Anti-tyrosinase, antioxidant and antimicrobial activities of hydroxycinnamoylamides

Article abstract of DOI:10.1007/s00044-012-0419-x

Synthetic hydroxycinnamoylamides of amino acids (precursors of aromatic amines) were studied for their antioxidant activity in vitro by two antioxidant assay systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and inhibition of lipid peroxidation (LPO). Furthermore, these compounds were tested and compared with their corresponding cinnamoylamides of aromatic amines for their inhibitory activity using mushroom tyrosinase. In addition, five hydroxycinnamoyl amino acid amides were investigated for their antimicrobial effect. Structure-activity relationships analysis disclosed that the presence of catechol rest at amino acid or at benzene moieties of substituted cinnamic acid amides significantly scavenged DPPH radical and inhibited LPO. The results obtained by LPO clearly expressed the positive influence of indole moiety on the activity. Moreover, the existence of p-hydroxy substituted cinnamic acid moiety leads to better tyrosinase inhibition. Amongst the tested compounds, amides of p-coumaroyldopamine or tyramine and their corresponding amino acid precursors are the most potent tyrosinase inhibitors.

Full text of DOI:10.1007/s00044-012-0419-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0419-x
ORIGINAL RESEARCH
Anti-tyrosinase, antioxidant and antimicrobial activities
of hydroxycinnamoylamides
•
Lyubomir Georgiev Maya Chochkova Iskra Totseva
•
•
•
Katya Seizova Emma Marinova Galya Ivanova
•
•
•
•
Mariana Ninova Hristo Najdenski Tsenka Milkova
Received: 10 August 2012 / Accepted: 12 December 2012
ꢀ Springer Science+Business Media New York 2012
Abstract Synthetic hydroxycinnamoylamides of amino
acids (precursors of aromatic amines) were studied for their
antioxidant activity in vitro by two antioxidant assay
systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical and inhibition of lipid peroxidation (LPO). Fur-
thermore, these compounds were tested and compared with
their corresponding cinnamoylamides of aromatic amines
for their inhibitory activity using mushroom tyrosinase. In
addition, five hydroxycinnamoyl amino acid amides were
investigated for their antimicrobial effect. Structure–
activity relationships analysis disclosed that the presence of
catechol rest at amino acid or at benzene moieties of
substituted cinnamic acid amides significantly scavenged
DPPH radical and inhibited LPO. The results obtained
by LPO clearly expressed the positive influence of indole
moiety on the activity. Moreover, the existence of
p-hydroxy substituted cinnamic acid moiety leads to better
tyrosinase inhibition. Amongst the tested compounds,
amides of p-coumaroyldopamine or tyramine and their
corresponding amino acid precursors are the most potent
tyrosinase inhibitors.
Keywords N-hydroxycinnamoylamides ꢀ
Mushroom tyrosinase ꢀ Monophenolase activity ꢀ
Antioxidant activity ꢀ Antimicrobial activity
Abbreviations
DPPH
ROS
1,1-Diphenyl-2-picrylhydrazyl radical
Reactive oxygen species
Dimethyl sulfoxide
Lipid peroxidation
1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide
1-Hydroxybenzotriazole
Hydroquinone
DMSO
LPO
EDC
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0419-x) contains supplementary
material, which is available to authorized users.
HOBT
HQ
L. Georgiev ꢀ M. Chochkova (&) ꢀ T. Milkova
Department of Chemistry, Faculty of Mathematics and Natural
Sciences, South-West University ‘‘Neofit Rilski’’,
66, Ivan Mihailov Str., 2700 Blagoevgrad, Bulgaria
e-mail: mayabg2002@yahoo.com
L-DOPA
Y
L-3,4-Dihydroxyphenylalanine
L-Tyrosine
F
L-Phenylalanine
W
L-Tryptophan
Tryp
TA
Tryptamine
I. Totseva ꢀ K. Seizova ꢀ E. Marinova
Tyramine
Institute of Organic Chemistry with Centre of Phytochemistry,
Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
PEA
DA
Phenylethylamine
Dopamine
G. Ivanova
Requimte, Departamento de Quımica, Faculdade de Ciencias,
CA
Cinnamic acid
´
Universidade do Porto, 4169-007 Porto, Portugal
ˆ
p-CoumA p-Coumaric acid
FA
Ferulic acid
SA
Sinapic acid
M. Ninova ꢀ H. Najdenski
The Stephan Angeloff Institute of Microbiology, Bulgarian
Academy of Sciences, Acad. G. Bonchev Str. Bl. 26,
1113 Sofia, Bulgaria
CafA
% RSA
Caffeic acid
Percentage of radical scavenging activity
123

Med Chem Res
Introduction
been developed both synthetically and from natural
resources. Recently, polyhydroxycinnamoylamides have
been reported to act as tyrosinase inhibitors (Masamoto
et al., 2004; Kwak et al., 2011; Fana et al., 2012).
