Synthesis and antioxidant capacities of hydroxyl derivatives of cinnamoylphenethylamine in protecting DNA and scavenging radicals

Article abstract of DOI:10.3109/10715762.2010.540576

Cinnamoylphenethylamine (CNPA) derivatives including feruloylphenethylamine (FRPA), caffeoylphenethylamine (CFPA), cinnamoyltyramine (CNTA), feruloyltyramine (FRTA) and caffeoyltyramine (CFTA) were synthesized in order to investigate the influence of the number and position of hydroxyl group on Cu²⁺/glutathione (GSH) and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. The radical-scavenging properties of these CNPA derivatives were also evaluated by trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate) cationic radical (ABTS), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and galvinoxyl radical. In addition, these CNPA derivatives were tested by linoleic acid (LH)-β-carotene-bleaching experiment. The chemical kinetic was employed to treat the results from AAPH-induced oxidation of DNA and gave the order of antioxidant ability as CFTA > CFPA > FRTA > FRPA. CFTA and CFPA also possessed high abilities to inhibit Cu²⁺/GSH-mediated degradation of DNA, whereas FRPA and FRTA can protect LH against the auto-oxidation efficiently. Finally, CFPA and FRPA exhibited high activity in trapping ABTS, DPPH and galvinoxyl radicals. Therefore, the cinnamoyl group bearing ortho-dihydroxyl or hydroxyl with ortho-methoxyl benefited for CNPA derivatives to protect DNA, while hydroxyl in tyramine cannot enhance the radical-scavenging abilities of CNPA derivatives.

Full text of DOI:10.3109/10715762.2010.540576

Free Radical Research, April 2011; 45(4): 445–453
Synthesis and antioxidant capacities of hydroxyl derivatives
of cinnamoylphenethylamine in protecting DNA and
scavenging radicals
YANGYANG, ZHI-GUANG SONG & ZAI-QUN LIU
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, PR China
(Received date: 12 October 2010; Accepted date: 11 November 2010)
Abstract
Cinnamoylphenethylamine (CNPA) derivatives including feruloylphenethylamine (FRPA), caffeoylphenethylamine (CFPA),
cinnamoyltyramine (CNTA), feruloyltyramine (FRTA) and caffeoyltyramine (CFTA) were synthesized in order to investi-
gate the influence of the number and position of hydroxyl group on Cu2ꢀ/glutathione (GSH) and 2,2’-azobis(2-amidinopro-
pane hydrochloride) (AAPH)-induced oxidation of DNA.The radical-scavenging properties of these CNPA derivatives were
also evaluated by trapping 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate) cationic radical (ABTSꢀ.), 2,2’-diphenyl-1-
picrylhydrazyl radical (DPPH) and galvinoxyl radical. In addition, these CNPA derivatives were tested by linoleic acid (LH)-
b-carotene-bleaching experiment.The chemical kinetic was employed to treat the results from AAPH-induced oxidation of
DNA and gave the order of antioxidant ability as CFTA ꢁ CFPA ꢁ FRTA ꢁ FRPA. CFTA and CFPA also possessed high
abilities to inhibit Cu2ꢀ/GSH-mediated degradation of DNA, whereas FRPA and FRTA can protect LH against the auto-
oxidation efficiently. Finally, CFPA and FRPA exhibited high activity in trapping ABTSꢀ., DPPH and galvinoxyl radicals.
Therefore, the cinnamoyl group bearing ortho-dihydroxyl or hydroxyl with ortho-methoxyl benefited for CNPA derivatives to
protect DNA, while hydroxyl in tyramine cannot enhance the radical-scavenging abilities of CNPA derivatives.
Keywords: Cinnamoylphenethylamine, Ceratostigma willmottianum, free radical, oxidation, antioxidant, DNA
Introduction
hainanesis [5], Physalis sordida [6] and Sparattanthe-
lium tupiniquinorum [7], etc. The pharmacological
activities of these herbs including anti-tubercular [8],
the anti-proliferation of HL-60, U937 and Jurkat
cells [9] and the inhibition of the superoxide anion
and elastase released from human neutrophils [10]
were proved to correlate with phenolic derivatives of
CNPA and their secondary metabolites [11]. More
phenolic hydroxyl groups attaching to benzene ring
A and B in CNPA enhance the ability to inhibit
P-selectin and cyclooxygenase-I and -II expression
[12], in which the mechanism of CFTA to inhibit
cyclooxygenase-II expression was regarded to inhibit
the activities of enhancer-binding protein and
activator protein-1 transcription factors [13]. As a
consequence, phenolic derivatives of CNPA even
became popular ingredients of some commercial
Ceratostigma willmottianum is widely used in Chinese
folk medicine to treat rheumatism, traumatic injury
and parotitis. The investigation on the chemical
constituents in this plant reveals that cinnamoyl-
phenethylamine (CNPA) is the major skeleton of
the bioactive compounds [1]. The cinnamoyl group
bridges with phenethylamine by amide linkage
and the hydroxyl groups attach to benzene ring
A and/or B to form a phenolic derivative of CNPA
(see Scheme 1). Meanwhile, phenolic derivatives of
CNPA such as feruloylphenethylamine (FRPA),
caffeoylphenethylamine (CFPA), cinnamoyltyramine
(CNTA), feruloyltyramine (FRTA) and caffeoyl-
tyramine (CFTA) are also found in many other
medicinal plants including Peperomia duclouxii [2],
Aristolochia constricta [3] and mollissima [4], Erycibe
Correspondence: Zai-Qun Liu, Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road,
Changchun 130021, PR China. Email: zaiqun-liu@jlu.edu.cn
ISSN 1071-5762 print/ISSN 1029-2470 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10715762.2010.540576

