Tyrosinase inhibitory effects and antioxidative activities of novel cinnamoyl amides with amino acid ester moiety

Article abstract of DOI:10.1016/j.foodchem.2012.03.021

Nine cinnamoyl amides with amino acid ester (CAAE) moiety were synthesized by the conjugation of the corresponding cinnamic acids (cinnamic acid, 4-hydroxy cinnamic acid, ferulic acid and caffeic acid) with amino acid esters, and their inhibitory effects on the activities of mushroom tyrosinase were investigated, using l-3,4-dihydroxyl-phenylalanine (l-DOPA) as the substrate. Among these CAAE amides, ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-1-yl]-l- phenylalaninate (b₄) showed the strongest inhibitory activity; the IC₅₀ was 0.18 μM. The IC₅₀ values, inhibition types, inhibition mechanisms and kinetics of all these CAAE amides were evaluated. A structure-activity relationship (SAR) study found that the inhibitory effects were potentiated with the increasing length of hydrocarbon chains at the amino acid esters and also influenced by the substituents at the styrene groups. Furthermore, the hydroxyl radical scavenging and anti-lipid peroxidation activities of four CAAE derivatives were also investigated. Among these compounds, b₃ (ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]- l-phenylalaninate) and b₄ exhibited potential antioxidant activities.

Full text of DOI:10.1016/j.foodchem.2012.03.021

Food Chemistry 134 (2012) 1081–1087
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Tyrosinase inhibitory effects and antioxidative activities of novel cinnamoyl
amides with amino acid ester moiety
Qian Fan a, Hong Jiang b,1, Er-dong Yuan a, Jian-xun Zhang c, Zheng-xiang Ning a, Sui-jian Qi a,
Qing-yi Wei a,
⇑
a College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, Guangdong, PR China
b Department of Chemistry, College of Science, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China
c Yichang Humanwell Pharmaceutical Co., Ltd., Yichang 443003, Hubei, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine cinnamoyl amides with amino acid ester (CAAE) moiety were synthesized by the conjugation of the
corresponding cinnamic acids (cinnamic acid, 4-hydroxy cinnamic acid, ferulic acid and caffeic acid) with
amino acid esters, and their inhibitory effects on the activities of mushroom tyrosinase were investigated,
Received 15 August 2011
Received in revised form 21 January 2012
Accepted 6 March 2012
using
(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-1-yl]-
inhibitory activity; the IC50 was 0.18 M. The IC50 values, inhibition types, inhibition mechanisms and
L
-3,4-dihydroxyl-phenylalanine (
L
-DOPA) as the substrate. Among these CAAE amides, ethyl N-[3-
Available online 16 March 2012
L
-phenylalaninate (b4) showed the strongest
l
Keywords:
kinetics of all these CAAE amides were evaluated. A structure–activity relationship (SAR) study found that
the inhibitory effects were potentiated with the increasing length of hydrocarbon chains at the amino
acid esters and also influenced by the substituents at the styrene groups. Furthermore, the hydroxyl rad-
ical scavenging and anti-lipid peroxidation activities of four CAAE derivatives were also investigated.
Cinnamoyl amides with amino acid ester
(CAAE) moiety
Tyrosinase inhibitory activity
Antioxidative activities
Among these compounds, b3 (ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-
nate) and b4 exhibited potential antioxidant activities.
L-phenylalani-
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
great importance in preventing browning of food products and
inhibiting the synthesis of melanin in medicinal and cosmetic
products. Development of tyrosinase inhibitors as food additives
and cosmetic products has interested more and more researchers,
and many novel tyrosinase inhibitors have been discovered (Huang
et al., 2006; Yi, Wu, Cao, Song, & Ma, 2009).
The inhibitory effects of hydroxylated cinnamic acid derivatives
(caffeic acid, ferulic acid, and p-coumaric acid) on tyrosinase have
been reported extensively (Gómez-Cordovés, Bartolomé, Vieira, &
Virador, 2001; Lee, 2002). Interestingly, some N-hydrox-
ycinnamoylphenalkylamides exhibited better inhibitory activities
on mushroom tyrosinase than cinnamic acid did (Okombi et al.,
2006; Shi et al., 2005). Furthermore, some amide derivatives of
these acids are naturally present in plants (Roh, Han, Kim, &
Hwang, 2004). Yoon-Sik Lee reported the tyrosinase inhibitory
activities of Kojic acid and hydroxyphenolic acid derivatives mod-
ified with amino acids, and found that compounds with phenylal-
anine moiety showed the strongest inhibitory activities (Noh & Lee,
2011; Noh et al., 2009).