Many efforts to find and develop novel and more
potent tyrosinase inhibitors, antioxidants without side
effects, and effective antimicrobial derivatives become
increasingly important in the food and the cosmetic
industries, and in medicine. A lot of these functions
have been connected to the phenols’ antioxidant activity
(Hollman, 2001).
Oxygen molecules are extremely essential for normal
metabolic process of all aerobes. These molecules possess
dual effects as follows: being essential for life, they can
also damage the cells, biological macromolecules like
DNA, lipids and proteins during oxidative events by
reactive oxygen species (ROS) (Diplock et al., 1998). An
important target of the free radicals is the poly-unsaturated
fatty acids in biomembranes. Their peroxidation, known as
lipid peroxidation, may disrupt membrane functions,
increase permeability, reduce the membrane fluidity, inhi-
bit the signal transduction over the membrane and inacti-
vate membrane-bound enzymes and receptors. To prevent
oxidative stress-induced pathologies, an antioxidant ther-
apy can be used. An important antioxidant pharmacophore
is the catechol moiety present in various antioxidants such
as catecholamines and numerous flavonoids like quercetin
(Heijnen et al., 2001). They are able to scavenge highly
reactive species such as peroxynitrite and the hydroxyl
radical (Heijnen et al., 2001; Ohshima et al., 1998).
Many researchers have shown that the antioxidants of
natural or synthetic origin could have a great importance as
therapeutic agents in several diseases, caused by oxidative
stress. Amongst many classes of compounds, phenolic
analogues such as hydroxycinnamic acids (e.g., caffeic-,
ferulic-, sinapic acids) are one group of antioxidants that
has received much attention. They are distributed ubiqui-
tously in plant kingdom, and shikimate pathway is the key
source for their biosynthesis. Frequently, these secondary
metabolites are present in their free or conjugated forms—
esters, amides or glycosides. Furthermore, amide deriva-
tives of phenolic acids (coupled with amino acids, poly- or
aromatic monoamines) are from natural origin or are
obtained synthetically. It is supposed that these bioactive
substances are involved in suppression of deleterious
effects of oxidative stress and possess a great range of
biological activities such as antioxidant (Wei et al., 2012a;
Kwak et al., 2012; Seo et al., 2010; Kwak et al., 2009; Son
and Lewis, 2002), anticancer (De Baltas and Bedos-Belval,
2011), antimicrobial (Wei et al., 2012b; Narsimhan et al.,
2004; Fu et al., 2010; Chochkova et al., 2012; Georgiev
et al., 2012) and etc.
Because of the constrained availability of natural
hydroxycinnamic acid analogues, the establishment of their
biological activities is still limited. Therefore, we syn-
thesised a series of hydroxycinnamoyl amino acid amides
and evaluated their antimicrobial effect, their antioxidant
activities against lipid peroxidation and their abilities to
scavenge free radical [1,1-diphenyl-2-picrylhydrazyl
(DPPH•)]. The inhibitory effect of hydroxycinnamoyl
amino acid amides and of previously synthesised hydrox-
ycinnamoylamides of biogenic amines (Georgiev et al.,
2012) on mushroom tyrosinase was investigated.
Materials and methods
Reagents
(E)-3-Phenylacrylic-(cinnamic), (E)-4-hydroxy-3-methox-
ycinnamic-(ferulic), (E)-3,4-dihydroxycinnamic-(caffeic),
(E)-3,5-dimethoxy-4-hydroxycinnamic (sinapic), (E)-4-
hydroxycinnamic (p-coumaric) acids, phenylalanine, tryp-
tophan, tyrosine, (S)-2-amino-3-(3,4-dihydroxyphenyl)
propanoic acid (L-DOPA), 1-[3-(dimethylamino)propyl]-3-
ethyl carbodiimide hydrochloride (EDC), triethylamine
(Et3N), mushroom tyrosinase (EC 1.14.18.1) and 1,1-
diphenyl-2-picrylhydrazyl (DPPH•) radical were purchased
from Sigma-Aldrich. Hydroquinone was obtained from
Ferak (Germany), and L-amino acids and their derivatives
were obtained from Merck (Germany). All solvents were
reagent grade and used without further purification. The
water used was deionized.