446 Y. Yang et al.
NH2
O
O
R1
R1
CH3OCH2Cl
malonic acid, benzene
pyridine, piperidine
H
R3
OH
K2CO3,CH3COCH3
R2
DCC, DMAP, dry CH2Cl2
O
O
1
2
R3
O
R3
O
R1
B
N
H
5% acetic acid
R1
R2
N
H
A
O
O
3
abbreviation R1
cinnamoylphenethylamine CNPA
R2
R3
H
name
H
H
CNPA and CNTAwere prepared
by cinnamic acid and corresponding
amine in the presence of DCC, DMAP,
and dry CH2Cl2.
feruloylphenethylamine
caffeoylphenethylamine
cinnamoyltyramine
feruloyltyramine
FRPA
CFPA
CNTA
FRTA
CFTA
OCH3 OH
H
OH
H
OH
H
H
OH
OH
OH
OCH3 OH
OH OH
caffeoyltyramine
Scheme 1. Synthesis routines of CNPA derivatives.
herb medicines [14]. In addition to phenolic hydroxyl
groups trapping radicals and/or reducing oxidative
species, N-H in the amide linkage in some drugs can
also scavenge radicals directly [15]. Many works
revealed the in vivo activities of CNPA derivatives,
but the antioxidant behaviour of phenolic hydroxyl
and amide groups in CNPA derivative were still nec-
essary to be clarified in vitro. Especially, the investiga-
tion of the structure–activity relationship between
hydroxyl group and radical-scavenging property was
beneficial for understanding the antioxidant mecha-
nism of CNPA derivatives. Therefore, as shown
in Scheme 1, various hydroxyl-substituted CNPA
derivatives were prepared in order to investigate
the effects of hydroxyl and amide groups on the
degradation of DNA, during which 2,2’-azobis
(2-amidinopropane hydrochloride) (AAPH, R-Nꢂ
N-R, R ꢂ ꢃCMe2C(ꢂNH)NH2) and Cu2ꢀ/gluta-
thione (GSH) acted as initiators of the oxidation of
DNA, respectively. Moreover, the radical-scavenging
properties of these compounds were also evaluated
by interacting with 2,2’-azinobis(3-ethylbenzothiazo-
line-6-sulphonate) cationic radical (ABTSꢀ.), 2,2’-
diphenyl-1-picrylhydrazyl (DPPH) and galvinoxyl
radical, respectively. In addition, the abilities of these
compounds to protect linoleic acid (LH) were
screened by b-carotene-bleaching test.
galvinoxyl radical, naked DNA sodium salt, glutathi-
one, linoleic acid and b-carotene were purchased
from ACROS ORGANICS (Geel, Belgium) and used
as received. Other reagents were of analytical grade
and purchased from Beijing Chemical Reagent Co. (Bei-
jing, China). The structures of the obtained com-
pounds were identified by NMR spectrometer (Varian
Mercury 300).The spectra of the obtained compounds
were listed in Supporting Information (Online version
only). The obtained compounds were analyzed by
HPLC, and the purities were larger than 98.0%.
Synthesis of cinnamoylphenethylamine derivatives
Synthesis of cinnamoylphenethylamine (CNPA) and cin-
namoyltyramine (CNTA). CNPA was prepared by the
amidation between 0.296 g (0.002 mol) of cinnamic
acid and 0.28 mL of phenethylamine in 50 mL of
dry CH2Cl2 with 0.452 g (0.0022 mol) of dicyclo-
hexylcarbodiimide (DCC) as the dehydrant and
10 mg of N,N-dimethylaminopyridine (DMAP) as
the catalyst.The aforementioned mixture was stirred
at room temperature for 4 h to afford crude product,
then the crude product was purified by silica chro-
matography (ethyl acetate:petroleum ether ꢂ 1:3) to
give 0.424 g of CNPA, yield 84% and melting point:
121–122°C. 1H NMR (300 MHz, DMSO-d6):
d (ppm) 2.80 (t, J ꢂ 7.2 Hz, 2H), 3.34–3.47 (m, 2H),
6.64 (dd, J ꢂ 3 Hz, J ꢂ 15.9 Hz, 2H), 7.18–7.33
(m, 5H), 7.36–7.47 (m, 4H), 7.55 (s, 1H), 7.69 (s,
1H), 8.20 (t, J ꢂ 4.8 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 35.1, 40.3, 122.2, 126.0, 127.4,
128.3, 128.6, 128.9, 129.3, 134.9, 138.5, 139.4,
164.9.CNTA was prepared by the amidation between
0.148 g (0.001 mol) of cinnamic acid and 0.206 g
Methods
Materials
2,2’-Azobis(2-amidinopropane hydrochloride), diam-
monium salt of 2,2’-azinobis(3-ethylbenzothiazo-
line-6-sulphonate), 2,2’-diphenyl-1-picrylhydrazyl,

Antioxidant property of cinnamoylphenethylamine derivatives 447
(t, J ꢂ 5.7 Hz, 1H), 9.40 (m, 1H); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 35.2, 40.3, 55.5, 110.8, 115.6,
119.0, 121.5, 126.0, 126.4, 128.3, 128.6, 138.9,
139.5, 147.8, 148.3, 165.4. CFPA was derived from
FRPA by demethylation. Briefly, 0.596 g (0.002 mol)
of FRPA was dissolved in 10 mL of dry CH2Cl2,
then 0.374 g of AlCl3 and 10 mg of triethylbenzyl
ammonium chloride (TEBA) were added and stirred
at 5°C in an ice-bath. Finally, 0.53 mL of (0.0066 mol)
pyridine was added dropwisely under stirring. The
mixture was refluxed for 28 h and cooled to room
temperature. The pH value of the mixture was