Pigmentation is one of the most obvious phenotypical charac-
teristics in the natural world. Enzymatic browning in plants,
animals and microorganisms is a typical pigmentation, which is
catalyzed by a copper-containing enzyme, tyrosinase (EC.1.14.18.
1; also known as catecholase or diphenol oxidase) (Huang et al.,
2006; Mayer, 1995). Tyrosinase is a multifunctional enzyme that
catalyzes both the hydroxylation of monophenols to o-diphenols
and the oxidation of o-diphenols to o-quinones (Lee, 2002). Tyros-
inase also plays an important role in the biosynthesis of melanin
(Okombi et al., 2006), which widely spreads in skin, hair and eyes
of mammals. On the other hand, melanin may involve in pigmen-
tation-related disorders of human. Moreover, the melanin-related
browning can cause undesirable quality loss in colour, flavour
and nutrition of food products, which may reduce their commer-
cial values. The degree of browning among fruits and vegetables
is variable because of the differences in the phenolic content and
tyrosinase activity (Martinez & Whitaker, 1995; Shi, Chen, Wang,
Song, & Qiu, 2005). Therefore, the inhibitors of tyrosinase are of
Inspired by these findings, two series of cinnamyl amides of
amino acid ester (CAAE) (series a and b, see Fig. 1 for structures)
were synthesized and their inhibitory effects on mushroom tyros-
inase were studied. Furthermore, the antioxidative activities of
series b compounds against hydroxyl radical and lipid peroxidation
were also investigated.
⇑
Corresponding author. Tel./fax: +86 02087112594.
E-mail address: feweiqingyi@scut.edu.cn (Q.-y. Wei).
Co-first author with equal contribution.
1
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.03.021

1082
Q. Fan et al. / Food Chemistry 134 (2012) 1081–1087
the resultant reaction mixture was stirred for 18 h at room temper-
ature. The mixture was then poured into water (100 ml), extracted
with ethyl acetate (4 Â 100 ml), washed with water (2 Â 100 ml)
and saturated brine (100 ml), and dried over MgSO4. After removal
of the solvent under reduced pressure, the residual paste was puri-
fied by column chromatography (silica gel, hexane/EtOAc (1:1)) to
give ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-L-
phenylalaninate (b3). Other compounds were synthesized in the
similar way with compound b3. The structures of the synthesized
compounds were established by IR, 1H NMR and EIMS. The results
were as follows:
Methyl N-[3-phenyl-1-oxo-2-propen-1-yl] glycinate (a1): Yield
90.2%, white solid, melting point: 121–122 °C; 1H NMR
(300 MHz, CDC13): 7.64 (d, 1H, J = 15.6 Hz, HC@C), 7.62–7.25 (m,
5H, C6H5), 6.46 (d, 1H, J = 15.6 Hz, C@CH), 6.22 (br s, 1H, NH),
4.19 (d, 2H, J = 4.8 Hz, CH2), 3.79 (s, 3H, OCH3); IR (KBr) t :
, cmÀ1
3297.3 (NAH), 3033.4 (ArAH), 3008.0 (C@CAH), 1726.2 (C@O),
1650.6 (O@CAN), 1612.5 (C@C), 1222.5 (CAOAC); MS (m/z, %):
219.9 (MÅ+, 100.0), 130.8 (45.3).
Ethyl N-[3-phenyl-1-oxo-2-propen-1-yl] glycinate (a2): Yield
89.5%, white solid, melting point: 102–104 °C; 1H NMR
(400 MHz, CDC13): 7.65 (d, 1H, J = 15.6 Hz, HC@C), 7.53–7.37 (m,
5H, C6H5), 6.47 (d, 1H, J = 15.6 Hz, C@CH), 6.15 (br s, 1H, NH),
4.25 (q, 2H, J = 7.6 Hz, OCH2), 4.19 (d, 2H, J = 4.8 Hz, CH2), 1.31 (t,
3H, J = 7.6 Hz, CH3); IR (KBr)
t
, cmÀ1: 3362.4 (NAH), 3083.4
(ArAH), 3035.9 (C@CAH), 1747.8 (C@O), 1655.3 (O@CAN),
1625.7 (C@C), 1074.9 (CAOAC); MS (m/z, %): 233.9 (MÅ+, 100.0),
130.8 (12.3).
Fig. 1. Chemical structures of CAAE derivatives.
