Thiaflavan scavenges radicals and inhibits DNA oxidation: A story from the ferrocene modification

Article abstract of DOI:10.1016/j.ejmech.2014.04.081

4-Thiaflavan is a sulfur-substituted flavonoid with a benzoxathiin scaffold. The aim of this work is to compare abilities of sulfur and oxygen atom, hydroxyl groups, and ferrocene moiety at different positions of 4-thiaflavan to trap radicals and to inhibit DNA oxidation. It is found that abilities of thiaflavans to trap radicals and to inhibit DNA oxidation are increased in the presence of ferrocene moiety and are further improved by the electron-donating group attaching to thiaflavan skeleton. It can be concluded that the ferrocene moiety plays the major role for thiaflavans to be antioxidants even in the absence of phenolic hydroxyl groups. On the other hand, the antioxidant effectiveness of phenolic hydroxyl groups in thiaflavans can be improved by the electron-donating group. The influences of sulfur and oxygen atoms in thiaflavans on the antioxidant property of para-hydroxyl group exhibit different manners when the thiaflavans are used to trap radicals and to inhibit DNA oxidation.

Full text of DOI:10.1016/j.ejmech.2014.04.081

European Journal of Medicinal Chemistry 81 (2014) 227e236
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thiaflavan scavenges radicals and inhibits DNA oxidation: A story from
the ferrocene modification
*
Hai-Wang Lai, Zai-Qun Liu
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Thiaflavan is a sulfur-substituted flavonoid with a benzoxathiin scaffold. The aim of this work is to
compare abilities of sulfur and oxygen atom, hydroxyl groups, and ferrocene moiety at different positions
of 4-thiaflavan to trap radicals and to inhibit DNA oxidation. It is found that abilities of thiaflavans to trap
radicals and to inhibit DNA oxidation are increased in the presence of ferrocene moiety and are further
improved by the electron-donating group attaching to thiaflavan skeleton. It can be concluded that the
ferrocene moiety plays the major role for thiaflavans to be antioxidants even in the absence of phenolic
hydroxyl groups. On the other hand, the antioxidant effectiveness of phenolic hydroxyl groups in thia-
flavans can be improved by the electron-donating group. The influences of sulfur and oxygen atoms in
thiaflavans on the antioxidant property of para-hydroxyl group exhibit different manners when the
thiaflavans are used to trap radicals and to inhibit DNA oxidation.
Received 29 September 2013
Received in revised form
27 April 2014
Accepted 29 April 2014
Available online 2 May 2014
Keywords:
Thiaflavan
Ferrocene
Antioxidant
Free radical
DNA oxidation
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
thiaflavans and to investigate positions of ferrocenyl group and
sulfur atom for being a mutual antioxidant.
Flavonoid (structure shown in Scheme 1) is an important
compound for natural polyphenols and is widely found in plants
[1]. The biological and pharmacological properties of flavonoids are
attributed to phenolic hydroxyl groups at different positions in
flavonoid [2]. It has been proved that the bond dissociation energy
(BDE) of OeH in hydroxyl group at B ring is about 10 kcal molꢀ1
lower than that at A ring. This result indicates that hydroxyl groups
at B ring play the key role in scavenging radicals and exhibiting
antioxidant effectiveness [3]. Some flavonoid derivatives are also
shown in Scheme 1 [4]. For example, when the double bond be-
tween C-2 and C-3 in flavonoid is converted into single bond,
the generated flavan is also natural polyphenol [5]. The antioxidant
properties of flavans have been estimated in various experimental
systems [6]. In addition, ferrocene-appended naphthoquinones
exhibit high antiplasmodial activities [7], and copper (II) complexes
of curcumin and N-ferrocenylmethyl-L-amino acids possess
high photocytotoxicities [8]. The aforementioned results reveal
that ferrocenyl group is able to improve the biological activities
of natural compounds, and this encourages us to explore whether
ferrocenyl group can also varies the antioxidant effects of
2. Chemistry
Scheme 1 outlines that sulfur atom can be applied to replace CH2
at 4-position in flavan, producing sulfur-contained benzoxathiin.
This sulfur-involved flavan is also called as 4-thiaflavan, which
behaves as a heterocyclic nucleus for designing many drugs [9].
The sulfur atom in thiaflavan can decrease the BDE of OeH
attaching to its para-position. As shown as the structure I in Scheme
1, the single electron at the oxygen atom in eOH may be transferred
to sulfur atom in order to use the sulfur atom for accommodating
the single electron and to stabilize the peroxyl radical [10]. Simi-
larly, the structure II in Scheme 1 outlines that the oxygen atom at
1-position can also stabilize the phenoxyl radical at 6-position.
Therefore, it is worthy to compare the ability of hydroxyl group at
6- and 7-position to scavenge radicals and to inhibit the oxidation
of biological species. Therefore, thiaflavan is an appropriate model
compound for the investigation on the effect of sulfur atom on
phenoxyl radical at ring A [11]. Although abilities of hydroxyl group
at 5-position to quench 2,20-diphenyl-1-picrylhydrazyl radical
(DPPH) [12] and to inhibit glutathione/ferric ion-induced oxidation
of DNA have been reported [13], the study on antioxidant abilities
of hydroxyl groups at 6- and 7-position are not usually found. A
straight carbon chain is used to attach to ring A of thiaflavan,
* Corresponding author.
E-mail address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
http://dx.doi.org/10.1016/j.ejmech.2014.04.081
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

228
H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
Scheme 1. Structures of flavonoid-related compounds and radicals deriving from 4-thiaflavan.
forming an amphiphilic antioxidant [14], and the carbon chain is
employed to attach to ring C, producing a tocopherol-like antioxi-
dant [15]. These results encourage us to apply an organometallic
group to attach ring B and to estimate the antioxidant property of
thiaflavan.
radicals. Hence, it is necessary to find a convenient way to express
the results from the aforementioned figures.
We have provided a method for expressing the relationship
between the concentration of a radical and the reaction period [22].
Briefly, when ABTSDꢁ is taken as the example, the rate constant (k)
of the reaction between an antioxidant and ABTSD can be calcu-
ꢁ
lated by equation (1) when [ABTSDꢁ], [antioxidant], and the cor-
3. Pharmacology
responding reaction rate (r) are known.
