferrocenyl group. But compared
6AR and 7AR with 6DH or compared 4DH and 5DH with 7DH, the
effect of ferrocenyl group becomes weaker as both hydroxyl group
and dihydro-structure exist in the same time with ferrocenyl group
in the molecule. The same phenomena when observing the effect of
hydroxyl group or dihydro-structure can also be found by seeing
about nOH,t and nDH,t. Such interesting structureeactivity relation-
ship can be concluded in brief that there is a greater contribution of
any one of the three factors (ferrocenyl group, dihydro-structure,
and hydroxyl group) to the antioxidant ability if only one of the
other two factors exists with it in the same time, or else there will
be a weaker contribution. Fig. 4 offers a visual expression.
values of n, as what has been done in radical scavenging, nDH, nOH
,
and nDNA were analogously defined in addition as the contribution
D
of dihydro-structure, hydroxyl group, and ferrocenyl group, re-
spectively. The values of nDH can be calculated by the n values of
dihydropyrazole derivatives minus those of the corresponding
pyrazole derivatives, while the values of nOH can be calculated by
the n values of hydroxylpyrazole derivatives minus those of the
corresponding non-hydroxylpyrazole derivatives. The values of
DnDNA can be calculated by the n values of ferrocenyl dihydropyr-
azole and pyrazole derivatives minus those of the corresponding
phenyl derivatives. Apparently, the strategy to calculate the values
of nDH, nOH, and DnDNA is similar with that in radical scavenging and
the results are listed in Tables 2 and 3.
3. Discussion
3.1. Radical-scavenging ability
As shown in Table 1, the nþnapp values of 1AR, 4AR, and 5AR are
larger than zero, in which no common effective group exists, in-
dicating that the electron pair in the N atom of pyrazole is able to
trap ABTS radicals by donating its electron. Dihydro-structure is an
effective group as all of the nDH,t values are positive except 6DH.
Hydroxyl group is another effective group for the same reason by
seeing about the values of nOH,t similarly, only with the exception of
2AR and 3AR. Compared 1AR with 2AR and 3AR, it can be found
that the nþnapp value of 1AR without hydroxyl group is 0.47, larger
than those of 2AR (0.40) and 3AR (0.41) with hydroxyl group,
showing a special negative effect of hydroxyl group only in 2AR and
3AR. The result of the nOH,t value of 3AR larger than that of 2AR
indicates hydroxyl group connected to the phenyl at 5-position of
the pyrazole ring is advantageous. Moreover, such phenomenon is
prevalent, which can be summarized as the contribution of hy-
droxyl group to the antioxidant ability depends on its position in
the molecule. It is greater when the hydroxyl group is on the phenyl
at 5-position of the pyrazole ring while it is weaker when the hy-
droxyl group is on the phenyl at 3-position of the pyrazole ring.
However, it is opposite for those with dihydropyrazole as the core.
Ferrocenyl
Group
Ferrocenyl
Group
S
S
S
S
H
r
H
r
A
S
HS
-
-
y
y
e
e
o
o
d
r
d
r
G
r
r
G
r
r
u
t
u
t
d
d
o
x
o
x
o
y
o
y
S/A
c
c
u
u
h
i
h
i
y
u
y
u
p
p
r
t
r
l
l
D
D
t
S
S
Scavenge ABTS radical
Protect DNA
S=synergetic relationship ; A=antagonistic relationship ; HS=highly synergetic relationship
Fig. 4. The relationships of ferrocenyl group, hydroxyl group, and dihydro-structure in
It should be noted that nearly all of the
higher than zero, as shown in Table 2, demonstrating that the
D
(nþnapp) values are much
their contributions to the antioxidant abilities of dendritic-like antioxidants in scav-
ꢀ
enging ABTSþ and protecting DNA.

P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
9903
3.2. Antioxidant effect on AAPH-induced oxidation of DNA
expression of the structureeactivity relationship between the three
factors in protecting DNA is also shown in Fig. 4.