Propyl N-[3-phenyl-1-oxo-2-propen-1-yl] glycinate (a3): Yield
94.3%, white solid, melting point: 66–67 °C; 1H NMR (400 MHz,
CDC13): 7.67 (d, 1H, J = 15.6 Hz, HC@C), 7.62–7.36 (m, 5H, C6H5),
6.44 (d, 1H, J = 15.6 Hz, C@CH), 6.19 (br s, 1H, NH), 4.19 (t, 2H,
J = 7.8 Hz, CH2), 4.15 (d, 2H, J = 4.0 Hz, CH2), 1.65–1.70 (m, 2H,
2. Materials and methods
2.1. Chemical reagents and instruments
CH2), 0.96 (t, 3H, J = 7.8 Hz, CH3); IR (KBr)
t
, cmÀ1: 3287.7
IR spectra were recorded on an Avatar 330 infra-red spectropho-
tometer (KBr pellet); only the most significant absorption bands
(NAH), 3061.6 (ArAH), 3027.6 (C@CAH), 1750.0 (C@O), 1661.4
(O@CAN), 1623.1 (C@C), 1208.2 (CAOAC); MS (m/z, %): 247.9
(MÅ+, 100.0), 130.8 (9.7).
were reported (m
max, cmÀ1). Melting points were determined on a
RY-2 melting apparatus. 1H NMR data were acquired at room tem-
perature on a Bruker AV 400-MHz operating at 400 MHz or 300-
MHz operating at 300 MHz. CDCl3 or DMSO-d6 was used as solvent.
Chemical shifts were expressed in d (parts per million) values rela-
tive to tetramethylsilane (TMS) as internal reference. Mass was per-
formed on a Saturn 2000 mass spectrometer. A Spectronic Genesys
8 UV/Vis spectrophotometer was used in the antioxidative assays.
Caffeic acid, ferulic acid, p-coumaric acid, mushroom tyrosinase
Isopropyl N-[3-phenyl-1-oxo-2-propen-1-yl] glycinate (a4): Yield
89.7%, white solid, melting point: 108–110 °C; 1H NMR
(400 MHz, CDC13): 7.66 (d, 1H, J = 15.6 Hz, HC@C), 7.52–7.37(m,
5H, C6H5), 6.47 (d, 1H, J = 15.6 Hz, C@CH), 6.14 (br s, 1H, NH),
5.08–5.14 (m, 1H, CH), 4.15 (d, 2H, J = 4.8 Hz, CH2), 1.29 (d, 6H,
J = 6.4 Hz, 2CH3); IR (KBr) t
, cmÀ1: 3448.1 (NAH), 3059.8 (ArAH),
3002.3 (C@CAH), 1758.6 (C@O), 1701.4 (O@CAN), 1634.93
(C@C), 1075.1 (CAOAC); MS (m/z, %): 247.9 (MÅ+, 100.0), 130.8
(9.5).
(EC.1.14.18.1), 3,4-dihydroxy-L-phenylalanine (L-DOPA) were pur-
chased from Sigma (St. Louis, MO, USA). 1-Hydroxybenzotriazole
(HOBt), 1-[3-(dimethylamino)propyl]-3-ethylcarbo diimidehydro-
chloride (DECÁHCl), and dimethylsulphoxide (DMSO) were pur-
chased from Shanghai Medpep Co., Ltd., China. All other reagents
were of analytical grade. The water used was re-distilled and
ion-free.
Butyl N-[3-phenyl-1-oxo-2-propen-1-yl] glycinate (a5): Yield
83.6%, white solid, melting point: 93–94 °C; 1H NMR (400 MHz,
CDC13): 7.64 (d, 1H, J = 15.6 Hz, CH@C), 7.52–7.37(m, 5H, C6H5),
6.43 (d, 1H, J = 15.6 Hz, C@CH), 6.04 (br s, 1H, NH), 4.78 (t, 2H,
J = 4.0 Hz, CH2), 4.16 (d, 2H, J = 4.8 Hz, ACH2), 1.67–1.59 (m, 2H,
CH2), 1.42–1.37(m, 2H, CH2ACH3), 0.98 (t, 3H, J = 6.4 Hz, ACH3);
IR (KBr) t
, cmÀ1: 3382.5 (NAH), 3060.2 (ArAH), 3027.0 (C@CAH),
2.2. Synthesis
1768.0 (C@O), 1701.6 (O@CAN), 1632.5 (C@C), 1207.7 (CAOAC);
MS (m/z, %): 261.9(MÅ+, 100).