It has been proved that oxidative stress plays a signifi-
cant role in carcinogenesis (Crohns, 2010). UV irradiation
causes UVA-induced oxidative stress and acts as a key
factor of many skin troubles. Skin hyperpigmentation can
be dependent on increased number of melanocytes or
activity of melanogenic tyrosinase enzyme. Tyrosinase (EC
1.14.18.1) is a key enzyme for melanin biosynthesis in
plants and animals, and its inhibition may be useful for the
treatment of disorders associated with melanin hyperpig-
mentation. Till now, numbers of tyrosinase inhibitors have
Apparatus
The structures and purity of all the amides were confirmed
1
by spectral methods—UV, H and 13C NMR spectroscopy
and ESI-MS. The H and 13C NMR spectra were acquired
1
by a Bruker Avance III 400 spectrometer, operating at
400.15 MHz for protons and 100.62 MHz for carbons. UV
spectra of the amides were measured with an ‘‘Agilent
8453’’ UV–Vis spectrophotometer. The ESI mass spectra
were obtained on an Esquire3000 plus instrument.
123

Med Chem Res
Synthetic procedures of amides
base), 118.2 (2 9 Ar–m-CH, base), 118.5 (o-Ar–CH,
base), 119. 3 (=CH), 120.8 (m-Ar–CH, base), 121.0 (m-Ar–
CH, base), 123.5 (HN–CH=), 123.7 (HN–CH=), 125.2
(Cq), 127.1 (Ar–Cq), 127.4 (Ar–Cq), 136.1 (Cq), 137.3
(Cq), 139.5 (=CH), 148.0 (2 9 C–OCH3), 165.1 (CONH),
171.8 (CONH), 172.1 (COOCH3).
The N-hydroxycinnamoylamides disclosed in Scheme 1,
Table 1 were prepared as in the previously described pro-
cedure (Spasova et al., 2006). Briefly, the amides were
synthesised by coupling of the corresponding cinnamic
acids with amino acid derivatives in DMF in the presence
of triethylamine and 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide (EDC), 1-hydroxybenzotriazole (HOBT) as
coupling reagents (Scheme 1). The reaction was monitored
by thin-layer chromatography on silica gel 60 F254
(Merck), and products were purified by column chroma-
tography. After purification, the yields were between 35
and 80 %.
CafA-W-W-OMe
UV kmax (abs.EtOH) nm: 221, 291, 284, 325. ESI-MS:
567.1 ([M?H]?), 589.4 ([M?Na]?). 1H NMR (DMSO-d6,
400 MHz, ppm): d 3.15 (m, 4H, 2 9 CH2), 3.56 (s, 3H,
COOCH3), 4.57 (m, 1H, HN–CH), 4.75 (m, 1H, HN–CH),
6.40 (d, J = 15.5 Hz, 1H, =CH), 6.73 (d, J = 7.9 Hz, 1H,
Ar–m-CH), 6.81 (dd, J = 7.9, 2.1 Hz, 1H, Ar–o-CH), 6.92
(d, J = 2.1 Hz, 1H, Ar–o-CH), 6.98 (m, 2H, Ar–m-CH),
7.06 (m, 2H, Ar–m-CH), 7.12 (s, 1H, =CH–NH), 7.17 (s,
1H, =CH–NH), 7.18 (d, J = 15.5 Hz, 1H, =CH), 7.32 (2H,
Ar–m-CH), 7.48 (d, J = 7.2 Hz, 1H, Ar–o-CH), 7.64 (d,
J = 8.1 Hz, 1H, Ar–o-CH), 8.13 (d, J = 8.1 Hz, 1H, NH),
8.52 (d, J = 6.3 Hz, 1H, NH), 9.2 (br s, 2H, OH), 10.79 (s,
1H, NH, base). 10.88 (s, NH, base). 13C NMR (DMSO-d6,
100.62 MHz, ppm): d 27.1 (CH2–CHNH), 28.2 (CH2–
CHNH), 51.6 (COOCH3), 53.0 (HN–CH), 53.1 (HN–CH),
109.3 (Ar–Cq), 110.1 (Ar–Cq), 111.2 (o-ArCH, base),
111.4 (o-ArCH, base), 113.8 (Ar–o-CH), 115.6 (Ar-m-
CH), 117.9 (Ar–o-CH, base), 118.2 (2 9 Ar–m-CH, base),
118. 3 (=CH), 118.4 (Ar–o-CH), 120.4 (Ar–o-CH), 120.8
(m-Ar–CH, base), 120.9 (m-Ar–CH, base), 123.5 (HN–
CH=), 123.6 (HN–CH=), 126.4 (Cq), 127.1 (Ar–Cq), 127.3
(Ar–Cq), 136.1 (Cq), 139.5 (=CH), 145.5 (C–OH), 147.4
(C–OH), 165.2 (CONH), 171.9 (CONH), 172.1
(COOCH3).