The inhibition effect on DNA oxidation and the ability to scav-
enge radicals are characteristic properties of an antioxidant. So, the
aim of this work is to clarify effects of hydroxyl groups at ring A and
B and ferrocene moiety at ring B on the antioxidant effectiveness of
thiaflavans. Therefore, as shown in Scheme 2, thiaflavans 1 to 11 are
synthesized following a description in a literature (protocol A) [16],
and thiaflavans 12 and 13 are prepared following the synthetic
ꢁ
d½ABTSþ ꢂ
ꢁ
ꢀ
¼ r ¼ k½ABTSþ ꢂ½antioxidantꢂ
(1)
dt
The chemical kinetic equation (1) is also available at t ¼ 0 and
can be expressed as equation (2), in which [ABTSD
]
t¼0
and [anti-
ꢁ
oxidant]t¼0 refer to concentrations of the antioxidant and ABTSDꢁ at
the beginning of the reaction.
protocol
B [17]. Then, the radical-scavenging properties are
compared by quenching 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) cationic radical (ABTSDꢁ), DPPH, and galvinoxyl radical,
respectively [18]. The antioxidant effectiveness of these thiaflavans
are also estimated inhibiting 2,20-azobis(2-amidinopropane hy-
drochloride) (AAPH)-induced oxidation of DNA [19].
ꢁ
r0 ¼ k½ABTSþ ꢂt¼0½antioxidantꢂt¼0
(2)
In view of the treatment of the experimental data in Fig. 1S, the
concentration of ABTSD and the corresponding time-point (t) are
ꢁ
input into a statistical software in order to find quantitative rela-
tionship of [ABTSDꢁ] w t. As a result, the double exponential
function is the most suitable function for expressing the relation-
ship of [ABTSDꢁ] w t and listed in Table 1S.
4. Results and discussion
4.1. Scavenging radicals
ꢁ
t
t
½ABTSþ ꢂ ¼ Aeꢀ þ Be þ C
(3)
ꢀb
a
ABTSDꢁ, DPPH, and galvinoxyl radical are radical resources,
which are usually employed to evaluate the property of an anti-
oxidant in scavenging radicals. The reaction of an antioxidant with
ABTSDꢁ, DPPH, and galvinoxyl radical reveals the ability of the
antioxidant to reduce radical and to donate its hydrogen to N- or O-
centered radicals, respectively. Some methods are designed to ex-
press the ability of the antioxidant to quench these radicals [20].
But kinetic methods are not usually found because the operation to
measure the rate constant (k) of the antioxidant to quench these
radicals are so complicated [21]. But Figs. 1S, 2S and 3S show that
some thiaflavans are able to decrease the concentrations of these
radicals rapidly, while other thiaflavans cannot react with these
Moreover, the differential operation is performed to equation
(3) for expressing the relationship between the reaction rate (r) and
the time (t), ꢀd[ABTSDꢁ]/dt w t (equation (4)).
ꢁ
d½ABTSþ ꢂ
dt
A
¼ r ¼ eꢀ
a
t
a
B
b
t
b
ꢀ
þ
eꢀ
(4)
The reaction rate at t ¼ 0 (r0) can be calculated following
equation (4) when the reaction time (t) is assigned to 0. The rate
constant (k) is thereby calculated by using equation (2) and
collected in Table 1. This method is also used to treat the

Synthetic protocol A
The hydoxyl group at 3- or 4-position in reagents was protected
by t-BuMe2SiCl before performing the following reaction,
or CH3O- attached to 3- or 4-position in the reagents.
2
O
t-BuMe2SiO
t-BuMe2SiO
3
4
1 OH
N-S-Cl
6
5
1
t-BuMe2SiO
t-BuMe2SiO
OH
S
O
O
2
(C2H5)3N
CHCl3, 600C
PhtNSCl
CHCl3, 00C
CH3O
CH3O
3
4
OH
N
or
6
O
5
thiophthalimide
5'
4'
3'
OH
OH
6'
1
O
8
B
HO
7
2
1'
2'
A
C
3
OSiMe2Bu-t
OSiMe2Bu-t
6
HO
S
5
4
Fe
t-BuMe2SiO
t-BuMe2SiO
O
S
(n-C4H9)4NF 3 H2O
styrene
O
HO
HO
or
00C
O
S
Fe
or
intermediate for Diels-Alder reaction
thiaflavan
vinyl ferrocene
Synthetic protocol B
O
TiCl4
CH2Cl2, 00C
O
S
HO
12
S
O
O
OH
VII
Br
OH
SH
O
PhPOCl2
CH3CN
reflux
S
O
(C2H5)3N, CH2Cl2
room temperature
V
O
KOH, C2H5OH,
VI
O
00C
S
O
O
Cl
O
O
O
S
H2N
NH2
O
O
S
O
CH3COOH, HCl
K2CO3, KI
HO
room temperature
then reflux
III
room temperature
IV
O
K2CO3, CH3I
O
room temperature
Br
O
OH
SH
KOH, C2H5OH
HO
O
S
00C
O
O
(C2H5)3N, CH2Cl2
room temperature
O
VIII
IX
OH
OH
S
OH
O
PhPOCl2
CH3CN
reflux
O
S
O
O
13
X
Scheme 2. Synthesis and structures of thiaflavans in the present work.

230
H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
Compounds 1 to 11 are produced following the synthetic protocol A, and compounds 12 and 13 are
produced following the synthetic protocol B. The total yields are included in parentheses. Trolox
and catechol are the reference compounds.
OH
OH
OH
OH
4'
4'
3'
4'
O
O
O
6
6
6
HO
S
HO
HO
S
O
S
3 (23%)
2 (7%)
1 (11%)
OH
OH
OH
OH
OH
4'
4'
3'
4'
3'
O
O
O
HO
7
7
6
S
S
S
O
O
4 (13%)
6 (7%)
5 (30%)
OH
4'
Fe
Fe
O
O
S
O
S
7
6
6
S
HO
O
7 (15%)
8 (11%)
9 (32%)
Fe
Fe
O
O
S
HO
O
O
S
2
3
7
6
7
S
HO
12 (13%)
10 (23%)
11 (14%)
OH
OH
OH
HO
4'
O
S
2
3
O
6
COOH
O
catechol
13 (9%)
trolox
Scheme 2. (continued).
experimental results from thiaflavan quenching other radicals, and
results are listed in Table 1 as well.
hydroxyl group attaches to 6- and 7-position, respectively, while
the ring B is a phenyl group in compound 12. These results reveal
that the ferrocene moiety at ring B remarkably enhances the ability
of thiaflavan to quench radicals.