Nine CAAE derivatives were synthesized from cinnamic acid or
substituted cinnamic acids and the corresponding amino acid es-
ters, according to literature with some modifications (Orlandi, Rin-
done, Molteni, Rummakko, & Brunow, 2001). The synthesis method
was exemplified by the preparation of compound b3. Generally, a
Ethyl N-[3-phenyl-1-oxo-2-propen-1-yl]-L-phenylalaninate (b1):
Yield 90.3%, white solid, melting point: 79–80 °C; 1H NMR
(400 MHz, CDC13): 7.64 (d, 1H, J = 15.6 Hz, HC@C), 7.52–7.12 (m,
10H, C6H5), 6.43 (d, 1H, J = 15.6 Hz, C@CH), 6.14 (br s, 1H, NH),
5.01(q, 1H, J = 4.0 Hz, CH), 4.23–4.18 (m, 2H, CH2), 3.21 (q, 2H,
solution of 3,4-dihydroxy cinnamic acid (1.0 g, 5.5 mmol),
L-ethyl
J = 7.2 Hz, CH2), 1.27 (t, 3H, J = 7.2 Hz, CH3); IR (KBr) t :
, cmÀ1
phenylalaninate hydrochloride (0.75 g, 5.5 mmol), and 1-hydroxy-
benzotriazole (0.74 g, 5.5 mmol) in N,N-dimethylformamide
(15 ml) was added to triethylamine (2.3 ml, 16.5 mmol) at 0 °C.
After 10 min, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide-
hydrochloride (EDCÁHCl 1.0 g, 5.5 mmol) was added at 0 °C and
3224 (NAH), 3061 (ArAH), 3034 (C@CAH), 1739 (C@O), 1654
(O@CAN), 1626 (C@C), 1209 (CAOAC); MS (m/z, %): 325.0 (MÅ+,
100.0).
Detailed characterization data of the compounds ethyl N-[3-(4-
hydroxyphenyl)-1-oxo-2-propen-1-yl]-
L
-phenylalaninate
(b2),

Q. Fan et al. / Food Chemistry 134 (2012) 1081–1087
1083
ethyl
nylalaninate (b3), and ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-
1-oxo-2-propen-1-yl]- -phenylalaninate (b4) were listed in our
N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-
L
-phe-
650
tube. Then, 100
was added to the tube. As the control, 50
l
l of 50 mM phosphate buffer (pH = 7.4) were mixed in a test
l of H2O2 solution (dissolved in deionised water)
l of DMSO was added in-
l
L
l
previous paper (Wei, Jiang, Zhang, Guo, & Wang, in press).
stead of sample solution to the tube. The mixture was left at 37 °C in
the dark for 1 h. Finally, the absorbance was measured at 536 nm.
2.3. Enzyme activity assay
2.5. Statistical analysis
The spectrophotometric assay for diphenolase activity of mush-
room tyrosinase was performed according to the method reported
by Yi et al. (2009) with slight modifications. In brief, L-DOPA was
used as substrate. All compounds were dissolved in dimethyl-
sulphoxide (DMSO) and the final concentration of DMSO in the test
Data were presented as mean standard deviations (SD) from
three independent analyses. All analyses were carried out with
the origin statistical and analysis software.
solution was less than 2.0% (v/v). First, 10
room tyrosinase with the indicated concentrations of compounds
was pre-incubated in 980 l 0.1 M K2HPO4–KH2PO4 buffer (pH
6.8) for 10 min at 25 °C. Ten microlitres of 0.26 mM -DOPA were
then added to initiate the reaction. The enzyme activities were
determined by following the increase of absorbance at 475 nm
for 1 min with a molar absorption coefficient of 3700 (MÀ1 cmÀ1),
ll (0.5 mg/ml) mush-
3. Results
l
3.1. Inhibition of tyrosinase activity by CAAE derivatives
The inhibitory effects on mushroom tyrosinase activities for the
L
oxidation of L-DOPA by nine CAAE derivatives (see Fig. 1 for struc-
tures), cinnamic acid and Kojic acid (as positive control) were
probed. All of the nine compounds have inhibitory effects on the
activities of the tyrosinase. The enzyme activities decreased dra-
matically with the increasing concentrations of the tested com-
pounds (data not showed). In order to compare the inhibitory
potencies among them, the IC50 values, which stand for the con-
centrations leading to a 50% activity loss of tyrosinase, of the nine
tested compounds that were determined. The results were summa-
rized and shown in Table 1. Among the nine compounds, the IC50
accompanying the oxidation of L-DOPA to dopachrome. The mea-
surement was performed in triplicate for each concentration and
averaged before further calculation. The inhibition type was as-
sayed by Lineweaver–Burk plot.
