Synthesis of licochalcones and inhibition effects on radical-induced oxidation of DNA

Article abstract of DOI:10.1007/s00044-012-0282-9

This study deals with the influence of phenolic hydroxyl groups on the antioxidant capacities of licochalcones. (E)-3-[5-(1,2-Dimethyl-2-propenyl)-4- hydroxy-3-methoxyphenyl]-1-(4-hydroxyphenyl)-2-propen-1-one (HMP), (E)-3-[5-(1,2-dimethyl-2-propenyl)-4-hydroxy-3-methoxyphenyl]-1-(2, 4-dihydroxyphenyl)-2-propen-1-one (DHM), and (E)-3-[5-(1,2-dimethyl-2-propenyl)- 4-hydroxy-3-methoxyphenyl]-1-(2,4,6-trihydroxyphenyl)-2-propen-1-one (HMT) were synthesized via abnormal Claisen rearrangement and Claisen-Schmidt condensation. The antioxidant capacities were evaluated in hydroxyl radical ( ^?OH)-, Cu²⁺/glutathione (GSH)-, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. It was found that only HMP possessed weak ability to inhibit ^?OH-induced oxidation of DNA, and DHM and HMT cannot protect DNA against the oxidation. All the licochalcones employed herein can retard Cu ²⁺/GSH-induced oxidation of DNA, the activity followed the order of HMP > DHM > HMT. Hence, more hydroxyl groups did not ameliorate the antioxidant effectiveness in the aforementioned experimental systems. On the other hand, HMT, DHM, and HMP can scavenge 5.61, 2.42, and 1.68 radicals, respectively, when they were applied to protect DNA against AAPH-induced oxidation. This result demonstrated that more hydroxyl groups enhanced the antioxidant capacities of licochalcones in this case. The obtained information was beneficial for designing novel licochalcones.

Full text of DOI:10.1007/s00044-012-0282-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2847–2854
DOI 10.1007/s00044-012-0282-9
ORIGINAL RESEARCH
Synthesis of licochalcones and inhibition effects on radical-
induced oxidation of DNA
•
Jianghua He Jizhen Li Zai-Qun Liu
•
Received: 11 March 2012 / Accepted: 15 October 2012 / Published online: 30 October 2012
ꢀ Springer Science+Business Media New York 2012
Abstract This study deals with the influence of pheno-
lic hydroxyl groups on the antioxidant capacities of lic-
ochalcones. (E)-3-[5-(1,2-Dimethyl-2-propenyl)-4-hydroxy
-3-methoxyphenyl]-1-(4-hydroxyphenyl)-2-propen-1-one
(HMP), (E)-3-[5-(1,2-dimethyl-2-propenyl)-4-hydroxy-3-
methoxyphenyl]-1-(2,4-dihydroxyphenyl)-2-propen-1-one
(DHM), and (E)-3-[5-(1,2-dimethyl-2-propenyl)-4-hydroxy
-3-methoxyphenyl]-1-(2,4,6-trihydroxyphenyl)-2-propen-
1-one (HMT) were synthesized via abnormal Claisen rear-
rangement and Claisen–Schmidt condensation. The anti-
oxidant capacities were evaluated in hydroxyl radical
(•OH)-, Cu2?/glutathione (GSH)-, and 2,20-azobis(2-ami-
dinopropane hydrochloride) (AAPH)-induced oxidation of
DNA. It was found that only HMP possessed weak ability
information was beneficial for designing novel
licochalcones.
Keywords Licochalcone ꢀ Antioxidant ꢀ Free radical ꢀ
Oxidation of DNA
Introduction
Modern medicine also advocates using natural products to
treat some diseases (Bhattacharya et al., 2009). After phar-
macological functions of traditional herbs have been proved
clinically (Udompataikul and Srisatwaja, 2011), active
ingredients are isolated in order to test whether these com-
poundscan be applied to treat other diseases (Qiu et al., 2010).
The roots and rhizomes of licorice (Glycyrrhiza) species
attract much research attention because they exhibit many
pharmacological properties such as antiinflammatory, anti-
viral, antimicrobial, antioxidation, anticancer, immunomod-
ulatory, hepatoprotective, and cardioprotective effects.