As shown in Fig. 2A, the slopes of the absorbance line of 4AR and
5AR are the same as that of the blank, indicating that the N atom in
pyrazole does not possess the inhibited effect in the oxidation of
DNA as the representation of 1AR in the literature,12 which is dif-
ferent from that in ABTS radical scavenging. Introducing ferrocenyl
group is still not able to change the result in this case. The phe-
nomenon that the absorbance of 4AR and 5AR is higher than that of
the blank may be attributed to the impact of their color on absor-
bance, not prooxidant effect. Thus, in order to differentiate no effect
from prooxidant effect, the impact of color should be eliminated.
Observing the incremental values of absorbance related to that at
the beginning of the reaction period instead of the absolute values
of absorbance is an available method and the results of using this
method are shown in Fig. 2B. From Fig. 2B, it is found that the in-
cremental values of 4AR and 5AR are nearly the same as that of the
blank, so it can be confirmed that it has no effect for 4AR and 5AR,
not prooxidant effect. In the presence of 1DH and 4DH, the rates of
oxidation of DNA are lower than that of the blank though the oxi-
dation process is not inhibited completely. As 1DH and 4DH do not
have the hydroxyl group, the results of their antioxidant effect are
due to the dihydro-structure. However, in the presence of 5DH, the
absorbance of TBARS does not increase at the beginning of the re-
action period, indicating that the dihydro-structure in 5DH can
inhibit the oxidation of DNA for a certain period (tinh), and so it
possesses nDH value of 2.22(>0). Other dihydropyrazole derivatives
except 3DH also possess positive nDH values as shown in Table 2,
demonstrating the dihydro-structure is also the effective group in
protecting DNA as that in scavenging ABTS radical. To all appear-
ances, hydroxyl group is alike according to the positive nOH values,
also with the exception of 3DH. Compared 2AR with 3AR first, the n
and nOH values of 3AR are larger than those of 2AR indicating the
hydroxyl group connected to the phenyl at 5-position of the pyr-
azole ring is advantageous. Furthermore, such phenomenon is
prevalent like that in ABTS radical scavenging as the effect of hy-
droxyl group still depends on its position in the molecule. There
will be a greater contribution when it on the phenyl at 5-position of
the heterocyclic core while there will be a weaker contribution at 3-
position, only opposite for 2DH and 3DH. Compound 3DH is
a special case, though it has both dihydro-structure and hydroxyl
group, its activity is very weak and the relationship between the
oxidation rates of DNA and its concentrations is also non-significant
as that of 1DH without hydroxyl group. But compared with 1DH
and 3DH, there is an evident relationship between concentrations
and oxidation rates for 4DH by observing the incremental values of
absorbance as shown in Fig. 2C, exhibiting the promotive effect of
ferrocenyl group. As shown in Table 2, nearly all of the values of
4. Conclusion
In conclusion, N atom in pyrazole is able to react with ABTS
radical but cannot react with peroxyl radical. Both hydroxyl group
and dihydro-structure are effective groups. The effect of hydroxyl
group is depending on its position in the molecule. For most
dendritic-like antioxidants with dihydropyrazole or pyrazole as the
core, they will possess much greater antioxidant abilities both in
protecting DNA and ABTS radical scavenging if phenyl is replaced
by ferrocenyl. This result exhibits the significant role of the ferro-
cenyl group. However, such role of ferrocenyl group is also related
to its position in molecule. In ABTS radical scavenging, the re-
lationship between any two factors of ferrocenyl group, dihydro-
structure, and hydroxyl group is synergetic but it is antagonistic
when all of the three factors exist in the molecule together. In
protecting DNA, the relationship between any two factors is syn-
ergetic only except what between hydroxyl group and dihydro-
structure is dubious. Especially, if all of the three factors exist in
the molecule together, it will give out a highly synergetic result,
which is quite different from that in ABTS radical scavenging.
5. Experimental section
5.1. Materials and instrumentations
ABTS radical was purchased from Fluka Chemie GmbH, Swit-
zerland. AAPH and naked DNA sodium salt were purchased from
Acros Organics, Belgium. Other reagents were of analytical grade
and used without further purification. Structures of ferrocenyl and
phenyl dendritic-like antioxidants with dihydropyrazole or pyr-
azole as the core were identified by 1H and 13C NMR (Varian Mer-
cury 300 NMR spectrometer).