adjusted to 1 by adding HCl aqueous solution to pro-
duce yellow solid.The mixture was extracted by ethyl
acetate. Ethyl acetate phase was washed by saturated
NaCl solution and dried over Na2SO4. After ethyl
acetate was evaporated under vacuum, the crude
product was purified by silica chromatography (ethyl
acetate:chloroform ꢂ 1:2) to afford 0.47 g of CFPA,
yield 81% and melting point: 148–150°C. 1H NMR
(300 MHz, DMSO-d6): d (ppm) 2.77 (t, J ꢂ 14.7
Hz, 2H), 3.39 (q, J ꢂ 13.2 Hz, 2H), 6.33 (dd, J ꢂ
3.6 Hz, J ꢂ 15.3 Hz, 1H), 6.74 (d, J ꢂ 8.1 Hz, 1H),
6.83 (m, 1H), 6.94 (d, J ꢂ 1.5 Hz, 1H), 7.17–7.32
(m, 6H), 8.05 (t, J ꢂ 5.6 Hz, 1H), 9.10 (s, 1H), 9.33
(s, 1H); 13C NMR (75 MHz, DMSO-d6): d (ppm)
35.2, 40.3, 113.8, 115. 7, 118.5, 120.3, 126.0, 126.4,
128.3, 128.6, 139.0, 139.5, 145.5, 147.2, 165.3.
(0.0015 mol) of tyramine in 150 mL of dry CH2Cl2
with 0.226 g (0.0011 mol) of DCC and 5 mg of
DMAP added. The reaction mixture was stirred at
room temperature for 4 h, and the crude product was
purified by silica chromatography (ethyl acetate:
chloroform ꢂ 1:4.5) to afford 0.165 g of CNTA,
yield 62% and melting point: 188–189°C. 1H NMR
(300 MHz, DMSO-d6): d (ppm) 2.65 (t, J ꢂ 7.35 Hz,
2H), 3.34 (t, J ꢂ 10.05 Hz, 2H), 6.59–6.69 (m, 3H),
7.00 (s, 1H), 7.03 (s, 1H), 7.34–7.43 (m, 4H), 7.54
(s, 1H), 7.56 (d, J ꢂ 1.5 Hz, 1H), 8.15 (t, J ꢂ 5.7
Hz, 1H), 9.18 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d (ppm) 34.4, 40.7, 115.1, 122.3, 127.5, 128.9,
129.4, 129.5, 134.9, 138.5, 155.7, 164.9.
Synthesis of feruloylphenethylamine (FRPA) and
caffeoylphenethylamine (CFPA). Vanillin (R1 ꢂ OCH3
and R2 ꢂ OH in compound 1, 4.56 g, 0.03 mol) and
K2CO3 (10.52 g, 0.076 mol) were mixed in acetone
(70 mL) and stirred at room temperature for 15 min,
to which 2.6 mL of CH3OCH2Cl was added drop-
wisely and stirred for at least 4 h. The reaction mix-
ture was filtered to remove the solid phase and the
solution was evaporated under vacuum to give inter-
mediate product. Then, the obtained intermediate
product was mixed with 5.86 g (0.056 mol) of malonic
acid, 60 mL of benzene, 30 mL of pyridine and 0.9 mL
of piperidine and refluxed for 8 h. After benzene was
evaporated under vacuum, the residual mixture was
acidified by HCl aqueous solution to pH ꢂ 1, a white
solid (compound 2) was yielded (6.24 g, 87%). The
compound 2 (2.38 g, 0.01 mol) was mixed with 1.4
mL of phenethylamine in 60 mL of dry CH2Cl2 with
2.26 g (0.011 mol) of DCC and 50 mg of DMAP
added. The mixture was stirred at room temperature
for 14 h and then cooled to precipitate DCC-H2O
complex. After the DCC-H2O complex was removed
by filtration and the solvent was evaporated under
vacuum, the crude product was purified by silica
chromatography (ethyl acetate:chloroform ꢂ 1:4.5)
to afford 3.04 g of compound 3, yield 89%.The com-
pound 3 was dissolved in 179 mL of 5% acetic acid
aqueous solution and refluxed for 12 h. After the mix-
ture was cooled to room temperature, the pH value
of the mixture was adjusted to 7 by adding saturated
NaHCO3 aqueous solution, then extracted by ethyl
acetate three times. The ethyl acetate phase was
washed with saturated NaCl solution and dried over
Na2SO4. Ethyl acetate was evaporated under vacuum
and the crude product was purified by silica chro-
matography (ethyl acetate:chloroform ꢂ 1:3) to
afford 2.1 g of FRPA, yield 79% and melting point:
33.5–35°C. 1H NMR (300 MHz, DMSO-d6): d (ppm)
2.69–2.79 (m, 2H), 3.36–3.41 (m, 2H), 3.74–3.84
(m, 3H), 6.43 (d, J ꢂ 15.9 Hz, 1H), 6.78 (dd, J ꢂ
2.7 Hz, J ꢂ 8.1 Hz, 1H), 6.98 (dd, J ꢂ 3 Hz, J ꢂ
8.1 Hz, 1H), 7.10 (s, 1H), 7.16–7.34 (m, 6H), 8.01
Synthesis of feruloyltyramine (FRTA) and caffeoyl-
tyramine (CFTA).Tyramine was used in the synthesis
of FRTA and CFTA and the other operation was
similar to the synthesis of FRPA and CFPA. Briefly,
0.238 g (0.001 mol) of compound 2, 0.226 g (0.0011
mol) of DCC and 5 mg of DMAP were dissolved in
10 mL dry CH2Cl2 and added to tyramine solution
(0.206 g of tyramine dissolved in 100 mL of dry
CH2Cl2) within 4 h. The mixture was stirred for
another 4 h and then cooled to precipitate DCC-
H2O complex. After the DCC-H2O complex was
removed by filtration and the solution was washed
by NaOH solution (1 M), the pH value of the water
phase was adjusted to 1 by adding HCl aqueous solu-
tion to yield white solid. The mixture was extracted
by ethyl acetate. Ethyl acetate phase was washed by
saturated NaCl solution and dried over Na2SO4.