Licorice species contain some bioactive ingredients such as
flavonoids, isoflavones, coumarins, stilbenoids, and miscel-
laneous compounds. Especially, they involve a series of spe-
cial chalcones, called licochalcones (Asl and Hosseinzadeh,
2008). Althoughbioactivitiesofchalconeshavebeenrevealed
by a large amount of experimental results (Dimmock et al.,
1999), as shown in Scheme 1, different positions of iso-pen-
tenyl and phenolic hydroxyl groups make licochalcones
exhibit various bioactivities (Nowakowska, 2007). For
example, licochalcone A inhibits TEL-Jak2-mediated trans-
formation through the specific inhibition of Stat3 activation
(Funakoshi-Tago et al., 2008) and induces cell cycle arrest or
apoptosis in cancer cells (Yo et al., 2009; Xiao et al., 2011;
Kim et al., 2010). Licocalchone C inhibits lipopolysacchar-
ide-mediated inflammation by improving antioxidant activity
•
to inhibit OH-induced oxidation of DNA, and DHM and
HMT cannot protect DNA against the oxidation. All the
licochalcones employed herein can retard Cu2?/GSH-
induced oxidation of DNA, the activity followed the order
of HMP [ DHM [ HMT. Hence, more hydroxyl groups
did not ameliorate the antioxidant effectiveness in the
aforementioned experimental systems. On the other hand,
HMT, DHM, and HMP can scavenge 5.61, 2.42, and 1.68
radicals, respectively, when they were applied to protect
DNA against AAPH-induced oxidation. This result dem-
onstrated that more hydroxyl groups enhanced the antiox-
idant capacities of licochalcones in this case. The obtained
J. He ꢀ J. Li (&) ꢀ Z.-Q. Liu (&)
Department of Organic Chemistry, College of Chemistry,
Jilin University, No. 2519 Jiefang Road,
Changchun 130021, China
e-mail: ljz@jlu.edu.cn
Z.-Q. Liu
e-mail: zaiqun-liu@jlu.edu.cn
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Med Chem Res (2013) 22:2847–2854
Scheme 1 Structures of some
typical licochalcones
OH
OH
OH
OH
B
B
B
HO
HO
HO
A
A
A
O
O
O
O
O
O
licochalcone A
licochalcone C
licochalcone B
OH
OH
B
HO
B
HO
OH
A
O
A
O
O
O
licochalcone E
licochalcone D
and regulating nitric oxide expression (Franceschelli et al.,
2011). Licochalcone E reduces chronic allergic contact der-
matitis and inhibits IL-12p40 production through down-reg-
ulation of NF-jB (Cho et al., 2010).
The lipid, DNA, membrane, and protein are susceptible to be
oxidized by radicals (Benigni and Bossa, 2011), thus, radicals
in the metabolism (Halliwell, 2009) and the environment
(Mena et al., 2009) are the major pathology of many diseases
(van Horssen et al., 2011). Therefore, antioxidant properties of
synthetic compounds are usually estimated in the experimental
system of radical-induced oxidation of DNA. 2,20-Azobis(2-
amidinopropane hydrochloride) (AAPH, R–N=N–R, R=–
CMe2C(=NH) NH2), Cu2?/glutathione (GSH), and hydroxyl
radical usually act as radical resources (Shao et al., 2010; Reed
and Douglas, 1991; Galano et al., 2010). Presented here is a
study on the abilities of licochalcones to protect DNA against
Cu2?/GSH-, •OH-, and AAPH-induced oxidation.
Licochalcones along with the glycoside of licochalcones
can be synthesized by biological or organic methods (Zhang
et al., 2011; Sharma et al., 2011; Deng et al., 2010). The
function of iso-pentenyl group and the position of phenolic
hydroxyl group at B ring are the major concern in the study
on the structure–activity-relationship of licochalcone (Dao
et al., 2011). The influence of phenolic hydroxyl group at A
ring is worthy to be explored in detail. Thus, the aim of this
study is to clarify the influence of the number of hydroxyl
group at A ring on the antioxidant capacity of licochalcone E.
