Y. Li et al. / Food Chemistry 158 (2014) 41–47
43
material. 1H and 13C NMR spectra were recorded using a Bruker AV
400 (Bruker Biospin Co. Ltd., Switzerland) spectrometer with deu-
terated chloroform (CDCl3) or deuterated dimethyl sulfoxide
(DMSO-d6) as a solvent. Mass spectra were recorded on a Bruker
Daltonics Esquire 6000 (Billerica, MA, USA) spectrometer (ESI-MS).
potentiostat (Chenhua Instruments Inc., Shanghai, China) following
the procedure described previously with some minor modification
(Yang et al., 2010). Briefly, the solutions of CA derivatives (1 mM)
were prepared in supporting electrolyte (tetra-n-butylammonium
perchlorate, TBAP, 0.1 M in methanol). The glassy carbon working
electrode (CH Instruments Inc., 3700 Tennison Hill Drive, Austin,
TX, USA) was polished with ultrafine alumina slurry and washed
thoroughly with water under sonication before use. A Pt wire
was employed as the counter electrode and an Ag/Ag+ electrode
(CH Instruments Inc., 3700 Tennison Hill Drive, Austin, TX, USA)
served as the reference electrode. The potential difference between
Ag/Ag+ and a saturated calomel electrode (SCE) was determined
2.2.1. (2E,4E)-5-(4-Hydroxy-3-methoxyphenyl)penta-2,4-dienoic acid
(11a, B1)
1H NMR (400 MHz, DMSO-d6): d = 3.81 (s, 3H), 5.90 (d, J = 15 Hz
1H), 6.77 (d, J = 8 Hz 1H), 6.92 – 6.98 (m, 3H), 7.16 (s, 1H), 7.31 (m,
J = 15 Hz, 1H), 9.43 (s, 1H), 12.12 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d = 55.6, 110.2, 115.6, 120.2, 121.5, 123.6, 127.6,
140.6, 145.0, 147.9, 148.0, 167.8; MS (EI): (m/z) = 219 [MꢀH]ꢀ.
þꢀSCE
ð
D
EAg=Ag
¼ 0:51 VÞ), and all the reported potentials were with
respect to SCE. All the CV measurements were carried out in the
TBAP electrolyte under an argon atmosphere using Ferrocene as
an internal standard for calibrating redox potentials against the
ferricenium/ferrocene (Fc+/Fc) couple (Epa = 0.43 V vs. SCE).
2.2.2. (2E,4E)-5-(4-Hydroxy-3,5-dimethoxyphenyl)penta-2,4-dienoic
acid (11b, B2)
1H NMR (400 MHz, DMSO-d6): d = 3.79 (s, 6H), 5.91 (d,
J = 15 Hz, 1H), 6.85 (s, 2H), 6.92 (d, J = 15 Hz, 1H), 7.00 (dd,
J = 15 Hz, J = 10 Hz, 1H), 7.31 (dd, J = 15 Hz, J = 10 Hz, 1H), 8,78
(s, 1H), 12.16 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d = 56.0
(2C), 105.0 (2C), 120.3, 124.0, 126.4, 137.1, 140.9, 144.9, 148.1
(2C), 167.7; MS (EI): (m/z) = 251 [M+H]+.
2.6. Assay for oxidative DNA strand breakage induced by AAPH
The inhibition of AAPH-induced DNA strand breakage by CAs
was assessed by measuring the conversion of supercoiled closed-
circular pBR322 plasmid DNA to its open-circular and linear forms
by gel electrophoresis following the procedure described previ-
ously (Qian et al., 2011).
2.2.3. (2E,4E)-5-(3,4-Dimethoxyphenyl)penta-2,4-dienoic acid (11c,
B3)
1H NMR (400 MHz, DMSO-d6): d = 3.77 (s, 3H), 3.80 (s, 3H), 5.94
(d, J = 15 Hz, 1H), 6.95 (d, J = 8 Hz, 1H), 6.92 – 7.01 (m, 2H), 7.07 (d,
J = 8 Hz, 1H), 7.20 (s, 1H), 7.33 (dd, J = 15 Hz, J = 10 Hz, 1H), 12.20
(s, 1H); 13C NMR (100 MHz, DMSO-d6): d = 55.8 (2C), 109.8,
112.0, 121.0, 121.6, 124.8, 129.2, 140.5, 145.1, 149.3, 150.2,
168.0; MS (EI): (m/z) = 257 [M+Na]+, 235 [M+H]+.