After ethyl acetate was evaporated under vacuum, the
crude product was dissolved in 50 mL of 5% acetic
acid and refluxed for 4 h. The following treatment
was similar to the synthesis of FRPA, affording 0.204 g
of FRTA, yield 57% and melting point: 59–61°C. 1H
NMR (300 MHz, DMSO-d6): d (ppm) 2.65
(t, J ꢂ 7.2 Hz, 2H), 3.34 (s, 2H), 3.80 (s, 3H), 6.43
(d, J ꢂ 15.6 Hz, 1H), 6.67 (s, 1H), 6.69 (s, 1H),
6.79 (d, J ꢂ 8.1 Hz, 1H), 6.97–7.03 (m, 3H), 7.11
(s, 1H), 7.31 (d, J ꢂ 15.6 Hz, 1H), 7.96 (t, J ꢂ 4.8 Hz,
1H), 9.15 (s, 1H), 9.39 (s, 1H); 13C NMR (75 MHz,

448 Y. Yang et al.
0.1 mL.This solution was dispensed into test tubes,
each one contained 2.0 mL. The test tubes were
incubated in a water bath (37°C) to initiate the oxi-
dation of DNA.Three tubes were taken out at every
2 h and cooled immediately. The following opera-
tion was the same as the oxidation of DNA medi-
ated by Cu2ꢀ/GSH except that the heating period
was 15 min after TBA and trichloroacetic acid
aqueous solution were added.
DMSO-d6): d (ppm) 34.4, 40.6, 55.5, 110.8, 115.1,
115.6, 119.1, 121.5, 126.4, 129.4, 129.5, 138.8,
147.8, 148.2, 155.6, 165.3. CFTA was derived from
FRTA by demethylation. Briefly, 0.314 g of FRTA,
0.187 g of AlCl3 and 5 mg of TEBA gave 0.254 g of
CFTA, yield 85% and melting point: 199.5–201°C.
1H NMR (300 MHz, DMSO-d6): d (ppm) 2.64
(t, J ꢂ 7.5 Hz, 2H), 3.28–3.30 (m, 2H), 6.31 (d, J ꢂ
15.6 Hz, 1H), 6.66–6.75 (m, 3H), 6.83 (dd, J ꢂ
21 Hz, J ꢂ 8.4 Hz, 1H), 6.93 (d, J ꢂ 2.1 Hz, 1H),
7.00 (d, J ꢂ 2.1 Hz, 1H), 7.02 (d, J ꢂ 1.8 Hz, 1H),
7.22 (d, J ꢂ 15.9 Hz, 1H), 8.02 (t, J ꢂ 5.7 Hz, 1H),
9.13 (s, 1H), 9.18 (s, 1H), 9.36 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d (ppm) 34.5, 40.7, 113.8,
115.1, 115.7, 118.6, 120.4, 126.4, 129.5, 129.5,
138.9, 145.5, 147.2, 155.6, 165.3.
b-Carotene-bleaching test
An emulsion was prepared by dissolving 5.0 mg of
b-carotene, 40 mg of LH and 400 mg of Triton X-100
in 5.0 mL of CHCl3. After CHCl3 was evaporated
under vacuum pressure, 100 mL of oxygen-saturated
water was added and the mixture was shaken under
ultrasonic vibration to form a homogeneous b-carotene-
LH emulsion (lmax ꢂ 460 nm) [17]. The ethanol
solutions of CNPA derivatives (0.1 mL) were mixed
with 1.9 mL of b-carotene-LH emulsion, in which
the final concentrations of CNPA derivatives were
20.0 μM. The decay of the absorbance at 460 nm
was measured.
Cu2ꢀ/GSH or AAPH-induced oxidation of DNA
The oxidation of DNA mediated by Cu2ꢀ and GSH
was following our previous description [16]. The
phosphate buffered solution (PBS1: 6.1 mM
Na2HPO4 and 3.9 mM NaH2PO4) was applied to
dissolve CuSO4, DNA and GSH. The final concen-
trations of DNA, Cu2ꢀ and GSH were 2.0 mg/mL,
5.0 mM and 3.0 mM, respectively, to which dime-
thyl sulphoxide (DMSO) solutions of CNPA deriva-
tives were added to a final concentration of 200 μM,
respectively.The total volume of the solution was 15 mL
and the volume of DMSO was 0.1 mL.This solution
was dispensed into test tubes, each one contained
2.0 mL. The test tubes were incubated in a water
bath (37°C) to initiate the oxidation of DNA.Three
tubes were taken out at every 30 min and cooled
immediately. To each tube, 1.0 mL of 30.0 mM
PBS1 solution of EDTA, 1.0 mL of thiobarbituric
acid (TBA) solution (1.00 g TBA and 0.40 g NaOH
dissolved in 100 mL of PBS1, the addition of NaOH
was beneficial for TBA dissolving in aqueous solu-
tion) and 1.0 mL of 3.0% trichloroacetic acid aque-
ous solution were added and heated in boiling water
for 30 min. After the solution was cooled to room
temperature, 1.5 mL of n-butanol was added and
shaken vigorously to extract TBA reactive substance
(TBARS). The absorbance of the n-butanol layer
was measured at 535 nm and is shown in Supporting
Information (Online version only).