5.2. Synthesis and structural characterization of dendritic-
like antioxidants
All of the ferrocenyl and phenyl dendritic-like antioxidants with
dihydropyrazole or pyrazole as the core were synthesized accord-
ing to the literature31,32 with some modifications as the following
general procedure:
One of chalcones 1e7 (1.0 equiv) and phenylhydrazine
(3.5 equiv) were added to a mixture of ethanol/water/acetic acid (v/
v/v¼3:1:1). The reaction mixture was refluxed by protecting from
light and monitored by TLC. After the reaction had finished, the
mixture was evaporated to give a dark brown solid or syrup. (A) The
solid was purified by recrystallization in suitable solvent to give
a pure product (1DHe5DH). (B) The syrup was dissolved in ethyl
acetate and washed with saturated brine. The organic phase was
dried with Na2SO4 and evaporated to give oily-solid crude product,
which was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate or benzene as eluant to give a pure
product as oil (6DH and 7DH), which can be crystallized slowly in
petroleum ether/ethyl acetate or methanol to give a crystal.
Compound 4DH or 5DH (1.0 equiv) was dissolved in toluene and
MnO2 (1.5 equiv) was added. The reaction mixture was refluxed for
10 min before the solvent was evaporated under reduced pressure.
The crude product as reddish-brown syrup with residual solid was
purified by column chromatography on silica gel using dichloro-
methane as eluant to give a pure product as oil (4AR or 5AR), which
can be crystallized slowly in ether or methanol to give crystal of
4AR or 5AR. Compounds 6AR and 7AR can be obtained by partial
spontaneous oxidation of 6DH and 7DH during the reactions and
DnDNA are higher than zero, especially it is 14.72 and 7.40 for 7DH
and 7AR. The absolute n values of the two dendritic-like antioxi-
dants are also much higher than those of the traditional antioxi-
dants, such as curcumin 8.40 and feruloylacetone 4.80.19 Such
results indicate that replacement of phenyl by ferrocenyl can also
improve the antioxidant ability of these dendritic-like antioxidants
on protecting DNA remarkably as that in scavenging ABTS radical.
Moreover, those of ferrocenyl group at 3-position will offer stronger
activity than those at 5-position. Like in ABTS radical scavenging,
the promotive effect of ferrocenyl group in protecting DNA is also
not independent but different from that in ABTS radical scavenging.
The situations of the other two factors (hydroxyl group and
dihydro-structure) are alike. In brief, the relationship between any
two factors is synergetic only except what between hydroxyl group
and dihydro-structure is dubious because it is synergetic for 7DH
but antagonistic for 6DH. If all of the three factors exist in the
molecule together, it will be a highly synergetic result, which is
quite different from that in ABTS radical scavenging. The visual

9904
P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
also can be separated from 6DH and 7DH by column chromatog-
raphy at the same time.
C
25H20FeN2 (%): C 74.27, H 4.99, N 6.93, found: C 74.63, H 4.91, N
7.05. 1H NMR (300 MHz, CDCl3)
: 7.346e7.273 (m, 10H), 6.552 (s,
1H), 4.797 (s, 2H), 4.326 (s, 2H), 4.160 (s, 5H). 13C NMR (75 MHz,
CDCl3) : 151.49, 143.66, 140.18, 130.71, 128.88, 128.73, 128.43,
d
5.2.1. 1,3,5-Triphenyl-4,5-dihydro-1H-pyrazole (1DH). Yellow nee-
dles, yield 70%, mp 134e135 ꢅC. Elemental analysis: calcd for
d
128.17, 127.17, 125.30, 105.83, 78.47, 69.69, 68.76, 66.96.
C
21H18N2 (%): C 84.53, H 6.08, N 9.39, found: C 84.86, H 6.09, N 9.53.