The data in Table 1 reveal that k values of all the thiaflavans in
trapping ABTSD are higher than those in trapping DPPH and gal-
ꢁ
vinoxyl radical. So, abilities of thiaflavans to reduce radicals are
higher than to donate hydrogen atom to N- and O-centered radi-
cals. Compounds 9 and 11 cannot react with DPPH and galvinoxyl
radical because of no phenolic hydroxyl group contained. Scheme 3
illustrates the comparison of k and their structures.
In trapping ABTSDꢁ, the k value of compound 12 is similar to
those of compounds 9 and 11. This result reveals that the single
hydroxyl group at 6-position in ring A is able to react with ABTSD
ꢁ
and DPPH, but cannot react with galvinoxyl radical. It can be found
by comparing k values of compounds 13 and 3 that the C]C bond
between 2- and 3-position in ring C is beneficial for the hydroxyl
group at 40-position in ring B of compound 13 to quench ABTSDꢁ
and DPPH. Moreover, the k value of compound 5 is higher than that
of compound 1 in trapping ABTSDꢁ, indicating that the hydroxyl
group at 7-poisiton in ring A (the para-position of sulfur atom)
exhibits higher ability to scavenge radical than that at 6-position
(the para-position of oxygen atom). This phenomenon is also
The low k values of compounds 3 and 7 in trapping ABTSD
ꢁ
indicate that a single hydroxyl group at 40-position in ring B only
exhibits weak ability to reduce radical and even cannot donate its
hydrogen atom to N- and O-centered radicals. On the contrary,
compounds 9 and 11 can react with ABTSDꢁ even in the absence of
hydroxyl group. The k values of compounds 8 and 10 are higher
than that of compound 12. In compounds 8 and 10, only one

H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
231
Table 1
The differential operation on the equation (5) reveals the for-
The rate constant (k) for thiaflavans, trolox, and catechol in scavenging ABTSþ
,
ꢁ
mation rate of TBARS as d[TBARS]/dt ¼ 7.1 nM minꢀ1
.
DPPH and galvinoxyl radicals.
Fig. 1 also illustrates that the addition of 100 mM compound 5
Compound
Rate constant for thiaflavans to scavenge
hinders the increase of the absorbance of TBARS, and equations of
[TBARS] w t together with d[TBARS]/dt in the presence of com-
pound 5 are listed in Fig. 1. Fig. 4S outlines the [TBARS] vs. t in the
presence of the same concentration of compounds 3, 12, and 13,
and Table 2 gives d[TBARS]/dt in the presence of compounds 3, 5,
12, and 13.
radicals, k (ꢄ 103 Mꢀ1 sꢀ1
)
ABTSþ
DPPH
Galvinoxyl radical
ꢁ
1
2
3
4
5
6
7
8
8.92
11.7
0.202
390
0.043
1.18
e
0.025
0.52
e
2.20
0.069
1.61
e
2.54
0.034
0.57
e
The d[TBARS]/dt value of compound 3 is lower than that of
compound 13, indicating that the C]C between 2- and 3-position
is not beneficial for enhancing the inhibitive effect of compound
13 on DNA oxidation. The d[TBARS]/dt value of compound 13 ap-
proaches to that of compound 12, implying that the inhibitive effect
of hydroxyl group in ring B of compound 13 is similar to that in ring
A of compound 12. On the other hand, compound 3 contains a
single hydroxyl at 40-position in ring B, while compound 5 contains
two hydroxyl groups at 7-position in ring A and 40-position in ring
B. But similar d[TBARS]/dt values of compounds 3 and 5 indicate
that hydroxyl group at 7-position does not increase the inhibitive
effect of compound 5 on DNA oxidation. So, hydroxyl group at ring
B plays the major role in hindering the formation of TBARS.
As shown in the panel A of Fig. 2, the addition of compound 1
hinders the increase of the absorbance of TBARS for a period, and
then the absorbance of TBARS increases as the blank experiment.
The inhibition period (tinh) can be measured from the beginning of
the reaction to the cross point of tangents for the inhibition and the
increase period in the absorbance line of TBARS. Then, as shown in
the panel B of Fig. 2, tinh increases with the concentration of com-
pound 1, and the linear relationship between tinh and the concen-
tration of compound 1 is given in the panel B of Fig. 2. Fig. 5S shows
the relationships between the absorbance of TBARS and the con-
centrations of compounds 1, 2, 4, 6, 7, 8, 9, 10, and 11, while Fig. 6S
outlines the linear relationships between tinh and the concentra-
tions of the aforementioned compounds. The equations of
10.8
16.7
0.168
22.3
5.54
22.9
5.03
4.89
8.14
29.2
2.58
0.070
e
0.039
e
9
10
11
12
13
Trolox
Catechol
0.124
e
0.084
e
0.0071
0.031
0.353
1.30
e
e
1.70
1.78
found in the comparison of k values of compounds 5 and 1 and of
compounds 6 and 2. As shown in Scheme 1, it can be concluded that
the radical resonance structure I is more beneficial for stabilizing
the single electronic species than structure II because the sulfur
atom accommodates single electron more sufficiently than oxygen
atom. This finding is in agreement with a previous report, in which
hydroxyl group at para-position of sulfur atom inhibits the oxida-
tion of styrene more efficiently than that at para-position of oxygen
atom [23].
Scheme 3 also illustrates that the k values of compound 6 in
trapping three radicals are higher than the corresponding values of
compound 5, revealing that ortho-dihydroxyl groups in ring B of
compound 6 play the key role in scavenging radicals [24]. But it is
not implied that the catechol structure feature itself can function as
the efficient radical-scavenger because the k value of catechol in
t
inh w [thiaflavans] are given in Table 3.