Scavenging radicals
The experiments of CNPA derivatives to trapABTSꢀ.,
DPPH and galvinoxyl radical were following the
description in the literature [18,19]. Briefly, ABTS
(2.00 mL, 4.0 mM) was oxidized by 1.41 mM K2S2O8
for 16 h to generate ABTSꢀ•, to which 100 mL of
ethanol was added to make the absorbance (Absref)
around 0.70 at 734 nm (e734 ꢂ 1.6 ꢄ 104 M–1cm–1,
the concentration of K2S2O8 was lower than that
of ABTS in order to verify complete exhaustion of
K2S2O8). DPPH and galvinoxyl radical were dissolved
in ethanol to make the absorbance (Absref) around
1.00 at 517 nm (e517 ꢂ 4.09 ꢄ 103 M–1cm–1) and
428 nm (e428 ꢂ 1.4 ꢄ 105 M–1cm–1), respectively.
The ethanol solution of CNPA derivatives were added
to the aforementioned radical solutions to the final
concentration of 20.0 μM. The absorbance of the
mixture (Absdetect) was recorded at room temperature
and the concentrations of the residual radical species
were calculated by the products of Absdetect and the
corresponding e.The concentrations of radical species
were plotted vs incubation time.
AAPH-induced oxidation of DNA was following
our previous description [16]. The phosphate buff-
ered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM
NaH2PO4, 10.0 μM EDTA) was applied to dissolve
DNA and AAPH. The final concentrations of DNA
and AAPH were 2.0 mg/mL and 40.0 mM, respec-
tively. CNPA derivatives were dissolved in DMSO
and added to the aforementioned solution to reach
various concentrations. The total volume of the
solution was 15 mL and the volume of DMSO was
Statistical analysis
All the data were the average values from at least three
independent measurements with the experimental
error within 10%.The data were analysed by one-way
ANOVA on Origin 6.0 professional Software and
p ꢅ 0.001 indicated a significance difference.

Antioxidant property of cinnamoylphenethylamine derivatives 449
Results
Antioxidant capacities of CNPA derivatives in
AAPH-induced oxidation of DNA
Inhibitive effects of CNPA derivatives on
Cu2ꢀ/GSH-induced degradation of DNA
AAPH is a resource of peroxyl radical (ROO.,
RꢂꢃCMe2C(ꢂNH)NH2) for abstracting H atom
Cu(II) is able to oxidize GSH to form Cu(I) and
GS. and then Cu(I) combines with DNA to form
DNA-Cu(I) complex. GS. may interact with the
complex, leading to the degradation of DNA to
form carbonyl species [16]. The carbonyl species
can be detected after reacting with thiobarbituric
acid (TBA). DNA (2.0 mg/mL), Cu2ꢀ (5.0 mM)
and GSH (3.0 mM) were mixed in PBS1 and incu-
bated at 37°C for 3 h.The absorbance of TBA reac-
tive substance (TBARS, lmax ꢂ 535 nm) was
assigned as 100% and the absorbances of TBARS
in the presence of 200 μM CNPA derivatives were
compared with that in the blank experiment and
outlined in Figure 1.
from the C-4’ atom of DNA, resulting in the cleavage
of DNA and the formation of carbonyl species even-
tually [16]. It can be found in Figure 2 that the absor-
bance ofTBARS increased with the incubation period
in the blank experiment, indicating that more carbo-
nyl species were generated from the degradation of
DNA with the increase of the incubation period.
The additions of various concentrations of CNPA
cannot influence the increase of the absorbance of
TBARS, demonstrating that N-H in CNPA was not
able to hinder AAPH-induced oxidation of DNA.The
absorbance of TBARS decreased remarkably when
other CNPA derivatives were added to the above
experimental system, revealing that the hydroxyl
group attaching to CNPA enhanced its antioxidant
ability to protect DNA against AAPH-induced oxida-
tion. In particular, as shown in Figure 2, when FRPA,
FRTA, CFPA and CFTA were added, the increase of
the absorbance of TBARS was retarded for a period
at the beginning of the incubation and then increased
as that in the blank experiment.This period was des-
ignated as the inhibition period (tinh) and was pro-
longed with the increase of the concentrations of
these four compounds. Figure 3 outlined the linear
relationship between the concentrations of these four
compounds and tinh and the equations of tinh ∼ [FRPA,
FRTA, CFPA and CFTA] were listed in Table I.
In the equations inTable I, the coefficient indicated
the sensitivity of tinh to the variety of the concentration
of CNPA derivatives and the constants were generated
in the linear regression to balance the equation. The
high coefficient meant that a little change of the con-
centration of CNPA derivatives led to remarkable vari-
ety of the tinh.Thus, the order of the coefficient, CFTA ꢁ
CFPA ꢁ FRTA ꢁ FRPA, indicated that the increase
of the concentration of CFTA resulted in the increase
of tinh more significantly than other compounds. This
may be that high concentration of CFTA can scavenge
the peroxyl radicals of AAPH and thereby cause inhi-
bition, while CNTA is not a peroxyl radical scavenger.