Three licochalcones including (E)-3-[5-(1,2-dimethyl-2-
propenyl)-4-hydroxy-3-methoxyphenyl]-1-(4-hydroxyl phenyl)
-2-propen-1-one (HMP), (E)-3-[5-(1,2-dimethyl-2-prope-
nyl)-4-hydroxy-3-methoxyphenyl]-1-(2,4-dihydroxy phenyl)
-2-propen-1-one (DHM), and (E)-3-[5-(1,2-dimethyl-2-
propenyl)-4-hydroxy-3-methoxyphenyl]-1-(2,4,6-trihydroxy-
phenyl)-2-propen-1-one (HMT) were synthesized as shown
in Scheme 2. The reaction applied to construct the skeleton
of chalcone is Claisen–Schmidt condensation under basic
condition (Montes-Avila et al., 2009). Because phenolic
hydroxyl groups are readily oxidized under basic condition,
they are protected before the Claisen–Schmidt condensation
(Liu et al., 2011). The key step in the synthesis of licochal-
cone E is to introduce iso-pentenyl group into B ring (Liu
et al., 2010). Recently, we reported a facile method to pre-
pare (dl)-licochalcone E via an abnormal Claisen rear-
rangement and Claisen–Schmidt condensation under acidic
condition (Li et al., 2010a).
Experimental
Materials and instrumentation
AAPH, glutathione (GSH), and the naked DNA sodium salt
were purchased from ACROS ORGANICS, Geel, Bel-
gium. Other reagents were of analytical grade and used
directly. The structures of the obtained products were
identified by 1H and 13C NMR (Varian Mercury 300 NMR
spectrometer). The purity and the molecular weight were
recorded on a high performance liquid chromatography
(HPLC) equipped with mass spectra (MS) system.
Synthesis of licochalcones
As shown in Scheme 2, the compound 2 was produced by
the condensation of 4-hydroxy-3-methoxybenzaldehyde (1)
and prenyl bromide. K2CO3 powder (2.8 g, 20.3 mmol)
was added to anhydrous acetone solution of 1 (2.0 g,
The following study is to select suitable experimental sys-
tem for estimating the antioxidant activities of licochalcones.
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Med Chem Res (2013) 22:2847–2854
2849
O
O
OH
OH
O
N
O
Cl
Br
H
H
H
185-1900C
CH3COCH3, K2CO3
O
CH3COCH3, K2CO3
r.t.
r.t.
O
O
O
2
3
1
R3
R2
OH
O
O
O
O
R3
R2
R1
O
1 M HCl-CH3OH
H
HMP, R1 = R2 = H, R3 = OH
DHM, R1 = R3 = OH, R2 = H
HMT, R1 = R2 = R3 = OH
HCl-C2H5OH, 00C-r.t.
or
O
R1
O
KOH-C2H5OH
licochalcones
4
Scheme 2 Synthetic routine of licochalcones
13.2 mmol) at room temperature, and then prenyl bromide
(1.80 mL, 15.3 mmol) was added dropwisely. The mixture
was kept stirring until 1 was not detected by thin layer
chromatography (TLC). K2CO3 was removed by filtration,
and the solvent was evaporated under vacuum. The residue
was recrystallized with petroleum ether to afford 2.4 g of
compound 2, yield 86 %.
Licochalcones were prepared by Claisen–Schmidt con-
densation. KOH solution (3.0 g KOH dissolved in 2.0 mL
of water and 2.5 mL of ethanol) was dropwisely added to
ethanolic solution (10 mL) of hydroxyl-protected aceto-
phenone (1.2 mmol) and compound 4 (263 mg, 1.0 mmol)
under nitrogen atmosphere and was kept in an ice bath until
compound 4 was not detected by TLC. The reaction mix-
ture was added to 5 g of crushed ice, neutralized with 3 M
HCl and extracted with ethyl acetate (3 9 10 mL). The
combined ethyl acetate layer was dried over anhydrous
Na2SO4, and the solvent was removed under vacuum. The
residual oil was purified over silica gel column with ethyl
acetate and petroleum ether being eluent (1:10, v:v) to
yield *75 % hydroxyl-protected licochalcones.
DHM and HMT were obtained by hydrolyzing the pro-
tective groups. HCl aqueous solution (1 M, 3 mL) was
added to metholic solution (10 mL) of hydroxyl-protected
licochalcones (1.0 mmol) and stirred at 35 ꢁC until hydro-
xyl-protected licochalcones was not detected by TLC.
Then, the reaction mixture was diluted with distilled water
(15 mL) and extracted with ethyl acetate (3 9 10 mL). The
combined ethyl acetate layer was dried over anhydrous
Na2SO4, and the solvent was removed under vacuum. The
residual oil was purified over silica gel column with ethyl
acetate and petroleum ether being eluent (2:5, v:v) to give
400 mg of DHM or 450 mg of HMT, yield *90 %.