As shown in equation (6), it has been demonstrated by chemical
kinetics that tinh correlates proportionally with the concentration of
trapping ABTSD is lower than that of compounds 2 and 6. In
ꢁ
thiaflavan, the ortho-dihydroxyl groups in ring B are the active site
for trapping radicals only in the case of the electron-donating group
attaching to ring A. Hence, when methoxyl group (a strong
electron-donating group) attaches to ring A, the radical-scavenging
ability of compound 4 increases to the highest level among thia-
flavans employed herein. It can be concluded that single hydroxyl
group at para-position of sulfur atom in ring A, adjacent double
hydroxyl groups in ring B with the aid of the electron-donating
group in ring A, and ferrocene moiety as ring B in thiaflavan play
the key role in scavenging radicals.
an antioxidant [25].
tinh ¼ (n/Ri) [antioxidant]
(6)
Ri refers to the initiation rate of the radical-induced reaction,
and the stoichiometric factor (n) is the number of radicals trapped by
one molecule of the antioxidant. It has been assumed that Ri is
equal to the generation rate (Rg) of radicals (Rg ¼ (1.4 ꢅ 0.2) ꢄ 10ꢀ6
[AAPH] sꢀ1 [25]) because the water-soluble AAPH and the sodium
salt of DNA locate at the same phase, and radicals generated from
AAPH attack DNA directly. Hence, Ri ¼ Rg ¼ 1.4 ꢄ 10ꢀ6
ꢄ
40 mM sꢀ1 ¼ 3.36
m
M minꢀ1 in this experimental system. The n
4.2. Inhibiting AAPH-induced oxidation of DNA
value is the product of the coefficient in the equation of
tinh w [thiaflavans] and Ri. So, n provides a quantitative index for
comparing the effect of an antioxidant on DNA oxidation. Accord-
ingly, the n values of thiaflavans are calculated and listed in Table 3,
in which the data are schematized in Scheme 4. We have measured
the tinh w [isoflavonoids] under the same experimental condition
[26]. The isoflavonoids include 3-(20-hydroxybenzylidene)-7-
methoxychroman-4-one (o-HBMC), 3-(30-hydroxybenzylidene)-7-
In the presence of oxygen at 37 ꢃC, the decomposition of AAPH
leads to the formation of peroxyl radical, which is able to oxidize
the guanine bases in DNA [19]. The products deriving from the
oxidation of DNA can be colorized after they react with thio-
barbituric acid. Thus, the oxidative products of DNA are also called
as thiobarbituric acid reactive species (TBARS) and can be deter-
mined by measuring the absorbance of at 535 nm (εTBARS
¼
1.56 ꢄ 105 Mꢀ1 cmꢀ1). The blank experiment in Fig. 1 shows a
continual increase of the absorbance, indicating that much more
TBARS are generated along with the increases of the reaction times.
The relationship between the concentration of TBARS and the re-
action period (t) is fitted by linear equation (5).
methoxychroman-4-one
(m-HBMC),
and
3-(40-hydrox-
ybenzylidene)-7-methoxychroman-4-one (p-HBMC). These iso-
flavonoids have the similar structures with thiaflavans, but the
coefficients of tinh w [o-HBMC or p-HBMC] are only 0.82 and 0.15,
respectively, lower than those of thiaflavans. So, the introduction of
sulfur atom into flavans improves the sensitivity of tinh towards the
variation of concentrations of thiaflavans.
[TBARS (nM)] ¼ 7.1 (ꢅ0.3) t (min) þ 3073 (ꢅ137)
(5)

232
H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
Scheme 3. The comparison of k values in trapping radicals with thiaflavan structures.
Similar n values of compounds 4 and 7 reveal that one or two
thatof compound 10, inwhich the hydroxyl group in ring A locates at
the para-position of sulfur atom. Thus, hydroxyl group at para-po-
sition of oxygen atom exhibits higher antioxidant effectiveness than
that at the para-position of sulfur atom.
hydroxyl groups in ring B exhibit similar antioxidant effect on DNA
oxidation. But compound 1 contains two hydroxyl groups, which
locate at ring A and B, respectively, while compound 7 contains only
one hydroxyl group in ring B. The n value of compound 1 is higher
than that of compound 7, indicating that two hydroxyl groups in ring
A and B, respectively, increase the ability of thiaflavan to inhibit DNA
oxidation. Moreover, the n value of compound 2 is much higher than
that of compound 1, revealing that double hydroxyl groups at
adjacent position in ring B together with one hydroxyl group in ring
A are beneficial for enhancing the antioxidant effect of thiaflavan.
The comparison of n values of compounds 2 and 6 reveals that hy-
droxyl group in ring A at the para-position of oxygen atom exhibits
higher antioxidant effectiveness than that at the para-position of
sulfur atom. In compound 8, the hydroxyl group in ring A locates at
the para-position of oxygen atom and possesses higher n value than
At present, modifying traditional drugs by organometallic
groups becomes a popular way to improve the pharmacological
properties [27], while ferrocene is usually applied to link with the
other organic structural features [28]. The cyclopentadiene moiety
in ferrocene can be used to enlarge the conjugation system of an
organic structure, and ferrocene moiety can also be a substituent in
an organic species [29]. We have explored the antioxidant effects of
ferrocene-appended curcumin on AAPH-induced oxidation of DNA.
It is found that the n value of ferrocene-appended curcumin is 9.5
[30], while that of curcumin is 8.2 [31]. So, the conjugation system
formed between carbon chain in curcumin and cyclopentadiene
moiety in ferrocene actually increases the antioxidant

H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
233
Table 3
The equations between inhibition period (tinh) and concentrations of thiaflavans in
AAPH-induced oxidation of DNA and n values of thiaflavans.a
[TBARS] (nM) = 7.1
[TBARS] / dt = 7.1
t (min) + 3073
1.2
0.8
0.4
control
5
Compound
tinh (min) ¼ (n/Ri) [concentration (
m
M)]
n
d
1
2
4
6
7
8
9
10
11
tinh ¼ 1.50 (ꢅ0.08) [1] þ 2.2 (ꢅ10.9)
tinh ¼ 2.57 (ꢅ0.03) [2] e 1.5 (ꢅ0.4)
tinh ¼ 1.15 (ꢅ0.12) [4] þ 62.1 (ꢅ21.7)
tinh ¼ 2.17 (ꢅ0.16) [6] þ 15.2 (ꢅ21.5)
tinh ¼ 1.18 (ꢅ0.06) [7] þ 43.6 (ꢅ1.5)
tinh ¼ 2.30 (ꢅ0.17) [8] þ 63.2 (ꢅ11.9)
tinh ¼ 3.76 (ꢅ0.34) [9] þ 40.9 (ꢅ31.5)
tinh ¼ 1.99 (ꢅ0.01) [10] þ 63.9 (ꢅ1.1)
tinh ¼ 3.74 (ꢅ0.23) [11] þ 21.1 (ꢅ21.1)
5.04 (ꢅ0.29)
8.64 (ꢅ0.01)
3.86 (ꢅ0.40)
7.29 (ꢅ0.54)
3.96 (ꢅ0.02)
7.73 (ꢅ0.57)
12.64 (ꢅ1.14)
6.69 (ꢅ0.03)
12.57 (ꢅ0.77)
[TBARS] (nM) = 4.1
t
(min) + 2759
d[TBARS] / dt = 4.1
a
The value of n is the product of the coefficient of tinh w [thiaflavans] and
Ri ¼ 1.4 ꢄ 10ꢀ6 ꢄ 40 mM sꢀ1 ¼ 3.36
m
M minꢀ1 when 40 mM AAPH is employed.