2.4. Determinations of antioxidative activity of b1, b2, b3 and b4
2.4.1. Preparation of rat brain homogenate
The preparation was carried out according to the method re-
ported by Wei, Chen, Zhou, Yang, and Liu (2006) with some modifi-
cations. In brief, female Wistar rats weighing 250 20 g were
starved overnight and sacrificed by cervical dislocation under ether
anaesthesia. Then the brain was rapidly removed and washed
extensively with 0.15 M NaCl. The tissue sample was homogenized
with 10 ml of ice-cold 0.15 M KCl, 10 mM Tris–HCl buffer, pH 7.4.
Later, the tissue homogenate was centrifuged at 800g for 10 min
to remove the residues. The supernatant was collected and stored
at À20 °C. The protein content of brain homogenate was determined
by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951).
values of compounds a3, a4, a5, and b1–4 were less than 100 lM.
Compounds a5 and b4 were the most potent inhibitors, while com-
pounds a1 and a2 were the weakest ones. For the series a, the inhib-
itory effects followed the sequence: a1 < a2 < a3 < a4 < a5, and the
inhibitory effects were potentiated with the increasing lengths of
the hydrocarbon chains at the amino acid esters. And for the series
b, the inhibition strength followed the order: b1 ꢀ b2 < b3 < b4,
with different substituted groups at the styrene groups.
3.2. The inhibitory effects of compounds a3, a4 and a5 on mushroom
tyrosinase following a non-competitive mechanism
2.4.2. Anti-lipid peroxidation assay
The inhibitory types of compounds a3, a4 and a5 on mushroom
tyrosinase, were determined during the oxidation of L-DOPA from
Lineweaver–Burk plots (Kubo & Kinst-Hori, 1999; Noh & Lee,
2011). Fig. 2 shows the double-reciprocal plots of the enzyme
inhibited by compound a3. The plots of 1/m versus 1/[S] gave a fam-
Anti-lipid peroxidation activity of the four compounds b1, b2, b3
and b4 were evaluated. The formation of thiobarbituric acid-reac-
tive substance (TBARS) was used to monitor the lipid peroxidation
product malondialdehyde (MDA), which was used as an indicator
of lipid peroxidation (Wei et al., 2006). Rat brain homogenate
was made up to a final protein concentration of 0.4 mg/ml in buffer
(10 mM Tris–HCl, 0.15 M KCl, pH 7.4). Antioxidants were dissolved
in DMSO and added to 0.5 ml of rat brain homogenate, in which the
final concentration of DMSO solution was less than 0.2% (v/v) that
ily of straight lines with different slopes, but they intersected one
another at the horizontal axis. The enhancement of the inhibitor
concentration could decrease the values of Vmax, but the values of
Km remained the same, which indicated that a3 was a non-compet-
itive inhibitor for the mushroom tyrosinase. This behaviour
showed that a3 could bind with both free enzyme and enzyme–
substrate (ES) complex, and their equilibrium constants were the
same. The inhibition constants for the inhibitor binding with the
free enzyme (KI) and enzyme–substrate (ES) complex (KIS) were
determined by the plot of vertical intercept (1/Vmax) versus the
inhibitor concentrations, which was a line as showed in the inset
of Fig. 2. Compounds a4 and a5 were studied with the same method
and similar results were obtained (data not showed). The inhibi-
tion constants were summarized in Table 1 and the results showed
that both of them were also non-competitives.
did not show appreciable interference to the reaction. Then, 5
ll of
10 mM ascorbic acid and 2 l of 2.5 mM FeCl2 were added to the
l
foresaid brain homogenate to initiate lipid peroxidation. The reac-
tion mixture was incubated for 1 h at 37 °C in a capped tube. After
incubation, 0.75 ml TBA reagent (15% TCA–0.375% TBA–0.25 M
BHT) was added and shaken vigorously. The mixture was heated
in a boiling water bath for 15 min. After cooling, the precipitate
was removed by centrifugation. TBARS in the supernatant was
determined at 532 nm using the extinction coefficient of
1.56 Â 105 MÀ1 cmÀ1
.
2.4.3. Hydroxyl radical-scavenging activity assay
The assay of hydroxyl radical-scavenging activity of the four
samples was carried out according to a previous research (Yi
3.3. The inhibitory effects of compounds b1, b2, b3 and b4 on mushroom
tyrosinase following a mixed-type non-competitive mechanism
et al., 2009), with minor changes. Briefly, 100
tion of 7.5 mM phenanthroline, 100 l of aqueous solution of
7.5 mM FeSO4, 50 l of sample solution (dissolved in DMSO) and
ll of ethanolic solu-
l
The inhibitory mechanisms of compounds b1, b2, b3 and b4 on
mushroom tyrosinase were also studied from Lineweaver–Burk
l

1084
Q. Fan et al. / Food Chemistry 134 (2012) 1081–1087
Table 1
Inhibitory effects and constants of mushroom tyrosinase by CAAE derivatives.