The compound 3 was produced from compound 2 by an
abnormal Claisen rearrangement. The compound 2 (2.2 g,
10.0 mmol) was dissolved in freshly distilled N,N-dimethyl-
aniline (8 mL) under nitrogen and then was heated at 185 ꢁC
for26 hinasealedtube. Aftercooledtoroomtemperature, the
reaction mixture was acidified with 10 % HCl aqueous solu-
tiontopH5–7andextractedwith ether. Theethersolutionwas
washed with saturated NaHCO3 and NaCl solution and then
dried over anhydrous Na2SO4. After the solvent was removed
under vacuum, the residue was purified over silica gel column
with petroleum ether and acetone (30:1, v:v) being eluant to
give 0.71 g of compound 3, yield 32 %.
The hydroxyl groups in compound 3 and acetophenone
were protected before Claisen–Schmidt condensation under
basic condition. For example, K2CO3 (1.04 g, 7.5 mmol)
was added to 15 mL of acetone solution of 3 (660 mg,
3 mmol) and stirred for 15 min. CH3OCH2Cl (290 mg,
3.6 mmol) was added dropwisely and stirred for 12 h until
compound 3 was not detected by TLC. The reaction mix-
ture was diluted with distilled water (20 mL) and then
extracted with ethyl acetate (3 9 10 mL). The combined
ethyl acetate layer was dried over anhydrous Na2SO4, and
the solvent was removed under vacuum. The residual oil
was purified over silica gel column with petroleum ether
and ethyl acetate being eluent (15:1, v:v) to give 821 mg of
compound 4, yield 91 %.
(E)-3-[5-(1,2-Dimethyl-2-propenyl)-4-hydroxy-3-
methoxyphenyl]-1-(2,4-dihydroxyphenyl)-2-propen-
1-one (DHM)
m.p. 161–163 ꢁC; MS: 355.1 (M?H?); 1H NMR
(300 MHz, DMSO-d6): d 1.28–1.30 (d, J = 6 Hz, 3H),
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Med Chem Res (2013) 22:2847–2854
1.60 (s, 3H), 3.77–3.84 (q, J = 21 Hz, 1H), 3.92 (s, 3H),
4.84 (s, 2H), 6.28–6.29 (d, J = 3 Hz, 1H), 6.41–6.45 (d,
J = 9 Hz, 1H), 7.07–7.08 (d, J = 3 Hz, 1H), 7.38–7.46
(dd, J = 9 Hz, J = 3 Hz, 1H), 7.73–7.74 (d, J = 3 Hz,
2H), 8.18–8.21 (d, J = 9 Hz, 1H), 9.25 (s, 1H), 10.65 (s,
1H), 13.64 (s, 1H); 13C NMR (75 MHz, CD3OD): d 19.05,
22.21, 38.67, 56.25, 90.46, 103.82, 107.63, 108.07, 110.40,
114.33, 117.31, 122.69, 126.58, 131.46, 132.03, 145.78,
146.44, 148.36, 163.36, 166.32, 192.19.
95.37, 113.74, 115.31, 120.72, 124.09, 127.75, 131.19,
134.92, 136.42, 136.79, 149.47, 152.03, 153.07, 153.60,
167.33, 192.52.