0
300
600
Reaction period (min)
effectiveness. Moreover, synthetic methods are recently reported to
prepare ferrocenyl-substituted flavones and flavonols [32]. This
encourages us to replace ring B of thiaflavan by ferrocene moiety
and to detect their antioxidant effects on DNA oxidation. It is found
herein that ferrocene moiety as B ring indeed enhances the n values
of compounds 8 and 10 although cyclopentadiene moiety in
ferrocene does not form a conjugation system with ring C of thia-
flavans. When an electron-donating group such as methoxyl group
attaches to ring A, compounds 9 and 11 possess the highest n values
and thus exhibit the strongest antioxidant effects on DNA oxidation
even in the absence of hydroxyl group. Hence, ferrocene group at
ring B together with electron-donating group at ring A remarkably
promote antioxidant effect of thiaflavans.
Fig. 1. The increase of the absorbance of TBARS in the mixture of 40 mM AAPH and
2.0 mg/mL DNA and in the presence of 100 mM compound 5.
Table 2
The formation rate of TBARS (d[TBARS]/dt) in the control experiment
and in the presence of compounds 3, 5, 12, and 13.
Compound
d[TBARS]/dt (nM minꢀ1
)
Control
3
5
12
13
7.1
4.2
4.1
5.2
5.8
5. Conclusion
Both radical-scavenging property and inhibition effect on DNA
oxidation are changed markedly when ferrocene moiety is applied
to modify the structure of thiaflavan. The ferrocene moiety at ring B
is beneficial for enhancing abilities of thiaflavans to trap radicals
and to inhibit DNA oxidation. The antioxidant effectiveness gener-
ated by hydroxyl groups or ferrocene moiety at ring B is enhanced
by the electron-donating group attaching to ring A. In addition,
sulfur and oxygen atom at ring C exhibit different effects on the
antioxidant property of hydroxyl group locating at their para-po-
sitions in ring A. The sulfur atom is beneficial for the hydroxyl group
in ring A to quench radicals, while the oxygen atom is beneficial for
the hydroxyl group in ring A to inhibit DNA oxidation. The struc-
tureeactivity relationship obtained from thiaflavans is useful for
the study on designing novel antioxidants.
6. Experimental section
6.1. Materials and instrumentation
ABTS, DPPH, and galvinoxyl radical were purchased from Fluka
Chemie GmbH, Switzerland, and AAPH and naked DNA sodium salt
were purchased from Acros Organics, Belgium. Other agents were
of analytical grade and used directly. The structures of the obtained
products were identified by 1H and 13C NMR (Varian Mercury
300 MHz NMR spectrometer and Bruker Avance III 400 MHz
spectrometer), and the spectral data were included in Supporting
Information.
6.2. Synthesis and structural characterization of thiaflavans
6.2.1. Thiaflavans prepared by the synthetic protocol A
As shown as the synthetic protocol A in Scheme 2, compounds
1e11 were synthesized following literature description [16]
with m-methoxyphenol, p-methoxyphenol, resorcinol, and
Fig. 2. (a) The variation of the absorbance of TBARS in the mixture of DNA (2.0 mg/mL)
and 40 mM AAPH at 37 ꢃC in the presence of various concentrations of compound 1. (b)
The relationship between tinh and the concentration of compound 1.

234
H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
Scheme 4. The d[TBARS]/dt and n values of thiaflavans in inhibiting DNA oxidation.
hydroquinone as reagents. One hydroxyl group in resorcinol and
hydroquinone was protected by tert-butyldimethylsilyl chloride.
Then, 17 mL of dry CHCl3 solution containing excess PhtNSCl was
added dropwisely to 8 mL of dry CHCl3 solution containing mono-
protected hydroquinone or resorcinol and stirred for 16 h at 0 ꢃC
until phenols cannot be detected by thin layer chromatography
(TLC). The mixture was diluted with CH2Cl2 and washed by satu-
rated NaHCO3 and water. The organic phase was dried over anhy-
drous Na2SO4, and the solvent was removed under vacuum. The
residue was purified by column chromatography with CH2Cl2 as the
eluent to afford thiophthalimides as colorless solid. The following
cycloaddition reactions were carried out in dry CHCl3 solution of
thiophthalimides (w0.1 M) and styrenes (2 equiv.) or vinyl ferro-
cene (2 equiv.) and freshly distilled (C2H5)3N (2 equiv.) at 60 ꢃC. The
reaction was finished with thiophthalimides not detected by TLC.