Thus, CFTA was a concentration-dependent antioxi-
dant with high efficiency in this case. The addition of
CNTA actually decreased the absorbance of TBARS,
but CNTA cannot generate tinh, even high concentra-
tion was applied. Comparing the structure of CNTA
with that of CFTA it can be concluded that more
hydroxyl groups enhanced the antioxidant effective-
ness of CNPA derivatives remarkably.
The percentages of TBARS in the presence of
CNPA derivative were CFTA ꢅ CFPA ꢅ CNTA ꢅ
FRTA ꢅ FRPA ꢅ CNPA ꢅ Blank.The percentages
of CNPA (98.4%), FRPA (97.7%) and FRTA
(96.0%) were higher than those of other CNPA
derivatives, revealing that the abilities of these
CNPA derivatives to protect DNA against Cu2ꢀ
/
GSH-induced degradation were lower than those
of CNTA, CFPA and CFTA. Moreover, the lowest
ability of CNPA implied that N-H in amide was not
active to Cu2ꢀ/GSH-induced degradation of DNA.
The ability of FRPA was similar to that of CNPA,
indicating that the hydroxyl group in combination
with ortho-methoxyl cannot improve the protective
capacity of CNPA derivatives in this case. CFPA
and CFTA possessed remarkably higher abilities
than other CNPA derivatives, demonstrating that
two hydroxyl groups at ortho-position along with
tyramine enhanced the inhibitive effect significantly.
This may be due to the two hydroxyl groups being
able to chelate Cu2ꢀ, resulting in less GS. and
DNA-Cu(I) complex being produced.Consequently,
DNA cannot be destroyed as much as in the blank
experiment.
100.0
100.0
98.4
97.7
98.0
96.0
96.0
94.7
94.0
91.3
92.0
90.4
90.0
88.0
A
A
A
A
A
A
T
Blank
FRP
CFP
FR
CFT
CNP
CNT
Effects of CNPA derivatives on b-carotene-bleaching test
Figure 1. Percentages ofTBARS in the mixture of DNA (2.0 mg/mL),
Cu2ꢀ (5.0 mM), GSH (3.0 mM) and CNPA derivatives (200 μM)
when the mixture was incubated at 37°C for 3 h.
LH and b-carotene can form a water-soluble emul-
sion in the presence of Triton X-100 or Tween as the

450 Y. Yang et al.
CNPA
CNTA
0 μM
260 μM
300 μM
340 μM
400 μM
600 μM
FRPA
0 μM
150 μM
180 μM
240 μM
300 μM
FRTA
0 μM
100 μM
120 μM
150 μM
200 μM
CFPA
CFTA
0 μM
100 μM
120 μM
140 μM
160 μM
0 μM
0 μM
1.2
0.8
0.4
150 μM
180 μM
240 μM
300 μM
100 μM
120 μM
150 μM
160 μM
tinh
300 600
0
300 600
0
300 600
0
300 600
0
300
600
0
300 600
0
Incubation period (min)
Figure 2. The absorbance of TBARS in the mixture of DNA (2.0 mg/mL), AAPH (40 mM) and various concentrations of CNPA
derivatives at 37°C.
surfactant [17]. The auto-oxidation of LH led to the
formation of peroxyl radical of LH (LOO.) that can
blench b-carotene. As can be seen in Figure 4, the
absorbance of b-carotene at 460 nm decreased con-
tinuously, indicating that much more LH was oxi-
dized by the ambient oxygen with the incubation
period increasing.
not powerful enough groups to inhibit the auto-
oxidation of LH.
Radical-scavenging abilities of CNPA derivatives
ABTSꢀ•, DPPH and galvinoxyl are stable radicals
at room temperature. The ability of the phenolic
hydroxyl group in an antioxidant to reduce radicals
can be measured by the interaction of the antioxi-
dant with ABTSꢀ•. The interactions of an antioxi-
dant with DPPH and galvinoxyl radicals show the
ability of the antioxidant to donate its hydrogen
atom to N- and O-centred radicals, respectively.
Figure 5 outlined the decay of the radical concentra-
tions when ethanol solutions of CNPA derivatives
(20.0 μM) were mixed with ABTSꢀ•, DPPH and
galvinoxyl solutions, respectively.
Recently, a novel method has been reported to treat
the data of antioxidants to trap ABTSꢀ• [18]. On the
basis of chemical kinetic, the decay of [ABTSꢀ•] along
with the incubation period (t) can be expressed by
equation (1).