•OH- and Cu2?/GSH-induced oxidation of DNA tests
•OH was produced by mixing tetrachlorohydroquinone
(TCHQ, dissolved in DMSO as the stock solution) and
H2O2 (Zhu et al., 2000). DNA and H2O2 were dissolved in
phosphate buffered solution (PBS1: 8.1 mM Na2HPO4,
1.9 mM NaH2PO4, 10.0 mM EDTA). A mixture of 2.0 mg/
mL DNA, 4.0 mM TCHQ, 2.0 mM H2O2, and 0.20 mM
licochalcones (dissolved in DMSO as the stock solution)
was dispatched into test tubes with each one containing
2.0 mL. The test tubes were incubated at 37 ꢁC for 30 min
and cooled immediately. Then, 1.0 mL of thiobarbituric
acid (TBA) solution (1.00 g of TBA and 0.40 g of NaOH
dissolved in 100 mL of PBS1) and 1.0 mL of 3.0 % tri-
chloroacetic acid aqueous solution were added, and the test
tubes were heated in boiling water for 30 min and cooled to
room temperature. n-Butanol (1.5 mL) was added and
shaken vigorously to extract thiobarbituric acid reactive
substance (TBARS) whose absorbance was measured at
535 nm. The absorbances in the control experiment and in
the presence of licochalcones were assigned as A0 and
(E)-3-[5-(1,2-Dimethyl-2-propenyl)-4-hydroxy-3-
methoxyphenyl]-1-(2,4,6-trihydroxyphenyl)-2-propen-
1-one (HMT)
m.p. 115–117 ꢁC; MS: 371.1 (M?H?); 1H NMR
(300 MHz, DMSO-d6): d: 1.25–1.27 (d, J = 6 Hz, 3H),
1.60 (s, 3H), 3.75–3.82 (q, J = 21 Hz, 1H), 3.86 (s, 3H),
4.85–4.88 (d, J = 9 Hz, 2H), 5.84 (s, 2H), 7.01–7.02 (d,
J = 3 Hz, 1H), 7.12–7.13 (d, J = 3 Hz, 1H), 7.61–7.67 (d,
J = 18 Hz, 1H), 7.96–8.01 (d, J = 18 Hz, 1H), 9.23 (s,
1H), 10.38 (s, 1H), 12.55 (s, 1H), 12.57 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d: 14.49, 18.90, 21.95, 37.59, 55.70,
66.31, 94.78, 104.15, 109.10, 109.80, 120.29, 123.93,
125.83, 131.54, 142.88, 146.43, 147.41, 148.04, 164.27,
164.59, 191.52.
A facile method was used to prepare HMP without
hydroxyl-protected. 4-Hydroxyacetophenone (0.27 g,
2.0 mmol) and compound 3 (0.40 g, 1.82 mmol) were
dissolved in anhydrous ethanol (2 mL) and cooled in an
ice-water bath, then 5 mL of 1.5–2.0 M HCl ethanolic
solution was added dropwisely under stirring. The mixture
was stirred at 0–5 ꢁC for 2 h until compound 3 was not
detected by TLC. The surplus HCl and ethanol were
removed under vacuum, and then the residue was extracted
with ethyl acetate and washed with water, saturated
NaHCO3, and water in turn. After the residue was dried
over Na2SO4, and the solvent was removed under vacuum,
the crude HMP was purified over silica gel column with
petroleum and acetone (10:1, v:v) being eluent to give
0.45 g of HMP, yield 72 %.
Adetect, respectively. The abilities of licochalcones to inhibit
the oxidation of DNA were indicated by Adetect/A0 9 100.
Cu2?/GSH-induced oxidation of DNA was carried out
following a previous report (Reed and Douglas, 1991) with
a little modification (Feng and Liu, 2011). In brief, DNA,
CuSO4, and GSH were dissolved in phosphate buffered
solution (PBS2: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4),
and licochalcones were dissolved in DMSO. Then, a
mixture of 2.0 mg/mL DNA, 5.0 mM Cu2?, 3.0 mM GSH,
and 0.2 mM licochalcones was dispatched into test tubes
with each one containing 2.0 mL. The test tubes were
incubated at 37 ꢁC. Three of them were taken out at
appropriate interval, and PBS2 solution of EDTA (1.0 mL,
30.0 mM) was added to chelate Cu2?, followed by adding
1.0 mL of TBA solution and 1.0 mL of 3.0 % trichloro-
acetic acid aqueous solution. The tubes were heated in
boiling water for 30 min and cooled to room temperature;
1.5 mL of n-butanol was added and shaken vigorously to
extract TBARS whose absorbance was measured at
535 nm. The absorbance of TBARS was plotted vs the
incubation period.