Compounds
IC50
(lM)
Inhibition type
Inhibition constants (lM)
KI
KIS
a1
a2
460 20
257.5 12.5
ND
ND
ND
ND
ND
ND
a3
a4
a5
b1
b2
b3
b4
20
14.5 1.5
1.8 0.2
74 8.5
80 20
31.2 1.9
0.185 0.005
22 2.45
475 65
2
Non-competitive
Non-competitive
Non-competitive
Mixed-type
Mixed-type
Mixed-type
Mixed-type
ND
40
1.25
1.43
30
193
110
5.64
0.076
ND
24
9.15
0.011
ND
Kojic acid
Cinnamic acid
The IC50 values represent means SD of three parallel experiments. ND: not determination.
the free enzyme, KI, and with ES, KIS, were determined by the sec-
ondary plots of Km/Vm and 1/Vmax versus the inhibitor concentra-
tions (inset in Fig. 3), respectively. Both of the secondary plots
are linear. Similar results were obtained with b2 and b4 and their
inhibition constants were summarized in Table 1. From the deter-
mined values, we noticed that the KI values were lower than KIS,
indicating that the affinity of the inhibitor for the free enzyme
was stronger than that for the enzyme–substrate complex. The va-
lue of KIS was about five times greater than that of KI, indicating
that the competitive effect was stronger than the uncompetitive
effect.
The inhibitory type of b3 on mushroom tyrosinase was deter-
mined by the same methods. The results showed that with the
increasing concentration of b3, a family of lines with different
slopes and intercepts intersect one another in the third quadrant
(Fig. 4). KI and KIS were obtained from the secondary plots by the
same methods mentioned above and summarized in Table 1. In
this case, the KIS values were lower than KI, which indicated that
b3 was a mixed-II type inhibitor (Chen et al., 2005).
Fig. 2. Lineweaver–Burk plots for inhibition of a3 on mushroom tyrosinase for the
catalysis of L-DOPA. Concentrations of a3 for curves 1–4 were 0, 4, 8 and 16 lM,
respectively. The inset represents the secondary plot of 1/Vmax versus concentra-
tions of a3 to determine the inhibition constant.
plots (Noh & Lee, 2011). Fig. 3 shows the double-reciprocal plots of
the enzyme inhibited by compound b1. The curves with different
slopes and intercepts intersect one another in the second quadrant.
This behaviour indicated that b1 could bind with both free enzyme
and enzyme–substrate (ES) complex and their equilibrium con-
stants were different, which indicated that b1 acted as a competi-
tive–uncompetitive mixed-I type inhibitor (Noh & Lee, 2011). The
inhibition equilibrium constants for the inhibitor binding with
3.4. Antioxidative activity of compounds b1, b2, b3 and b4
The inhibitory effects of CAAE derivatives (b1, b2, b3 and b4) on
TBARS production in rat brain homogenates induced by FeCl2–
ascorbic acid were shown in Fig. 5. Caffeic acid was taken as a po-
sitive control. The results indicated that when the compounds
were of the same concentration (80 lM), b3 showed the strongest
antioxidative activity among the four compounds. Its inhibition ra-
tio of TBARS production was 88.7%, approaching the level of caffeic
acid, 93.4%. And b4 came in the second place, with the inhibition
ratio of 50%. The compounds b1 and b2 had the weakest antioxida-
tive activities, with the inhibition ratios of TBARS production 16%
and 34.6%, respectively. The IC50 values of the compounds b1, b2,
b3 and b4 for the hydroxyl radical-scavenging activity, were also
determined. The IC50 values of the compounds b1, b2 were more
than 2 mM, while the IC50 values of the compounds b3, and b4 were
0.13 and 0.58 mM, respectively. The scavenging strength followed
the order: b1 < b2 < b4 < b3 < caffeic acid. The combination of these
antioxidative activity results showed that, b3 and b4 were good
antioxidants against hydroxyl radical, and that rat brain homoge-
nates lipid peroxidation was induced by hydroxyl radical.
4. Discussion
Fig. 3. Lineweaver–Burk plots for inhibition of b1 on mushroom tyrosinase for the
catalysis of L-DOPA. Concentrations of b1 for curves 1–4 were 0, 0.025, 0.05 and
0.075 mM, respectively. Insets (b) and (c) represent the secondary plots of the
slopes and the intercepts versus concentrations of b1 to determine the inhibition
constants.