Other lines in Figure 4 illustrated the decrease of
the absorbance at 460 nm in the presence of 20 μM
CNPA derivatives. It can be found that the lines of
CNPA and CNTA were even below the line of the
blank experiment, implying that the addition of
CNPA and CNTA accelerated the auto-oxidation
of LH and CNPA and CNTA played a pro-oxidative
role in this case. The lines of CFPA and CFTA
(superposition) located above the blank line, indi-
cating that CFPA and CFTA showed similar activ-
ityininhibitingtheauto-oxidationofLH.Furthermore,
the additions of FRTA and FRPA efficiently hin-
dered the decay of the absorbance, revealing that
FRTA, especially FRPA, protected LH against the
auto-oxidation significantly. This may be ascribed
to hydroxyl with an ortho-methoxyl group. The
present data also implied that ortho-dihydroxyl
groups and the hydroxyl group in tyramine were
ab
[ABTSꢀ• ]ꢂ[ABTSꢀ• ]∞ ꢀ
(1)
bꢀt
whereaꢂnapp[AH]0 andbꢂ1/(k2app[AH]0).[ABTSꢀ•]∞
360
is the residual concentration of ABTSꢀ• when the
FRTA
CFTA
CFPA
270
Table I. The relationships between the concentrations of CFTA,
CFPA, FRTA and FRPA and tinh and stoichiometric factor (n) of
the aforementioned compounds.a
FRPA
180
tinh (min) ꢂ (n/Ri) [CNPA
Antioxidant
derivatives (μM)] ꢀ constant
n
CFTA
CFPA
FRTA
FRPA
10.8
7.1
3.0
1.0
tinh ꢂ 3.20 [CFTA] – 202.4
tinh ꢂ 2.12 [CFPA] – 85.1
tinh ꢂ 0.88 [FRTA] – 10.4
tinh ꢂ 0.29 [FRPA] ꢀ 56.6
90
100
200
300
400
Concentration (μM)
Figure 3. The relationship between the concentrations of CFTA,
aRi ꢂ Rg ꢂ 1.4 ꢄ 10ꢃ6 ꢄ 40 mM/s ꢂ 3.36 μM/min when 40 mM
CFPA, FRTA and FRPA and inhibition period (tinh).
AAPH was employed to initiate the oxidation of DNA.

Antioxidant property of cinnamoylphenethylamine derivatives 451
0.9
0.6
0.3
0.0
Discussion
CNPA derivatives attracted much research attention
because they were found in many folk medicinal
herbs. As a kind of simple molecule, hydroxyl-sub-
stituted CNPA derivatives were also applied to
FRPA
FRTA
CFTA
CFPA
Blank
CNTA
CNPA
explore the interactions between drug and receptor
[20] or proteins [21]. In addition to the study on the
biosynthetic pathway of CNPA derivatives [22],
some artificial synthetic routines were designed on
the basis of the amidation of phenethylamine by
using cinnamic acid [23] or cinnamoyl anhydride
[24]. In the direct amidation of phenethylamine and
cinnamic acid, some tertiary amines were added
to ionize the acid. For example, benzotriazol-1-
yloxy-tri(dimethylamino)phosphonium hexafluoro-
phosphate was used to catalyse the amidation of
p-hydroxycinnamic acid derivatives with phenylal-
kylamines[25].N,N-Dimethylaminopyridine(DMAP),
a simple tertiary amine, was employed herein because
of its high activity to couple phenethylamine or
tyramine with cinnamic acid, as well as dicyclohexy-
lcarbodiimide (DCC) was used to improve the dehy-
dration between -COOH and -NH2 according to the
literature [23]. In our work, the hydroxyl in ferulic
acid was etherized by CH3OCH2Cl before the ami-
dation because it was very sensitive to the oxidation
and the synthesis was carried out at room tempera-
ture to obtain satisfied yield.
0
60
120
180
Incubation period (min)
Figure 4. Decay of the absorbance at 460 nm of β-carotene-LH
emulsion containing 20.0 μM CNPA derivatives.
reaction time (t) approaches to ∞ and [AH]0 refers to
the initial concentration of the antioxidant. Thus, napp
the apparent stoichiometric factor, means the number
of electrons that the antioxidant traps radicals, while
k2 is the apparent rate constant of the limiting step
,
app
of the reaction between the antioxidant and ABTSꢀ•.
Traditionally, effective concentration (EC50) refers to
the concentration of the antioxidant applied to scavenge
50% concentration of the radical, while TEC50 means
the time needed to reach the steady state at EC50.
According to the kinetic deduction, 1/(k2app[AH]0)
represents the time necessary for the radicals to be
decreased to 50% and, thus, has the same physical
meaning as TEC50 [18]. Therefore, napp and TEC50 can
be obtained when t and the corresponding [ABTSꢀ•] are
input into software to obtain equation (1).The obtained
The N-H bond in CNPA exhibited very low activ-
ity to scavenge radicals (data shown in Table II) and
the data of CNTA showed that the radical-scaveng-
ing ability was not remarkably improved when the
benzene ring B (see Scheme 1) was replaced by
tyramine.The antioxidant behaviour of tyramine, as
pointed out in the literature [26], was owing to the
deamination to form p-hydroxyphenylacetaldehyde
and/or to couple with another tyramine at ortho-
position of hydroxyl group to form dimmer of
tyramine. On the other hand, as shown in Table II,
the values of napp of FRPA and CFPA were higher
than those of FRTA and CFTA. This result was in
agreement with the traditional conclusion that
hydroxyl with an ortho-methoxyl group and ortho-
dihydroxyl group played the major role in scaveng-
ing radicals. On the other hand, our results indicated
that the introduction of tyramine decreased the
abilities of CNPA derivatives to trap radicals. How-
ever, as pointed out in the literature [27], the abili-
ties of CNPA derivatives to inhibit U937 and Jurkat
cells may be enhanced by replacing tyramine by
dopamine to form caffeoyldopamine because ortho-
dihydroxyl group attached to two benzene rings.