(E)-3-[5-(1,2-Dimethyl-2-propenyl)-4-hydroxy-3-
methoxyphenyl]-1-(4-hydroxylphenyl)-2-propen-1-one
(HMP)
m.p. 170–172 ꢁC; MS: 339.1 (M?H?); 1H NMR (300 MHz,
DMSO-d6): d: 1.27–1.30 (d, J = 9 Hz, 3H), 1.60 (s, 3H),
3.77–3.83 (q, J = 18 Hz, 1H), 3.91 (s, 3H), 4.84 (s, 2H),
6.88–6.91 (d, J = 9 Hz, 2H), 7.03–7.04 (d, J = 3 Hz, 1H),
7.33–7.41 (dd, J = 9 Hz, J = 3 Hz, 1H), 7.55–7.72 (dd,
J = 15 Hz, J = 15 Hz, 2H), 8.04–8.08 (dd, J = 3 Hz,
J = 3 Hz, 2H), 9.14 (s, 1H), 10.33 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d: 24.33, 27.38, 43.19, 50.63, 61.51,
AAPH-induced oxidation of DNA test
AAPH-induced oxidation of DNA was carried out fol-
lowing a previous report (Zhao and Liu, 2009). In brief, a
mixture of 2.0 mg/mL DNA, 40 mM AAPH, and various
concentrations of licochalcones (dissolved in DMSO as the
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Med Chem Res (2013) 22:2847–2854
2851
stock solution) was dispatched into test tubes with each one
containing 2.0 mL. The following operation was the same
as in Cu2?/GSH-induced oxidation of DNA test except the
heating period was 15 min after TBA and trichloroacetic
acid were added. The absorbance of TBARS was plotted
versus the incubation period.
110.0
100.0
90.0
105.8
100.0
98.2
91.3
Statistical analysis
All the data were the average value from at least three
independent measurements with the experimental error
within 10 %. The equations were analyzed by one-way
ANOVA using Origin 7.5 professional software, and
p \ 0.001 indicated a significance difference.
Fig. 1 Percentages of TBARS in the mixture of 2.0 mg mL-1 DNA,
4.0 mM tetrachlorohydroquinone, 2.0 mM H2O2, and 0.20 mM
licochalcones after incubated for 30 min
unsubstituted positions. The obtained result implies that
more phenolic hydroxyl groups are not beneficial for
increasing the antioxidant activity of licochalcones to
Results and discussion
Effects of licochalcones on OH- and Cu2?/GSH-
•
•
inhibit OH-induced oxidation of DNA.
induced oxidation of DNA
Cu(II) can oxidize GSH to form corresponding radical
(GS.) when the concentration of Cu(II) is close to that of
GSH. GS. is able to destroy DNA to form carbonyl species,
thus, the absorbance of TBARS in the blank experiment
increases with the incubation period as shown in Fig. 2.
Although the addition of 0.20 mM licochalcones cannot
completely inhibit the increase of the absorbance of
TBARS, the position of absorbance lines are lower than
that of the blank experiment. Thus, licochalcones are able
to hinder Cu2?/GSH-induced oxidation of DNA. Further-
more, the position of the absorbance line from top to bot-
tom is HMT, DHM, and HMP, revealing that the ability to
retard Cu2?/GSH-induced oxidation of DNA follows
HMP [ DHM [ HMT. The mechanism of DNA damage
caused by Cu2? and GSH is divided into two processes. In
addition to the formation of GS., Cu(I) deriving from the
•
Cu2?/GSH and OH can destroy the supercoiled structure
of DNA, leading to the formation of carbonyl species (Ohta
et al., 2000; Battin and Brumaghim, 2008). Thus, the
experimental systems of Cu2?/GSH- and •OH-induced
oxidation of DNA are usually applied to evaluate the
antioxidant capacity. Because the produced carbonyl spe-
cies can be conveniently detected after reacting with
thiobarbituric acid (Dedon, 2008), they are also called
thiobarbituric acid reactive substance (TBARS). As can be
seen in Fig. 1, the absorbance of TBARS was assigned as
100 % when 2.0 mg mL-1 DNA is mixed with 4.0 mM
TCHQ and 2.0 mM H2O2 for 30 min. The absorbance in
the presence of 0.20 mM licochalcones was compared with
that in the blank experiment to express the effects of lic-
•
ochalcones on OH-induced oxidation of DNA.
Figure 1 reveals that HMT does not exert inhibitive
effect on •OH-induced oxidation of DNA because the
percentage of TBARS in the presence of HMT (98.2 %) is
closed to that of the blank experiment (100 %). More
hydroxyl groups cannot enhance the antioxidant ability of
blank
HMP
DHM
HMT
0.6
•
licochalcones to inhibit OH-induced oxidation of DNA.