The chemical structures of all known compounds have
been assigned on the basis of their spectral data and
compared with published data. The detailed UV, NMR and
ESI-MS spectral data of the newly hydroxycinnamoyla-
mides are as follows:
SA-W-W-OMe
UV kmax (abs.EtOH) nm: 205, 221, 291, 322. ESI-MS:
611.4 ([M?H]?). 1H NMR (DMSO-d6, 400 MHz, ppm): d
3.5 (m, 4H, 2 9 CH2), 3.56 (s, 3H, COOCH3), 3.78 (s, 6H,
Ar–OCH3), 4.57 (m, 1H, HN–CH), 4.76 (m, 1H, HN–CH),
6.56 (d, J = 16.1 Hz, 1H, =CH), 6.82 (s, 2H, Ar–o-CH),
6.98 (m, 2H, Ar–m-CH), 7.05 (m, 2H, Ar–m-CH), 7.12 (d,
J = 2.0 Hz, 1H, =CH–NH), 7.17 (d, J = 2.0 Hz, 1H,
=CH–NH), 7.26 (d, J = 16.1 Hz, 1H, =CH), 7.31 (2H, Ar–
m-CH), 7.48 (d, J = 8.1 Hz, 1H, Ar–o-CH), 7.61 (d,
J = 8.1 Hz, 1H, Ar–o-CH), 8.0 (d, J = 7.5 Hz, 1H, NH),
8.51 (d, J = 7.5 Hz, 1H, NH), 8.77 (s, 1H, OH), 10.79 (s,
1H, NH, base). 10.88 (s, NH, base). 13C NMR (DMSO-d6,
100.62 MHz, ppm): d 27.3 (2 9 CH2–CHNH), 51.6
(COOCH3), 51.9 (HN–CH), 52.2 (HN–CH), 56.1
(2 9 OCH3), 105.3 (2 9 Ar–o-CH), 109.3 (Ar–Cq), 110.0
(Ar–Cq), 111.3 (2 9 o-ArCH, base), 117.8 (Ar–o-CH,
Mushroom tyrosinase inhibition assay
Tyrosinase inhibitory activity was determined spectropho-
tometrically by the modified dopachrome method using
O
O
X
Ar
R'
R'
X
OH
N
H
EDC/ HOBt
Et3N, DMF
H
Ar
+
N
H
R''
R''
R'''
R'''
R'=R''=R'''=H, cinnamic acid (CA);
R'=R"'=H, R"=OH, p-coumaric acid (p-CoumA)
R'=H, R"=OH, R'"= OCH3, ferulic acid (FA);
R'=R'''=OCH3 , R"=OH, sinapic acid (SA);
R'=H, R"= R'"=OH, caffeic acid (CafA)
Scheme 1 Synthetic route of cinnamoyl- and hydroxycinnamoylamides
123

Med Chem Res
Table 1 Synthesised cinnamoyl- and hydroxycinnamoylamides
O
X
Ar
R'
N
H
R''
R'''
Compound
Substituents
R0
R00
R000
X
Ar
(A) Amides of aromatic amino acids
CA-DOPA-OMe
CA-F-OMe
H
H
H
H
H
H
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOC(CH3)3
COOCH3
COOCH3
COOCH3
COOC(CH3)3
COOCH3
COOCH3
COOCH3
COOC(CH3)3
COOCH3
COOCH3
COW-OCH3
COOCH3
COOCH3
COOCH3
COOCH3
COW-OCH3
–C6H3-3,4(OH)2
–C6H5
H
H
CA-Y-OMe
H
H
–C6H4-4-OH
3-Indolyl
CA-W-OMe
H
H
p-CoumA-DOPA-OMe
p-CoumA-F-OBut
p-CoumA-W-OMe
p-CoumA-Y-OMe
FA-DOPA-OMe
FA-F-OBut
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
–C6H3-3,4(OH)2
–C6H5
H
H
H
H
3-Indolyl
H
H
–C6H4-4-OH
–C6H3-3,4(OH)2
–C6H5
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
H
FA-Y-OMe
H
–C6H4-4-OH
3-Indolyl
FA-W-OMe
H
SA-DOPA-OMe
SA-F-OBut
OCH3
OCH3
OCH3
OCH3
OCH3
H
–C6H3-3,4(OH)2