1H NMR (300 MHz, CDCl3)
d: 7.74e7.71 (m, 2H), 7.47e7.32 (m, 5H),
5.2.8. 4-(5-Ferrocenyl-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenol
(6DH). Khaki solid, yield 53%, mp 110e111 ꢅC. Elemental analysis:
calcd for C25H22FeN2O (%): C 71.10, H 5.25, N 6.63, found: C 69.99, H
7.27e7.15 (m, 5H), 7.07 (dd, J¼9.0 Hz, J¼1.2 Hz, 2H), 6.84e6.75 (m,
1H), 5.27 (dd, J¼12.3 Hz, J¼7.2 Hz, 1H), 3.84 (dd, J¼17.1 Hz, J¼12.3
Hz, 1H), 3.14 (dd, J¼17.1 Hz, J¼7.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
5.68, N 6.10. 1H NMR (300 MHz, DMSO-d6)
d: 9.780 (s, 1H), 7.687 (d,
d
: 146.65, 144.79, 142.53, 132.67, 129.08, 128.85, 128.53, 128.48,
J¼8.7 Hz, 2H), 7.179e7.062 (m, 4H), 6.866 (d, J¼8.7 Hz, 2H), 6.680 (t,
J¼7.2 Hz,1H), 5.120 (dd, J¼11.1 Hz, J¼5.4 Hz, 1H), 4.417 (s, 1H), 4.206
(s, 5H), 4.146 (s, 1H), 4.096 (s, 1H), 3.850 (dd, J¼17.1 Hz, J¼11.1 Hz,
1H), 3.689 (dd, J¼17.1 Hz, J¼5.4 Hz, 1H). 13C NMR (75 MHz, DMSO-
127.50, 125.81, 125.68, 119.03, 113.32, 64.42, 43.50.
5.2.2. 4-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenol
(2DH). Khaki sheets, yield 40%, mp 132e133 ꢅC. Elemental analysis:
calcd for C21H18N2O (%): C 80.23, H 5.77, N 8.91, found: C 78.18, H
d6) d: 163.44, 153.35, 150.21, 133.82, 132.60, 128.72, 123.27, 120.76,
118.65, 94.82, 74.40, 73.78, 73.04, 72.43, 71.23, 63.54, 46.80.
5.78, N 8.73. 1H NMR (300 MHz, DMSO-d6)
d: 9.79 (s, 1H), 7.58 (d,
J¼8.4 Hz, 2H), 7.36e7.21 (m, 5H), 7.12 (t, J¼8.1 Hz, 2H), 6.95 (d,
J¼8.1 Hz, 2H), 6.81 (d, J¼8.4 Hz, 2H), 6.67 (t, J¼7.5 Hz, 1H), 5.38 (dd,
J¼12.0 Hz, J¼6.3 Hz, 1H), 3.86 (dd, J¼17.4 Hz, J¼12.0 Hz, 1H), 3.03
5.2.9. 4-(5-Ferrocenyl-1-phenyl-1H-pyrazol-3-yl)phenol
(6AR). Orange solid, yield 6%, mp 225e226 ꢅC. Elemental analysis:
calcd for C25H20FeN2O (%): C 71.44, H 4.80, N 6.67, found: C 71.40, H
(dd, J¼17.4 Hz, J¼6.3 Hz, 1H). 13C NMR (75 MHz, DMSO-d6)
d:
4.93, N 6.64. 1H NMR (300 MHz, CDCl3)
d
: 7.771 (d, J¼7.2 Hz, 2H),
7.410 (s, 5H), 6.861 (d, J¼7.8 Hz, 2H), 6.727 (s, 1H), 4.207 (s, 2H),
4.188 (s, 2H), 4.099 (s, 5H). 13C NMR (75 MHz, CDCl3)
: 155.57,
158.24, 147.49, 144.69, 142.70, 128.82, 128.67, 127.29, 127.19, 125.76,
123.28, 118.02, 115.44, 112.68, 63.04, 43.27.
d
151.41, 143.01, 140.38, 128.74, 127.98, 127.26, 126.32, 126.02, 115.50,
103.46, 77.12, 70.11, 68.85.