However, DHM even increases the percentages of TBARS
•
0.4
to 105.8 %, indicating that DHM improves OH-induced
oxidation of DNA. On the other hand, HMP decreases the
percentage of TBARS to 91.3 %. Thus, more hydroxyl
groups convert licochalcones into prooxidants to promote
•OH-induced oxidation of DNA. High activity of •OH may
oxidize multi-hydroxyl-substituted licochalcones to form
radicals, which are able to initiate additional oxidation of
0.2
0
50
100
150
Incubation period (min)
•
DNA. On the other hand, OH can add to benzene ring by
Fig. 2 The increase of the absorbances at 535 nm in the absence and
presence of 0.20 mM licochalcones in the mixture of 2.0 mg/mL
DNA, 5.0 mM Cu2?, and 3.0 mM GSH
substituting one hydrogen atom of benzene (Lewis et al.,
1988). HMP may react with •OH because it possesses more
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Med Chem Res (2013) 22:2847–2854
Fig. 3 The increase of the
absorbance of TBARS in the
mixture of 40 mM AAPH,
2.0 mg mL-1 DNA, and various
concentrations of licochalcones
blank
50 µM
100 µM
150 µM
200 µM
blank
DHM
HMT
HMP
blank
50 µM
100 µM
150 µM
200 µM
100 µM
200 µM
300 µM
400 µM
1.2
0.8
0.4
1.2
1.2
0.8
0.4
0.8
0.4
tinh
0
300
600
0
300
600
0
300
600
Incubation period (min)
reaction between Cu2? and GSH generates a complex with
DNA (DNA-Cu(I)-OOH) in the presence of oxygen. The
oxidative products are generated from the reaction between
DNA-Cu(I)-OOH and GS. (Reed and Douglas, 1991). As
reported in a literature, hydroxycinnamic acids show pro-
oxidant activities in the mixture of Cu(II) and DNA
because more hydroxyl groups reduce Cu(II) to form Cu(I),
and Cu(I) improves the formation of DNA-Cu(I)-OOH
(Zheng et al., 2008). Thus, more hydroxyl groups may
decrease the antioxidant effectiveness of licochalcones in
Cu2?/GSH-induced oxidation of DNA.
HMT
375
250
HMP
DHM
125
125
250
375
Concentration (µM)
Effects of licochalcones on AAPH-induced oxidation
of DNA
Fig. 4 Relationships between tinh and concentrations of licochal-
cones in AAPH-induced oxidation of DNA
The radical generated from the decomposition of AAPH is
able to oxidize guanine bases in DNA, leading to the for-
mation of carbonyl species (Niki, 2010). As shown in
Fig. 3, the increase of the absorbance of TBARS in the
blank experiment indicates that the formation of carbonyl
species is linearly related to the incubation period when
DNA undergoes the oxidation induced by AAPH.
Table 1 Quantitative relationships between the inhibition period
(tinh) and the concentrations of licochalcones in AAPH-induced oxi-
dation of DNA
Licochalcone
tinh (min) = (n/Ri) [licochalcone (lM)]
n
HMP
DHM
HMT
tinh = 0.50 ( 0.03) [HMP] ? 8.5 ( 0.43)
tinh = 0.72 ( 0.04) [DHM] ? 60.5 ( 3.03)
tinh = 1.67 ( 0.08) [HMT] ? 61.0 ( 3.05)
1.68 ( 0.08)
2.42 ( 0.12)
5.61 ( 0.28)
Figure 3 also outlines that the increase of the absorbance
of TBARS is hindered by adding licochalcones, thus, lic-
ochalcones are able to retard the formation of TBARS.
Especially, with the increase of the concentration of lic-
ochalcones, the absorbance lines of TBARS bend at the
beginning of the reaction. The cross-point between two
tangents indicates a period of the oxidation of DNA
inhibited by licochalcones, called inhibition period (tinh).
Figure 4 outlines the relationship between tinh and the
concentrations of licochalcones, and the quantitative
equations are listed in Table 1.
The value of n is the product of the coefficient of tinh * [licochalcone] and
Ri = 1.4 9 10-6 9 40 mM s-1 = 3.36 lM min-1 when 40 mM AAPH is
employed
the concentration of HMT. In view of chemical kinetics,
tinh correlates proportionally with the concentration of the
antioxidant as shown in Eq. (1) (Zennaro et al., 2007).