–C6H5
SA-Y-OMe
–C6H4-4-OH
3-Indolyl
SA-W-OMe
SA-W-W-OMe
CafA-DOPA-OMe
CafA-F-OMe
CafA-Y-OMe
CafA-W-OMe
CafA-W-W-OMe
(B) Amides of aromatic amines
CA-TA
3-Indolyl
–C6H3-3,4(OH)2
–C6H5
OH
H
OH
H
–C6H4-4-OH
3-Indolyl
OH
H
OH
H
3-Indolyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–C6H4-4-OH
–C6H4-4-OH
–C6H4-4-OH
3-Indolyl
SA-TA
OCH3
OH
OCH3
OCH3
OCH3
OH
OCH3
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OCH3
CafA-TA
H
SA-Tryp
OCH3
H
FA-DA
–C6H3-3,4(OH)2
3-Indolyl
FA-Tryp
H
CafA-DA
H
–C6H3-3,4(OH)2
–C6H3-3,4(OH)2
3-Indolyl
SA-DA
OCH3
H
CafA-Tryp
p-CoumA-Tryp
CA-Tryp
H
3-Indolyl
H
H
3-Indolyl
FA-TA
OCH3
H
OH
H
H
–C6H4-4-OH
–C6H3-3,4(OH)2
–C6H5
CA-DA
H
p-CoumA-PEA
p-CoumA-DA
p-CoumA-TA
H
OH
OH
OH
H
H
H
–C6H3-3,4(OH)2
–C6H4-4-OH
H
H
123

Med Chem Res
L-tyrosine as a substrate (Kwak et al., 2011). The reaction
media (3 ml) contained: phosphate buffer (1.0 ml, 0.1 M,
pH 6.8); tyrosine (1.0 ml, 1.5 mM) dissolved in deionized
water; inhibitor (0.350 ml, 0.2 mM) dissolved in DMSO;
deionized water (0.350 ml); mushroom tyrosinase aqueous
solution (0.300 ml, 192 U/ml). The reference solution was
prepared with 0.300 ml of DMSO instead of inhibitor.
After adding mushroom tyrosinase solution, the reaction
mixture was incubated at 37 ꢁC for 30 min. The UV
absorbance of the reaction mixture was measured at
475 nm. The percentage of mushroom tyrosinase inhibitory
activity was calculated by the following equation:
The effectiveness of the additives in the lipid systems
was estimated on the basis of the induction period (IP) i.e.,
the time in which the concentration of the antioxidants is
fully consumed, was determined by the method of the
tangents. That means the tangents are applied to the two
parts of the kinetic curves. The coefficient of variation
ranged from 6 to 13 % and was inversely related to the
induction period. The reported IP values result from three
independent experiments. The effectiveness was expressed
as stabilization factor F:
F ¼ IPadd= IPo
where IPadd is the induction period in the presence of the
tested additive, and IPo is the induction period of the
control sample.
Mushroom tyrosinase activity % ¼ B=A ꢁ 100;
where A is the absorbance of the reference solution and B is
the absorbance of the test sample solution.
Each experiment was performed in triplicate and aver-
aged. Hydroquinone, p-coumaric and caffeic acids were
used as positive controls.