Then, the solvent was evaporated under vacuum pressure, and the
residual solid was purified with column chromatography to afford
silylated adducts. The desilylation operation was performed in dry
tetrahydrofuran (THF) solution containing 0.04 M aforementioned
adducts at 0 ꢃC, to which a solution of (n-C4H9)4NF$3H2O in THF
(1 equiv. for each protective group) was added. The reaction was
finished with the reagent not detected by TLC, and then the mixture
was diluted with ethyl acetate and washed with saturated NH4Cl
and water. The organic layer was dried over anhydrous Na2SO4, and
the solvent was evaporated under vacuum pressure. The residue
was purified with column chromatography to afford thiaflavans.
by methyl or benzyl group before the following operation. In brief,
50 mL of acetone was used to dissolve reagent III and potassium
iodide, which was not needed when the methylation was carried
out. The solution was cooled to 0 ꢃC and then benzyl chloride or
methyl iodide was added. The reaction mixture was stirred for
30 min, and potassium carbonate was added. Then, the mixture was
stirred for 36 h in nitrogen atmosphere. After the insoluble material
was filtered, the liquid was concentrated and purified by column
chromatography with ethyl acetateepetroleum ether as eluent to
afford compound VIII or IV as white solids. The alcoholysis of
compound VIII or IV was performed in ethanol and THF solution
(1:1, v:v) containing w1.0 M compound VIII or IV. An ethanolic
solution of KOH (2.5 equiv.) was added to the aforementioned so-
lution and stirred for 6 h in nitrogen atmosphere and then 2 M
hydrochloride acid was added. The mixture was extracted with
ethyl acetate, and the organic layer was washed with water and
dried over anhydrous Na2SO4. The organic layer was concentrated
under vacuum pressure, and the obtained oil (compound V or IX)
was used without further purification. The compound V and 2-
bromo-1-phenylethanone (1.5 equiv.) (or compound IX and 2-
bromo-1-(p-hydroxyphenyl)ethanone (1.5 equiv.)) were dissolved
in 20 mL of CH2Cl2, followed by adding (C2H5)3N and stirring for
12 h. The solvent was evaporated under vacuum pressure, and the
residual solid was purified with column chromatography to afford
compound VI or X. The annulation of compound VI or X to afford
compound VII or 13 was performed in a solution of CH3CN with
PhPOCl2 (2.0 equiv.) as the dehydrant in nitrogen atmosphere. The
reaction mixture was refluxed for 16 h and then quenched by
adding ethanol. The mixture was poured into water and extracted
with ethyl acetate. The organic layer was dried over anhydrous
Na2SO4, and the solvent was evaporated under vacuum pressure.
The residual solid was purified with column chromatography to
afford compound VII or 13. The compound VII (0.4 g, 1.2 mmol) was
6.2.2. Thiaflavans prepared by the synthetic protocol B
As shown as the synthetic protocol B in Scheme 2, compounds
12 and 13 were prepared by the following operation [17]. The
glacial acetic acid (90 mL) solution of quinone (5.4 g, 50.0 mmol)
was added to hydrochloride acid (100 mL) solution of thiourea
(5.7 g, 75.0 mmol) and stirred at room temperature for 4 h, then
refluxed for 1 h and cooled to room temperature. The solid was
filtered to give crude product, which was recrystallized with
ethanol-water to afford the regent III for the synthesis of com-
pounds 12 and 13. The hydroxyl group in reagent III was protected
added to 5 mL of CH2Cl2, to which TiCl4 (600 mL, 5.5 mmol, in 10 mL
of CH2Cl2) was added dropwisely at 0 ꢃC. After stirring overnight,
methanol was added to quench the reaction. The solvent was
removed under vacuum pressure, and the residual solid was

H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
235
purified with column chromatography to afford compound 12 as
colorless oil. The systematic nomenclature, isolation method, and
total yields for various thiaflavans were given as following.
6.2.3.7. 4-(7-Methoxy-2,3-dihydro-benzo[1,4]oxathiin-2-yl)-phenol
(7). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 15%. Colorless oil. 1H NMR (DMSO-d6,
400 MHz)
d
: 9.51 (s, 1H, OH), 7.26 (d, J ¼ 7.2 Hz, 2H, aromatic), 7.00
(d, J ¼ 7.2 Hz, 1H, aromatic), 6.78 (d, J ¼ 6.0 Hz, 2H, aromatic), 6.49e
6.53 (m, 2H, aromatic), 5.10 (d, J ¼ 8.0 Hz, 1H, CH), 3.68 (s, 3H,
6.2.3. The characterization of structures of thiaflavans
6.2.3.1. 2-(4-Hydroxyphenyl)-2,3-dihydro-benzo[1,4]oxathiin-6-ol
(1). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 11%. m.p. 179e182 ꢃC. 1H NMR (DMSO-
OCH3), 3.15e3.24 (m, 2H, CH2). 13C NMR (DMSO-d6, 100 MHz)
d:
157.6, 157.4, 152.9, 130.4, 127.8, 127.6, 115.1, 108.7, 107.7, 103.6, 76.2,
55.2, 30.2.
d6, 400 MHz)
d
: 9.49 (s, 1H, OH), 9.04 (s, 1H, OH), 7.24 (d, J ¼ 7.6 Hz,
2H, aromatic), 6.77 (d, J ¼ 7.6 Hz, 2H, aromatic), 6.68 (d, J ¼ 8.4 Hz,
1H, aromatic), 6.48 (s, 1H, aromatic), 6.42 (d, J ¼ 8.4 Hz, 1H, aro-
matic), 4.94 (d, J ¼ 9.2 Hz, 1H, CH), 3.12e3.28 (m, 2H, CH2). 13C NMR
6.2.3.8. 2-Ferrocenyl-2,3-dihydro-benzo[1,4]oxathiin-6-ol
Purified by column chromatography with CH2Cl2 as eluent. Yield
11%. m.p. 170e172 ꢃC. 1H NMR (CDCl3, 400 MHz)
: 6.78 (d,
(8).
d
(DMSO-d6, 100 MHz) d: 157.3, 151.5, 145.1, 130.7, 127.7, 119.0, 117.5,
J ¼ 8.8 Hz, 1H, aromatic), 6.59 (s, 1H, aromatic), 6.51 (d, J ¼ 8.8 Hz,
1H, aromatic), 4.89 (d, J ¼ 9.2 Hz, 1H, CH), 4.39 (s, 1H, OH), 4.32 (s,
1H, ferrocenyl), 4.29 (s, 1H, ferrocenyl), 4.22 (br, 7H, ferrocenyl),
115.1, 112.7, 112.1, 75.5, 30.6.
6.2.3.2. 4-(6-Hydroxy-2,3-dihydro-benzo[1,4]oxathiin-2-yl)-ben-
zene-1,2-diol (2). Purified by column chromatography with petro-
leum ether/ethyl acetate as eluent. Yield 7%. m.p. 172e176 ꢃC. 1H
3.15e3.26 (m, 2H, CH2). 13C NMR (CDCl3, 100 MHz)
d: 153.9, 153.0,
127.9, 109.6, 108.3, 105.6, 87.5, 74.1, 69.1, 68.5, 68.3, 67.1, 66.5, 30.7.