2.7. Assay for haemolysis of RBCs
Human red blood cells (RBCs) were provided by the Red Cross
Center for Blood (Gansu, China). The extent of haemolysis was
determined spectrophotometrically at 540 nm and compared with
that of complete haemolysis by treating with distilled water as de-
scribed previously (Qian et al., 2011).
2.2.4. (2E,4E)-5-(4-Hydroxyphenyl)penta-2,4-dienoic acid (11d, B4)
1H NMR (400 MHz, DMSO-d6): d = 5.91 (d, J = 15 Hz, 1H), 6.77
(d, J = 8 Hz, 2H), 6.87 (dd, J = 15 Hz, J = 10 Hz, 1H), 6.94 (d,
J = 15 Hz, 1H), 7.32 (dd, J = 15 Hz, J = 10 Hz, 1H), 7.39 (d, J = 8 Hz,
2H), 9.82 (s, 1H), 12.12 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d = 115.8 (2C), 120.2, 123.3, 127.1, 128.9 (2C), 140.3, 145.1, 158.6,
167.8; MS (EI): (m/z) = 189 [MꢀH]ꢀ.
3. Results and discussion
3.1. Synthesis of CAs with the conjugated chain elongation
The overall strategy for synthesis of the CAs (B1–4) with the
conjugated chain elongation is outlined in Scheme 1. Horner–
Wadsworth–Emmons reactions of appropriate aldehydes with a
prior-synthesized carbomethoxy-methylenetriphenylphosphorane
were performed under a strong base (NaH) condition in absolute
anhydrous THF to give the corresponding methyl ester 8a–d and
10. The desired CAs B1–4 was obtained after an alkaline hydrolysis
of the methyl esters with NaOH in methanol–water. Additionally,
the acetyl protection groups can be removed by base in the last
step to furnish B1–2 and B4 bearing a phenolic hydroxyl group.
2.3. Assay for DPPHÅ-scavenging activity
The EC50 values of CAs (A1–4 and B1–4) in the scavenging of
DPPHÅ were determined by monitoring the absorbance change of
DPPHÅ (100
lM) at 517 nm in methanol after 1 h incubation in
the dark with different concentrations of compounds at 25 °C,
using a TU-1901 UV/Vis spectrometer (Beijing Purkinje General
Instrument Co. Ltd., Beijing, China). The percentage of radical scav-
enging activity was calculated as follows: radical scavenging rate
(RSR, %) = (A0 ꢀ As)/A0 ꢁ 100, where A0 is the absorbance of
100
l
M DPPHÅ only and As is the absorbance of the reaction mix-
3.2. Antioxidant activity of CAs evaluated by the DPPHÅ-scavenging
and FRAP assays
ture after 1 h incubation. The stoichiometry (the equivalent of
DPPHÅ scavenged by one equivalent of antioxidant) n was calcu-
lated follow the equation: n = 100 (
Williams, Cuvelier, & Berset, 1995).
l
M)/(EC50
(
l
M) ꢁ 2) (Brand-
The DPPHÅ-scavenging activity was measured at 25 °C in meth-
anol as a commonly accepted characteristic responsible for antiox-
idant capability. The EC50 (concentration for 50% radical
scavenging) and stoichiometric factor n values as well as radical
scavenging rate (RSR) of CAs and Trolox C (a reference compound)
are represented in Table 1. According to the EC50 and n values, the
DPPHÅ-scavenging activity decreased in the order B2 > A2 > B1 >
A1 > Trolox C > B4 > A4 > B3 > A3. All the studied SA and FA deriv-
atives presented higher activity than Trolox C, while A3 and B3
without hydroxyl group had the poorest activity. These results
clearly indicate that p-hydroxyl group on the aromatic ring is not
the only factor responsible for the DPPHÅ-scavenging activity,
and the substitution of electron-donating methoxy group in
2.4. Assay for ferric reducing/antioxidant power (FRAP)
The FRAP assay (Benzie & Strain, 1996) was used to evaluate the
reducing capability of CAs, and the procedure has been previously
described in detail (Tang et al., 2011).
2.5. Assay for the electrochemistry behaviour
Cyclic voltammetry, as a widely used electrochemical tech-
nique, was performed using a computer controlled CHI-660C