5.2.3. 4-(1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-yl)phenol
(3DH). Light olivine needles, yield 60%, mp 146e147 ꢅC. Elemental
analysis: calcd for C21H18N2O (%): C 80.23, H 5.77, N 8.91, found: C
5.2.10. 4-(3-Ferrocenyl-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)phe-
nol (7DH). Orange sheets, yield 74%, mp 186e187 ꢅC. Elemental
analysis: calcd for C25H22FeN2O (%): C 71.10, H 5.25, N 6.63, found: C
79.52, H 5.54, N 8.86. 1H NMR (300 MHz, DMSO-d6)
d: 9.39 (s, 1H),
7.74 (dd, J¼7.8 Hz, J¼1.5 Hz, 2H), 7.45e7.34 (m, 3H), 7.17e7.08 (m,
4H), 7.01 (d, J¼7.8 Hz, 2H), 6.72e6.68 (m, 3H), 5.35 (dd, J¼12.0 Hz,
J¼6.3 Hz, 1H), 3.86 (dd, J¼17.4 Hz, J¼12.0 Hz, 1H), 3.06 (dd, J¼17.4
69.33, H 5.29, N 6.62. 1H NMR (300 MHz, DMSO-d6)
d: 9.368 (s, 1H),
7.133e7.080 (m, 5H), 6.896 (d, J¼7.8 Hz, 2H), 6.728 (d, J¼8.4 Hz, 2H),
6.646 (t, J¼7.2 Hz, 2H), 5.239 (dd, J¼11.7 Hz, J¼5.7 Hz, 1H), 4.694 (s,
1H), 4.598 (s, 1H), 4.388 (s, 2H), 4.133 (s, 5H), 3.757 (dd, J¼17.1 Hz,
J¼11.7 Hz,1H), 2.909 (dd, J¼17.1 Hz, J¼5.7 Hz,1H). 13C NMR (75 MHz,
Hz, J¼6.3 Hz, 1H). 13C NMR (75 MHz, DMSO-d6)
d: 156.68, 147.14,
144.39, 132.83, 132.47, 128.86, 128.71, 128.68, 127.13, 125.69, 118.57,
115.74, 113.08, 62.93, 43.10.
DMSO-d6) d: 161.72, 153.65, 149.86, 138.11, 133.92, 132.13, 122.84,
5.2.4. 5-Ferrocenyl-1,3-diphenyl-4,5-dihydro-1H-pyrazole
(4DH). Brown needles, yield 81%, mp 158e159 ꢅC. Elemental
analysis: calcd for C25H12FeN2 (%): C 73.90, H 5.46, N 6.89, found: C
120.79, 117.74, 82.55, 74.67, 74.58, 74.18, 72.00, 71.49, 67.28, 49.86.
5.2.11. 4-(3-Ferrocenyl-1-phenyl-1H-pyrazol-5-yl)phenol
(7AR). Yellow solid, yield 7%, mp 256e257 ꢅC. Elemental analysis:
calcd for C25H20FeN2O (%): C 71.44, H 4.80, N 6.67, found: C 70.80, H
73.94, H 5.53, N 7.00. 1H NMR (300 MHz, CDCl3)
d: 7.822 (d,
J¼7.2 Hz, 2H), 7.458e7.329 (m, 3H), 7.234e7.160 (m, 4H),
6.834e6.780 (m, 1H), 5.045 (dd, J¼11.1 Hz, J¼6.0 Hz, 1H), 4.223 (s,
4.93, N 6.14. 1H NMR (300 MHz, CDCl3)
d
: 7.316e7.276 (m, 5H),
7.083 (d, J¼8.4 Hz, 2H), 6.714 (d, J¼8.4 Hz, 2H), 6.485 (s, 1H), 4.767
(s, 2H), 4.297 (s, 2H), 4.125 (s, 5H). 13C NMR (75 MHz, CDCl3)
1H), 4.148 (s, 8H), 3.856e3.674 (m, 2H). 13C NMR (75 MHz, CDCl3)
d:
147.36, 145.39, 132.90, 128.83, 128.68, 128.61, 125.77, 119.23, 114.12,
90.59, 68.73, 68.46, 68.14, 67.74, 67.01, 59.34, 42.13.
d:
156.23, 151.50, 143.83, 139.90, 130.09, 128.86, 127.25, 125.42, 122.47,
115.48, 105.14, 77.21, 69.62, 68.77, 66.95.