NMR (DMSO-d6, 400 MHz) d: 9.08 (s, 1H, OH), 9.00 (br s, 2H, OH),
6.2.3.9. 6-Methoxy-2-ferrocenyl-2,3-dihydro-benzo[1,4]oxathiine
(9). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 32%. m.p. 140e142 ꢃC. 1H NMR (CDCl3,
6.84 (s, 1H, aromatic), 6.74e6.78 (m, 2H), 6.71 (d, J ¼ 8.8 Hz, 1H,
aromatic), 6.52 (s, 1H, aromatic), 6.46 (d, J ¼ 8.8 Hz, 1H, aromatic),
4.91 (d, J ¼ 8.8 Hz, 1H, CH), 3.15e3.25 (m, 2H, CH2). 13C NMR
300 MHz)
d
: 6.81 (d, J ¼ 8.7 Hz, 1H, aromatic), 6.63 (d, J ¼ 3.0 Hz,1H,
(DMSO-d6, 100 MHz) d: 151.5, 145.2, 145.1, 145.0, 131.3, 119.0, 117.6,
aromatic), 6.58 (dd, J ¼ 8.7 Hz, 3 Hz, 1H, aromatic), 4.84 (d, 1H,
J ¼ 9.0 Hz, CH), 4.26e4.36 (m, 9H, ferrocenyl), 3.75 (s, 3H, OCH3),
117.4, 115.3, 113.8, 112.7, 112.1, 75.6, 30.8.
3.16e3.29 (m, 2H, CH2). 13C NMR (CDCl3, 100 MHz)
d: 154.1, 146.6,
6.2.3.3. 4-(6-Methoxy-2,3-dihydro-benzo[1,4]oxathiin-2-yl)-phenol
(3). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 23%. m.p.160e163 ꢃC. 1H NMR (DMSO-
119.2, 118.1, 112.2, 111.1, 87.7, 73.4, 69.1, 68.4, 68.2, 67.1, 66.5, 55.8,
31.0.
d6, 400 MHz)
d
: 9.50 (s, 1H, OH), 7.25 (d, J ¼ 7.6 Hz, 2H, aromatic),
6.2.3.10. 2-Ferrocenyl-2,3-dihydro-benzo[1,4]oxathiin-7-ol
Purified by column chromatography with CH2Cl2 as eluent. Yield
23%. m.p. 134e136 ꢃC. 1H NMR (CDCl3, 400 MHz)
: 6.78 (d,
(10).
6.78 (s, 3H, aromatic), 6.69 (s, 1H, aromatic), 6.59 (d, J ¼ 8.0 Hz, 1H,
aromatic), 4.99 (d, J ¼ 8.8 Hz, 1H, CH), 3.68 (s, 3H, OCH3), 3.17e3.28
d
(m, 2H, CH2). 13C NMR (DMSO-d6, 100 MHz)
d: 157.4, 153.6, 146.2,
J ¼ 8.8 Hz, 1H, aromatic), 6.58 (s, 1H, aromatic), 6.50 (d, J ¼ 8.8 Hz,
1H, aromatic), 4.87 (d, J ¼ 9.2 Hz, 1H, CH), 4.33 (s, 1H, ferrocenyl),
4.30 (s, 1H, ferrocenyl), 4.23 (br, 7H, ferrocenyl), 3.15e3.26 (m, 2H,
130.6, 127.7, 119.1, 117.9, 115.1, 111.9, 110.8, 75.6, 55.4, 30.5.
CH2). 13C NMR (CDCl3, 100 MHz)
d: 153.8, 153.0, 127.9, 109.5, 108.4,
6.2.3.4. 4-(6-Methoxy-2,3-dihydro-benzo[1,4]oxathiin-2-yl)-ben-
zene-1,2-diol (4). Purified by column chromatography with petro-
leum ether/ethyl acetate as eluent. Yield 13%. m.p. 134e137 ꢃC. 1H
105.6, 87.4, 74.1, 69.0, 68.4, 68.2, 67.0, 66.4, 30.7.
NMR (DMSO-d6, 400 MHz) d: 9.02 (s, 1H, OH), 8.99 (s, 1H, OH), 6.85
6.2.3.11. 7-Methoxy-2-ferrocenyl-2,3-dihydro-benzo[1,4]oxathiine
(11). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 14%. m.p. 94e97 ꢃC. 1H NMR (CDCl3,
(s, 1H, aromatic), 6.81 (s, 1H, aromatic), 6.73e6.79 (m, 3H, aro-
matic), 6.63 (d, J ¼ 8.8 Hz, 1H, aromatic), 4.96 (d, J ¼ 8.0 Hz, 1H, CH),
3.72 (s, 3H, OCH3), 3.19e3.28 (m, 2H). 13C NMR (DMSO-d6,100 MHz)
300 MHz)
d
: 6.97 (d, J ¼ 8.4 Hz, 1H, aromatic), 6.51 (d, J ¼ 8.4 Hz, 1H,
d: 153.6, 146.2, 145.3, 145.2, 131.2, 119.0, 117.9, 117.4, 115.3, 113.8,
aromatic), 6.49 (s, 1H, aromatic), 4.99 (s, 1H, CH), 4.23e4.33 (m, 9H,
ferrocenyl), 3.76 (s, 3H, OCH3), 3.19 (s, 2H, CH2). 13C NMR (CDCl3,
111.9, 110.8, 75.6, 55.4, 30.7.
100 MHz) d: 158.2, 153.0, 127.7, 108.7, 108.2, 103.8, 87.5, 74.1, 69.0,
68.4, 68.2, 67.1, 66.4, 55.5, 30.6.
6.2.3.5. 2-(4-Hydroxyphenyl)-2,3-dihydro-benzo[1,4]oxathiin-7-ol
(5). Purified by column chromatography with petroleum ether/
ethyl acetate as eluent. Yield 30%. Colorless oil. 1H NMR (DMSO-d6,
6.2.3.12. 2-Phenylbenzo[1,4]oxathiin-6-ol (12). Purified by column
400 MHz)
d
: 9.51 (s, 1H, OH), 9.32 (s, 1H, OH), 7.24 (d, J ¼ 8.4 Hz, 2H,
chromatography with petroleum ether/ethyl acetate as eluent.