Antimicrobial assay
The microorganisms were originated from the Bulgarian
National Collection for Microorganisms and Cell Cultures
(Sofia, Bulgaria). Antimicrobial activities were assayed
against the Gram-positive strains: Staphylococcus aureus
209 and Streptococcus pyogenes 10535. The minimal
inhibitory concentration of each compound was determined
by the method of serial dilutions in tubes with liquid media
inoculated with standardised number of microorganisms
(1.0 9 105 CFU/ml) as described by Hindler (1992). After
incubation at 37 ꢁC for 24 h, the lowest concentration
(highest dilution) of each sample that visibly inhibited
microbial growth was taken as the MIC (Table 2). The
results are shown in triplicate. For a positive control, a
commercially available antibiotic tobramycin was used that
was bought from the local pharmacy. The solvent—96 %
EtOH was tested as negative control.
Antioxidant activity assays
Radical scavenging activity
The DPPH assay was performed according to a method
described by Nenadis and Tsimidou (2002). Briefly, an
ethanolic DPPH solution (0.1 mM, 2.96 ml) was added to
0.04 ml of the antioxidant solution in abs. ethanol at the
following concentrations: 0.9, 1.8, 3.6 mM. The decrease
in absorbance at 516 nm was measured at 25 ꢁC after 10
and 20 min of reaction. The results were expressed in
triplicate as the percentage of radical scavenging activity
(RSA %), calculated as follows:
ÈÀ
Á
É
ꢂ
ꢂ
RSA % = AbsðDPPH Þ ꢃ Abssample =AbsðDPPH Þ ꢄ 100
Statistical analysis
In vitro inhibition of lipid peroxidation
The values are presented as mean value of three mea-
surements confidence interval, calculated at level of
significance 0.05.
The antioxidative activities of some synthetic hydroxycin-
namoyl amino acid amides were evaluated in a concentra-
tion range (2.5 9 10-4–20 9 10-4 M) by autoxidation of
triacylglycerols of sunflower oil. Oxidation was carried out
at 100 ꢁC ( 0.2 ꢁC) in the dark by blowing air through the
samples (2 g) at a rate of 50 ml/min. Under these conditions,
the process took place in a kinetic regime i.e., at a suffi-
ciently high oxygen concentration at which the diffusion
rate did not influence the oxidation rate. The process was
followed by withdrawing samples (ca. 0.1 g) at various time
intervals and subjecting them to iodometric determination of
the primary oxidation products (peroxides) concentration
i.e., the peroxide value (PV) (Yanishlieva et al., 1978), and
kinetic curves of peroxide accumulation were plotted.
Results
Effects of hydroxycinnamic acids and their derivatives
on the monophenolase activity of mushroom tyrosinase
In order to elucidate the role of amino component on
tyrosinase activity, the inhibitory activity of 18 N-hy-
droxycinnamoyl aromatic amino acids esters (precursors of
aromatic amines) and of recently synthesised hydrox-
ycinnamoylamides of biogenic amines (Georgiev et al.,
2012) on mushroom tyrosinase was studied (Table 1).
123

Med Chem Res
Our results might reinforce previously reported data of
the importance of p-hydroxyphenyl moiety in the mole-
cules of tyrosinase inhibitors. p-Hydroxycinnamoylamides
(with DOPA, tyrosine methyl esters or with dopamine or
tyramine) showed inhibitory activities similar to hydro-
quinone and better one, even though, than p-coumaric
acid, used as standard tyrosinase inhibitors. Most of the
other compounds were less active than the referent
hydroquinone.
Table 2 Antimicrobial activity of cinnamoyl amides, expressed as
minimal inhibitory concentration [MIC (lg/ml)]
Compound
Test microorganisms
Solvent
S. aureus
MIC
S. pyogenes
SA-W-OMe
SA-Tryp*
N.d.
250
125
62.5
125
62.5
125
62.5
N.d.
125
125
125
125
15.62
250
62.5
250
62.5
N.d.
125
N.d.
125
250
62.5
125
125
125
7.81
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
96 % EtOH
Water
SA-Y-OMe
SA-TA*
FA-Y-OMe
FA-TA*
Antioxidant activity
FA-DOPA-OMe
FA-DA*
In order to carry out a structure–antioxidant activity rela-
tionship study, a series of amides of hydroxycinnamic acid
amides were tested for both DPPH scavenging and lipid
peroxidation-inhibiting activity. It must be emphasised that
one compound may not just be classified as an antioxidant
on the basis of a single antioxidant experiment (Frankel,
2001). Therefore, a series of synthetic phenolic amides and
referent antioxidants: caffeic, sinapic and ferulic acids
were tested for both DPPH scavenging and lipid peroxi-
dation-inhibiting activities.