Moreover, CNPA derivatives with ortho-dihydroxyl
group attached to benzene ring A benefitted for
chelating metal ions and, consequently, showed
relative high activities to inhibit metal ion-mediated
oxidation of DNA (see Figure 1).
n
app is a thermodynamic parameter to express the abil-
ity of the antioxidant in trapping radicals and TEC50
is a kinetic parameter to indicate the reaction rate
between the antioxidant and radicals. Because the same
initialconcentrationofCNPAderivativeswereemployed
herein, napp and TEC50 as thermodynamic and kinetic
parameters can be used to characterize the radical-
scavenging activities of CNPA derivatives.Furthermore,
the results from the interactions between CNPA
derivatives and DPPH or galvinoxyl radical were also
treated by the aforementioned method and napp and
TEC50 in scavenging three kinds of radical were listed
in Table II.
The high value of napp indicates that the antioxidant
is able to provide many electrons to trap radicals and
low value of TEC50 implicates that the antioxidant can
trap radicals much more rapidly. Taking napp and
T
EC50 into consideration, one can find that FRPA and
CFPA were able to trap ABTSꢀ• and DPPH effi-
ciently, while CFPA and CFTA showed high effec-
tiveness to scavenge galvinoxyl radicals. In particular,
the lowest value of napp of CNPA indicated that N-H
in CNPA possessed very weak ability to trap radicals.
The relative low value of napp of CNTA revealed that
hydroxyl group in tyramine was not active to trap
radicals, whereas hydroxyl with an ortho-methoxyl
group and ortho-dihydroxyl groups exhibited power-
ful effectiveness to trap radicals.

452 Y. Yang et al.
Table II. The apparent stoichiometric factor (napp) and TEC50 of
CNPA derivatives in trapping ABTSꢀ•, DPPH and galvinoxyl
radicals.
45
CNPA
CNTA
FRTA
CFTA
CFPA
FRPA
Reacting with
ABTSꢀ•
Reacting with
DPPH
Reacting with
galvinoxyl
30
15
TEC50
TEC50
TEC50
Antioxidant
napp
(min)
napp
(min)
napp
(min)
CNPA
CNTA
FRPA
FRTA
CFPA
CFTA
0.23
1.10
2.03
1.37
2.01
1.57
0.22
3.95
0.21
1.16
0.12
0.12
1.52
1.68
5.78
2.69
6.61
4.71
0.15
0.13
2.11
0.78
0.45
0.09
0.01
0.03
0.25
0.17
0.30
0.29
—
13.36
5.88
25.60
0.02
0.24
0
0
60
120
180
Incubation period (min)
240
180
120
assumed that the rate of initiating the radical propaga-
tion was equal to that of the radical generated from the
decomposition of AAPH (Rg ꢂ 1.4 ꢄ 10ꢃ6 [AAPH] sꢃ1
[29]), viz., Ri ꢂ Rg ꢂ 1.4 ꢄ 10ꢃ6 ꢄ 40 mM/s ꢂ 3.36
μM/min when 40 mM AAPH was employed to initiate
the oxidation of DNA [30]. Thus, n values of CNPA
derivatives can be calculated because the coefficients in
the equations of tinh ∼ [CNPA derivatives] were n/Ri, and
the results were involved in Table I. Higher values of n
of CFTA and CFPA revealed that the ortho-dihydroxyl
group benefitted for CNPA derivatives to protect DNA
against AAPH-induced oxidation.The n value of CFTA
was higher than that of CFPA and the same order of the
n values was also found in FRTA and FRPA, indicating
that replacing the benzene ring by tyramine benefitted
for the enhancement of the antioxidant effectiveness in
this case. All the CNPA derivatives possess radical-scav-
enging properties, thus, the protective effects of these
compounds may be due to the scavenge peroxyl radical
deriving from the decomposition of AAPH in protecting
DNA against AAPH-induced oxidation. Since two
hydroxyl groups attach to ring A in CFTA and CFPA,
and ring A forms a conjugation system with the CꢂC
bond in the carbon chain, radicals deriving from the
ortho-dihydroxyl group in CFTA and CFPA may be sta-
bilized by the conjugation system via resonance struc-
ture, as shown in equation (3). But the radical deriving
from the hydroxyl group at ring B cannot be stabilized
by the conjugation system.Thus, hydroxyl groups at ring
A play main role in trapping radicals.
CNPA
CNTA
FRTA
CFTA
FRPA
CFPA
60
0
50
100
150
Incubation period (min)
6
4
2
0
CNPA
CNTA
FRTA
FRPA
CFTA
CFPA
80
160
240
Incubation period (min)
Figure 5.Decay of the absorbance ofABTSꢀ.,DPPH and galvinoxyl
radical solution containing 20.0 μM CNPA derivatives.
The chemical kinetic deduction reveals the relation-
ship between the inhibition period (tinh) and the concen-
tration of the antioxidant (AH) as equation (2) [28].
O
O
O
HO
O
HO
O
HO
O
N
N
N
H
H
A
A
H
A
(3)
O
O
tinh ꢂ (n/Ri) [AH]
(2)
HO
HO
N
N
H
A
H
A
O
O
where n is stoichiometric factor to express the ability of
the antioxidant to terminate the radical-chain propaga-
tion and Ri refers to the initiation rate of the radical-
induced reaction. It has been proved that this equation
was also available for evaluating the antioxidant capaci-
ties in biological experimental system [29]. We have
Conclusion
More hydroxyl groups enhanced the antioxidant capac-
ities of CNPA derivatives. Especially, ortho-dihydroxyl
or hydroxyl with ortho-methoxyl attaching to the

Antioxidant property of cinnamoylphenethylamine derivatives 453
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The authors report no conflicts of interest. The
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This paper was first published online on Early Online on 9
December 2010.
Supplementary material available online
Figure S1.