Yield 13%. Pale orange oil. 1H NMR (DMSO-d6, 400 MHz)
d: 9.13 (s,
aromatic), 6.87 (d, J ¼ 8.4 Hz, 1H, aromatic), 6.77 (d, J ¼ 8.4 Hz, 2H,
aromatic), 6.36 (dd, J ¼ 2.0 Hz, 8.4 Hz, 1H, aromatic), 6.28 (d,
J ¼ 2.0 Hz, 1H, aromatic), 5.07 (d, J ¼ 7.2 Hz, 1H, CH), 3.11e3.22 (m,
1H, OH), 7.57 (s, 1H, CH), 7.55 (s, 1H, aromatic), 7.41e7.49 (m, 4H,
aromatic), 6.87 (d, J ¼ 8.0 Hz, 1H, aromatic), 6.51 (d, J ¼ 8.0 Hz, 1H,
2H, CH2). 13C NMR (DMSO-d6, 100 MHz)
d: 157.4, 155.6, 152.8, 130.6,
aromatic), 6.50 (s, 1H, aromatic). 13C NMR (DMSO-d6, 100 MHz)
d:
127.7, 127.5, 115.1, 109.7, 105.6, 105.1, 76.2, 30.3.
152.4, 141.9, 140.1, 129.15, 129.07, 129.0, 126.7, 120.2, 119.0, 113.6,
112.5, 96.1.
6.2.3.6. 4-(7-Hydroxy-2,3-dihydro-benzo[1,4]oxathiin-2-yl)-ben-
zene-1,2-diol (6). Purified by column chromatography with petro-
leum ether/ethyl acetate as eluent. Yield 7%. Colorless oil. 1H NMR
6.2.3.13. 4-(6-Methoxybenzo[1,4]oxathiin-2-yl)-phenol
(13).
Purified by column chromatography with petroleum ether/ethyl
(DMSO-d6, 400 MHz) d: 9.30 (s, 1H, OH), 8.98 (s, 1H, OH), 8.96 (s, 1H,
acetate as eluent. Yield 9%. Pale orange oil. 1H NMR (DMSO-d6,
OH), 6.87 (d, J ¼ 8.4 Hz, 1H, aromatic), 6.79 (s, 1H, aromatic), 6.73 (d,
J ¼ 8.4 Hz, 1H, aromatic), 6.69 (d, J ¼ 8.0 Hz, 1H, aromatic), 6.36 (dd,
J ¼ 2.0 Hz, 8.4 Hz, 1H, aromatic), 6.28 (d, J ¼ 2.0 Hz, 1H, aromatic),
5.00 (t, J ¼ 5.2 Hz, 1H, CH), 3.12 (d, J ¼ 5.2 Hz, 2H, CH2). 13C NMR
400 MHz)
d
: 7.59 (s, 1H, OH), 7.58 (s, 1H, CH), 7.54 (d, J ¼ 8.0 Hz, 2H,
aromatic), 7.24 (d, J ¼ 8.0 Hz, 2H, aromatic), 6.93 (d, J ¼ 8.8 Hz, 1H,
aromatic), 6.70 (d, J ¼ 2.8 Hz, 1H, aromatic), 6.66 (dd, J ¼ 8.8 Hz,
J ¼ 2.8 Hz, 1H, aromatic), 3.70 (s, 3H, OCH3). 13C NMR (DMSO-d6,
(DMSO-d6, 100 MHz)
d
: 155.6, 152.8, 145.3, 145.2, 131.2, 127.5, 117.4,
100 MHz)
d: 159.2, 158.0, 152.5, 149.5, 128.9, 128.2, 126.0, 113.8,
115.3, 113.7, 109.7, 105.6, 105.1, 76.2, 30.4.
112.6, 111.5, 96.2, 58.7.

236
H.-W. Lai, Z.-Q. Liu / European Journal of Medicinal Chemistry 81 (2014) 227e236
6.3. Scavenging radicals
Acknowledgment
The tests for thiaflavans, trolox, and catechol to scavenge
ABTSþꢁ, DPPH, and galvinoxyl radicals were performed following
previous description [22]. ABTS salt and K2S2O8 were dissolved in
2.0 mL of water, in which the final concentration of ABTS and
K2S2O8 were 4.0 mM and 1.41 mM, respectively. The aforemen-
tioned solution was kept for 16 h and then diluted by 100 mL of
Financial support from Jilin Provincial Science and Technology
Department, China, is acknowledged gratefully (20130206075GX).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.081.
ethanol to give ABTSD solution, whose absorbance was around
ꢁ
0.70 at 734 nm (εABTS ¼ 1.6 ꢄ 104 Mꢀ1 cmꢀ1). DPPH and galvinoxyl
þ
ꢁ
radical were dissolved in 50 mL of ethanol, and their absorbances
were around 1.00 at 517 nm (εDPPH ¼ 1.09 ꢄ 104 Mꢀ1 cmꢀ1) and
428 nm (εgalvinoxyl ¼ 1.4 ꢄ 105 Mꢀ1 cmꢀ1), respectively. Dimethyl
sulfoxide (DMSO) solutions of thiaflavans, trolox, and catechol
(0.1 mL) were added to 1.9 mL of radical solutions. The final con-
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AAPH-induced oxidation of DNA was performed following pre-
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in phosphate buffered solution (PBS: 8.1 mM Na2HPO4, 1.9 mM
NaH2PO4,10.0 mM EDTA) with the final concentration as 2.0 mg/mL
and 40 mM, respectively. The aforementioned solution also con-
tained various concentrations of thiaflavans (dissolved in 0.1 mL of
DMSO). The same volume of DMSO was also contained in the blank
experiment as the control. The solution was then dispatched into
test tubes with each one containing 2.0 mL. These tubes were
heated at 37 ꢃC in a water bath for initiating the oxidation of DNA.
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solution (1.00 g TBA and 0.40 g NaOH dissolved in 100 mL of PBS)
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heating in a boiling water bath for 15 min. After the tubes were
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added and shaken vigorously to extract thiobarbituric acid reactive
species (TBARS). Finally, the tubes were centrifuged for a few mi-
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