CafA-W-OMe
CafA-Tryp*
Ferulic acid (FA)
Caffeic acid (CafA)
Sinapic acid (SA)
Tobramicyn
N.d. not determined
* The data were presented previously and were given for comparison
(Georgiev et al., 2012)
According to the established DPPH scavenging activity
(above 60 %; Table 3), the studied amides could be
ordered as follows: N-p-coumaroyl-DOPA methyl
ester \ sinapic acid \ D,L-a-tocopherol \ quercetin \ N-
sinapoyl-DOPA methyl ester \ N-feruloyl-DOPA methyl
ester & N-caffeoyl-tryptophan methyl ester & N-caf-
feoyl-DOPA methyl ester & N-cinnamoyl-DOPA methyl
ester & N-caffeoyl-tyrosine methyl ester & N-caffeoyl-
The percentage of monophenolase tyrosinase activities
(using L-tyrosine as a substrate) of amides was outlined in
Fig. 1. Amongst the tested compounds, both types of
amides with para-hydroxyphenyl moiety in their structures
possessed the notable inhibitory activity which might be
due to their structural resemblance to L-tyrosine.
Amides with aromatic amino acids
Amides with aromatic amines
Reference compounds
150
100
50
0
HQ
CafA
SA-TA
CA-TA
FA-TA
CA-DA
FA-DA
SA-Tryp FA-Tryp
SA-DA
CA-Tryp
CafA-TA
CafA-DA
p-CoumA
CafA-Tryp
FA-YS-AO-MY-eOMe
CA-Y-OMe
CafA-Y-OMe
FA-DOPA-OMe
SA-W-OMFAe -W-OMe
CA-W-OMe
p-CoumA-TA
p-CoumA-DA
CafA-W-OMe
SA-DOPA-OMe
SA-W-W-OMe
CafA-DOPA-OMe
p-CoumA-PEA
p-CoumA-Tryp
CA-DOPA-OMe
CafA-W-W-OMe
p-CoumA-F-OBut
p-CoumA-Y-OMe
p-CoumA-W-OMe
p-CoumA-DOPA-OMe
Fig. 1 Mushroom tyrosinase activity with cinnamoyl- and hydroxycinnamoylamides, and reference samples
123

Med Chem Res
Table 3 DPPH radical scavenging activity of N-cinnamoyl- and hydroxycinnamoyl amino acid amides
Compound
% (RSA)
0.9 mM
100
1.8 mM reaction time (min)
3.6 mM
100
200
100
200
200
Quercetin
40.43 – 0.32
23.24 – 0.10
16.10 – 0.02
13.67 2.55
N.d.
45.18 – 0.52
24.75 – 0.22
17.22 – 0.03
14.63 2.61
N.d.
46.68 – 0.15
40.82 – 0.20
26.50 – 0.05
25.97 2.04
N.d.
58.36 – 0.15
46.84 – 0.17
31.91 – 0.02
27.30 1.59
N.d.
76.01 – 0.20
62.91 – 0.22
69.03 – 0.13
48.14 2.22
N.d.
78.05 – 0.15
73.31 – 0.15
69.62 – 0.10
D,L-a-Tocopherol
Sinapic acid
SA-W-OMe
49.92 1.57
N.d.
SA-W-W-OMe
SA-Y-OMe
14.17 2.11
23.80 – 3.00
13.72 0.50
2.10 0.05
1.92 0.61
0.64 0.43
17.99 – 1.54
1.97 0.70
12.64 0.07
7.96 0.87
7.04 1.22
24.26 – 4.62
10.91 2.24
33.50 – 0.99
27.11 – 5.22
N.d.
15.55 2.09
25.37 – 2.16
16.70 0.99
2.90 0.07
2.13 0.88
0.71 0.34
19.10 – 1.26
2.30 0.29
13.81 0.05
9.76 0.95
8.79 1.43
28.47 – 5.17
13.51 1.74
34.10 – 0.57
28.70 – 5.47
N.d.
23.14 1.84
43.94 – 1.77
25.00 2.24
3.70 0.05
1.69 0.63
1.04 0.30
33.32 – 4.49
1.89 1.12
21.02 0.27
14.52 1.37
13.65 1.00
44.82 – 6.53
19.50 0.25
62.11 – 2.52
44.62 – 4.22
N.d.