tinh ¼ ðn=RiÞ ½antioxidantꢁ
ð1Þ
The stoichiometric factor (n) refers to the number of the
radical-propagation terminated by one molecule of the
antioxidant, and Ri is the radical-initiation rate. We have
applied this equation to estimate the antioxidant abilities of
homoisoflavonoids in AAPH-induced oxidation of DNA (Li
et al., 2010b). It is assumed that Ri is equal to the radical-
The linear relationship between concentrations of lic-
ochalcones and tinh indicates that licochalcones protect
DNA against AAPH-induced oxidation of DNA concen-
tration dependently. The high coefficient in the equation of
HMT implies that tinh is much sensitive to the variation of
123

Med Chem Res (2013) 22:2847–2854
2853
generation rate (Rg = (1.4 0.2) 9 10-6 [AAPH] s-1
(Bowry and Stocker, 1993)) because water-soluble sodium
salt of DNA and AAPH make AAPH attack DNA at the same
phase. The values of n of HMP, DHM, and HMT are the
Dimmock JR, Elias DW, Beazely MA, Kandepu NM (1999)
Bioactivities of chalcones. Curr Med Chem 6:1125
Feng J-Y, Liu Z-Q (2011) Feruloylacetone as the model compound of
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Franceschelli S, Pesce M, Vinciguerra I, Ferrone A, Riccioni G,
Patruno A, Grilli A, Felaco M, Speranza L (2011) Licocalchone-
C extracted from Glycyrrhiza Glabra inhibits lipopolysacchar-
ide-interferon-c inflammation by improving antioxidant condi-
tions and regulating inducible nitric oxide synthase expression.
Molecules 16:5720
Funakoshi-Tago M, Tago K, Nishizawa C, Takahashi K, Mashino T,
Iwata S, Inoue H, Sonoda Y, Kasahara T (2008) Licochalcone A
is a potent inhibitor of TEL-Jak2-mediated transformation
through the specific inhibition of Stat3 activation. Biochem
Pharmacol 76:1681
product of the coefficients in the equation of tinh
*
[licochalcones] and
Ri = Rg = 1.4 9 10-6 9 40 mM
s-1 = 3.36 lM min-1. The results are 1.68, 2.42, and 5.61,
respectively. HMT can terminate 5.61 radical-propagation;
DHM and HMP can trap 2.42 and 1.68 radicals, respectively.
Hence, more hydroxyl groups enhance the ability of
licochalcone to inhibit AAPH-induced oxidation of DNA.
´
Galano A, Macıas-Ruvalcaba NA, Campos ONM, Pedraza-Chaverri J
Conclusion
(2010) Mechanism of the OH radical scavenging activity of
nordihydroguaiaretic acid: a combined theoretical and experi-
mental study. J Phys Chem B 114:6625
The behaviors of licochalcones depend upon the kinds of
initiators for causing the oxidation of DNA. More hydroxyl
groups do not increase the antioxidant capacities of licoch-
alcones in the case of •OH as the initiator. Furthermore, few
hydroxyl group exhibits relative high capacity when lic-
ochalcones protect DNA against Cu2?/GSH-induced oxi-
dation. On the contrary, more hydroxyl groups increase the
abilities of licochalcones to inhibit AAPH-induced oxidation
of DNA. The obtained information is beneficial for designing
the structure in the synthesis of licochalcones.
Halliwell B (2009) The wanderings of a free radical. Free Radical
Biol Med 46:531
Kim YH, Shin EK, Kim DH, Lee HH, Park JHY, Kim J-K (2010)
Antiangiogenic effect of licochalcone A. Biochem Pharmacol
80:1152
Lewis JG, Stewart W, Adams DO (1988) Role of oxygen radicals in
induction of DNA damage by metabolites of benzene. Cancer
Res 48:4762
Li J, Mi Y, He J, Deng X (2010a) A short and efficient synthesis of
licochalcone E. Synlett 15:2289
Li Y-F, Liu Z-Q, Luo X-Y (2010b) Properties of synthetic homoi-
soflavonoids to reduce oxidants and to protect linoleic acid and
DNA against oxidations. J Agric Food Chem 58:4126
Liu Z, Yoon G, Cheon SH (2010) An enantioselective total synthesis
of (S)-(-)-licochalcone E: determination of the absolute config-
uration. Tetrahedron 66:3165
Acknowledgments Financial support from Jilin Provincial Foun-
dation for Natural Science, China, (201115017) is acknowledged
gratefully.
Liu Z, Lee W, Kim S-N, Yoon G, Cheon SH (2011) Design,
synthesis, and evaluation of bromo-retrochalcone derivatives as
protein tyrosine phosphatase 1B inhibitors. Bioorg Med Chem
Lett 21:3755
Mena S, Ortega A, Estrela JM (2009) Oxidative stress in environ-
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