5.2.5. 5-Ferrocenyl-1,3-diphenyl-1H-pyrazole (4AR). Red grains,
yield 60%, mp 100e101 ꢅC. Elemental analysis: calcd for C25H20FeN2
(%): C 74.27, H 4.99, N 6.93, found: C 74.27, H 4.98, N 6.97. 1H NMR
ꢀ
5.3. Scavenging of ABTSD by dendritic-like antioxidants
ꢀ
(300 MHz, CDCl3)
d
: 7.931e7.902 (m, 2H), 7.453e7.331 (m, 8H),
ABTSþ was formed in a mixture of 2.0 ml aqueous solution of
6.821 (s,1H), 4.197 (s, 4H), 4.091 (s, 5H). 13C NMR (75 MHz, CDCl3)
d:
4.0 mM ABTS and 1.41 mM K2S2O8 for 16 h. Then, 100 ml ethanol
ꢀ
151.65, 143.09, 140.43, 133.21, 128.84, 128.62, 128.11, 127.90, 126.32,
125.80, 103.79, 74.87, 69.88, 68.73, 68.67.
was added to make the absorbance of ABTSþ around 0.800 at
þꢀ
734 nm (
3
¼ 1:6 ꢄ 104 Mꢂ1 cmꢂ133). The ethanol solutions of
ferrocenylABaTnSd phenyl dendritic-like antioxidants with dihy-
dropyrazole or pyrazole as the core were added to the aforemen-
5.2.6. 3-Ferrocenyl-1,5-diphenyl-4,5-dihydro-1H-pyrazole
(5DH). Brown grains, yield 56%, mp 244e245 ꢅC. Elemental analy-
sis: calcd for C25H12FeN2 (%): C 73.90, H 5.46, N 6.89, found: C 73.79,
tioned radical solution with the final concentrations being 10.0
mM.
ꢀ
The decay of the absorbance of ABTSþ was recorded at room
temperature.
H 5.45, N 6.81. 1H NMR (300 MHz, CDCl3)
d: 7.36e7.34 (m, 4H),
7.29e7.27 (m, 1H), 7.17 (dd, J¼8.4 Hz, J¼7.5 Hz, 2H), 6.96 (d, J¼7.8 Hz,
2H), 6.71 (t, J¼7.2 Hz, 1H), 5.26 (dd, J¼11.4 Hz, J¼6.3 Hz, 1H), 4.70 (s,
1H), 4.56 (s, 1H), 4.35 (s, 2H), 4.11 (s, 5H), 3.76 (dd, J¼16.8 Hz, J¼12.0
Hz,1H), 2.99 (dd, J¼16.8 Hz, J¼6.3 Hz,1H). 13C NMR (75 MHz, CDCl3)
5.4. DNA protection against AAPH-induced oxidation by
dendritic-like antioxidants
d
: 147.93, 145.08, 142.84, 129.06, 128.78, 127.45, 125.75, 118.47,
The experiment of AAPH-induced oxidation of DNA was per-
formed as described in the literature34 with a little modification.
Briefly, DNA and AAPH were dissolved in phosphate-buffered so-
113.03, 69.63, 69.47, 69.18, 66.78, 66.51, 63.81, 45.02.
5.2.7. 3-Ferrocenyl-1,5-diphenyl-1H-pyrazole (5AR). Yellow nee-
dles, yield 58%, mp 175e176 ꢅC. Elemental analysis: calcd for
lution (PBS0: 8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 10.0
Then, 2.0 mg/ml DNA, 40 mM AAPH, and a certain concentration of
mM EDTA).

P.-Z. Li, Z.-Q. Liu / Tetrahedron 69 (2013) 9898e9905
9905
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dropyrazole or pyrazole as the core (dissolved in DMSO as the stock
solution) were mixed to form a solution. The solution was poured
into test tubes, and each test tube contained 2.0 ml. All the tubes
were incubated in a water bath at 37 ꢅC to initiate the reaction.
Three tubes were taken out at appropriate interval and cooled
immediately, to which 1.0 ml thiobarbituric acid (TBA) solution
(1.00 g TBA and 0.40 g NaOH dissolved in 100 ml PBS0) and 1.0 ml
trichloroacetic acid (3.0% aqueous solution) were added. The tubes
were heated in a boiling water bath for 15 min. After cooling to
room temperature, 1.5 ml n-butanol was added and shaken vigor-
ously to extract thiobarbituric acid reactive substance (TBARS,
lmax¼535 nm). The absorbance of n-butanol phase was measured
and plotted versus incubation time.
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