Wine phenolic compounds, such as caffeic acid, ferulic acid, and
p-coumaric acid have been reported to act as tyrosinase inhibitors
(Gómez-Cordovés et al., 2001). Aromatic carboxylic acids of cin-
namic acid and their analogues, p-coumaric, ferulic, and sinapic

Q. Fan et al. / Food Chemistry 134 (2012) 1081–1087
1085
Fig. 4. Lineweaver–Burk plots for inhibition of b3 on mushroom tyrosinase for the catalysis of L-DOPA. Concentrations of b3 for curves 1–4 were 0, 0.01, 0.02 and 0.04 mM,
respectively. Insets (b) and (c) represent the secondary plots of the slopes and the intercepts versus concentrations of b3 to determine the inhibition constants.
Huang et al. (2006) reported that p-octylbenzoic acid was the
most activity one in the inhibition activity of mushroom tyrosinase
by alkylbenzoic acid. Moreover, they concluded that the inhibitory
effects were potentiated with the increasing lengths of the hydro-
carbon chains. The present work assesses the inhibitory activities
of mushroom tyrosinase of nine cinnamoyl amino acid ester
(CAAE) derivatives, and enables a structure–activity relationship
(SAR) of these compounds. As Table 1 showed, among the a series
the a1–a5 compounds bear different lengths of hydrocarbon chains
at the amino acid esters. Compounds a1 and a2 which bear meth-
oxy- and ethoxy- at the amino acid ester respectively were less ac-
tive ones (IC50 > 200 lM), while a3, a4 and a5 which bear propoxy-,
isopropoxy- and butoxy- at the amino acid ester, respectively,
were more active ones. Noteworthily, a5 was the most active. It
is suggested that the inhibitory activities of the CAAE derivatives
on the diphenolase activity of mushroom tyrosinase were en-
hanced with the increasing lengths of hydrocarbon chains at the
amino acid esters. Similarly, when cinnamic acid was modified
with phenylalanine (b1) instead of glycine (a2), the inhibitory ef-
fects were also enhanced. When Kojic acid and hydroxyphenolic
acid derivatives were modified with amino acids, the compounds
with phenylalanine moiety were found to have the strongest inhib-
itory activities of mushroom tyrosinase (Noh & Lee, 2011; Noh
et al., 2009). Butoxy- side chain at the amino acid ester and the
phenylalanine (b1) residue may contribute to the hydrophobic ef-
fect of these compounds, which could increase the hydrophobic
interactions with the hydrophobic side chains located at the tyros-
inase active site (Noh & Lee, 2011). The substituents at the styrene
groups were also important for the activity when the activities of
b3 and b4were compared with that of b2 or b1 as Noh and Lee
(2011) found for the inhibitory activities of hydroxyphenolic
acid–amino acid conjugates on tyrosinase. However, in our study,
when a methoxyl group appeared in the meta-position (compared
b4 with b3), the inhibitory effect was strengthened significantly.
The difference in the inhibitory activities between these two com-
pounds (b4 and b3) could be explained by the lower electron-
donating capacity of methoxy- compared with hydroxy- at the sty-
rene group.
Fig. 5. Inhibitory effects of different CAAE derivatives (b1, b2, b3 and b4) on lipid
peroxidation in rat brain homogenates induced by FeCl2–ascorbic. The concentra-
tions of the inhibitors are 80 lM, respectively. Data were presented as the
percentage of inhibition on the lipid peroxidation, means SD (n = 3).
acids are competitive inhibitors of polyphenoloxidase (PPO) due to
their structural similarities to the phenolic substrates (Kim, Mar-
shall, & Wei, 2000). Nirmal and Benjakul (2009) reported the ef-
fects of ferulic acid (FA) on polyphenoloxidase (PPO) of Pacific
white shrimp (Litopenaeus vannamei) and the concentration of
50% inhibition was about 1% (w/v) (Nirmal & Benjakul, 2009). Fur-
thermore, cinnamic acid and its derivatives were inhibitors of
tyrosinase (Qiu et al., 2009; Shi et al., 2005) and their IC50 values
were estimated to be more than 0.4 mM for the oxidation of L-
DOPA initiated by mushroom tyrosinase. In this study, several cin-
namic acid derivatives (cinnamic acid, 4-hydroxy cinnamic acid,
ferulic acid and caffeic acid) were modified by conjugating with
amino acid esters, and their effects on the oxidation of L-DOPA ini-
tiated by mushroom tyrosinase were screened. Among the nine
modified cinnamyl amino acid esters, the IC50 values of a5 and b4
was less than 2 lM and 0.2 lM, respectively, which were more
effective inhibitors of diphenolase activity than Kojic acid (Table
1). As shown in Table 1, each of the nine cinnamoyl amino acid es-
ter (CAAE) derivatives revealed an enhanced inhibitory activity
compared with cinnamic acid.
Recently, the crystallographic structure of tyrosinase has been
reported, enabling a closer look at its three-dimensional structure
and a better understanding of its mechanism of action (Khatib
et al., 2007; Matoba, Kumagai, Yamamoto, Yoshitsu, & Sugiyama,
2006; Noh & Lee, 2011; Noh et al., 2009). Within the structures,
there are two copper ions in the active centre of tyrosinase and a

1086
Q. Fan et al. / Food Chemistry 134 (2012) 1081–1087
lipophilic long-narrow gorge near to the active centre. Our results
showed that all of the CAAE derivatives were reversible inhibitors
and they could be mainly divided into two groups: a series, includ-
ing a3, a4 and a5, were non-competitive inhibitors, while b series
which included b1, b2, b3 and b4 were mixed-type inhibitors. Shi
et al. (2005) summarized the inhibitory types of cinnamic acid
and its derivatives on tyrosinase and they found that cinnamic acid
was non-competitive, while 4-hydroxy cinnamic acid was compet-
itive. In our research, when cinnamic acid was modified with gly-
cine ester (a3, a4 and a5), their inhibitory types were the same as
that of cinnamic acid. We suggest that the modification of cin-
namic acid by conjugating with glycine ester (a3, a4 and a5) may
help insert the tested compounds into the lipophilic long-narrow
gorge near to the active centre, which may hinder the binding of
the substrate to the enzyme through steric hindrance or by chang-
ing the protein conformation. Interestingly, when cinnamic acid,
4-hydroxy cinnamic acid, ferulic acid and caffeic acid were conju-
gated with phenylalanine ester (Table 1), they became mixed-type
inhibitors. As for b1, b2 and b4, the value of KIS was about five times
greater than that of KI, indicating that the binding of inhibitors to
the free enzyme was stronger than to the enzyme–substrate com-
plex, which may be induced by hydrophobic interaction near the
active site of tyrosinase. Noh et al. (2009) docked Kojic acid–amino
acid conjugates into the active site of tyrosinase using the AUTO-
DOCK Tools program, and found that quite a few hydrophobic ami-
no acids were located around the copper active site of tyrosinase.
The fact that hydrophobic interactions existed between the aro-
matic rings of Kojic acid–amino acid conjugates and the hydropho-
bic side chains in the tyrosinase active site was confirmed, and it
was concluded that these interactions blocked the accessibility of
the substrate to the active site (Noh & Lee, 2011; Noh et al.,
2009). Our results were similar to these reports. The different inhi-
bition types of b series compounds from a series may be attributed
to the aromatic rings of phenylalanine. However, in the b series,
compound b3 was a different one. Compound b3 could bind with
the enzyme–substrate complex more easily and tightly, which
may be induced by the hydrogen-bonding interactions between
the two adjacent positions of hydroxyls and the amino groups in
the tyrosinase. A previous study reported that hydrogen-bonding
interactions could also influence the stability of the oxy-form of
tyrosinase (Kubo & Kinst-Hori, 1999). The inhibition mechanism
of compound b3 needs to be more studied.
homogenates activity induced by hydroxyl radical increased
accordingly. The conclusion was identical with what Yi et al.
(2009) summarized in their study.
In summary, in this paper, nine cinnamoyl amino acid ester
derivatives, a series and b series were designed, synthesized and
their inhibitory effects on mushroom tyrosinase were investigated.
Their inhibition mechanisms and inhibition kinetics were also
studied. Furthermore, their antioxidant activities of hydroxyl radi-
cal scavenging and anti-lipid peroxidation of homogenates induced
by hydroxyl radical were also revealed. All the data suggested that
b3, b4, and a5 might serve as new potent preservatives for the food
industry or skin-whitening agents in cosmetics. Further develop-
ment of such compounds may be of interest.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant Nos. 20702016 and 21002034), the Funda-
mental Research Funds for the Central Universities, SCUT (Grant
Nos. 2012ZM0070 and 2011ZM0097W) and the National Key Tech-
nology R&D Program of China in the 12th five-year period (Grant
No. 2012BAD33B11).
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