25.01 1.56
45.75 – 2.43
28.24 0.25
4.70 0.10
2.37 0.51
1.42 0.42
34.83 – 4.66
2.23 1.11
25.10 0.30
17.40 1.47
16.25 1.64
50.60 – 6.62
23.63 0.25
64.28 – 2.89
50.43 – 4.22
N.d.
42.96 2.38
80.92 – 3.39
46.82 0.25
4.50 0.07
2.42 1.18
1.44 0.46
64.41 – 5.02
2.46 0.49
36.71 0.12
26.45 2.54
24.84 2.33
83.76 – 4.85
30.42 0.50
88.04 – 1.78
80.11 – 1.99
N.d.
45.28 1.90
82.75 – 3.17
50.60 0.25
6.10 0.10
2.88 2.15
1.88 0.27
67.08 – 5.47
2.92 0.25
44.32 0.20
30.91 2.89
29.25 2.20
86.95 – 1.43
36.90 0.55
91.83 – 1.61
87.90 – 0.75
N.d.
SA-DOPA-OMe
SA-F-OBut
p-Coumaric acid
p-CoumA-W-OMe
p-CoumA-Y-OMe
p-CoumA-DOPA-OMe
p-CoumA-F-OBut
Ferulic acid
FA-W-OMe
FA-Y-OMe
FA-DOPA-OMe
FA-F-OBut
Caffeic acid
CafA-W-OMe
CafA-W-W-OMe
CafA-Y-OMe
CafA-DOPA-OMe
CafA-F-OMe
Cinnamic acid
CA-W-OMe
27.39 – 1.07
44.81 – 6.11
28.80 – 1.16
0.30 0.11
0.79 0.29
1.74 0.13
24.42 – 0.70
2.00 1.10
28.66 – 0.99
50.48 – 6.71
29.74 – 0.86
0.32 0.10
0.69 0.34
2.12 0.43
26.60 – 0.61
2.52 0.75
51.01 – 2.85
82.46 – 6.50
53.48 – 3.12
0.32 0.10
1.20 0.48
2.05 1.36
45.53 – 3.19
2.39 1.44
52.53 – 3.23
86.01 – 2.48
54.65 – 3.54
0.35 0.2
89.61 – 0.83
88.73 – 0.23
90.34 – 0.27
0.32 0.2
90.20 – 0.44
88.81 – 0.59
90.49 – 0.38
0.35 0.3
1.41 0.06
2.30 1.43
48.42 – 3.16
2.60 1.79
1.18 0.94
2.12 0.52
84.99 – 4.30
2.18 1.22
1.48 0.61
2.57 0.45
88.11 – 4.08
2.58 0.88
CA-Y-OMe
CA-L-DOPA-OMe
CA-F-OMe
% RSA for typical antioxidants are given for comparison. All tests were performed in triplicate. The values presented in this table are the mean value of three
measurements confidence interval, calculated at level of significance 0.05
N.d. insoluble above this concentration
Significance of bold means that the compounds are the most active
phenylalanine methyl ester & caffeic acid. Results showed
that seven of the tested amides have better activity than
sinapic acid, D,L-a-tocopherol, quercetin and, six of them
have commensurable activity with caffeic acid.
of peroxide accumulation obtained the values for the sta-
bilization factor (F) which were determined. A value of
F = 1 corresponds to no antioxidant activity. The results
obtained are presented in Fig. 2. A comparison with the
effectiveness of caffeic, sinapic and ferulic acids at the
same concentrations is given. Cinnamic and p-coumaric
acids were inactive during these oxidation conditions.
The antioxidant activity decreases in the following order:
N-caffeoyl-phenylalanine methyl ester [ N-cinnamoyl-
DOPA methyl ester [ caffeic acid & N-caffeoyl-trypto-
phan methyl ester [ N-p-coumaroyl-DOPA methyl ester &
N-caffeoyl-tyrosine methyl ester & N-caffeoyl-DOPA
methyl ester & N-feruloyl-DOPA methyl ester [ N-
sinapoyl-DOPA methyl ester [ N-sinapoyltryptamine [
Besides the free radical scavenging assays, all the
compounds were tested in the lipid peroxidation assay
which is one of the biomimetic antioxidant tests. We chose
to test the compounds with high value % RSA and some
tryptophan and tyramine derivatives for evaluation of
indole ring influence in the antioxidant capability of the
amides. Ten of them were evaluated for their antioxidant
effectiveness at the concentration 1 9 10-3 M during
autoxidation of pure triacylglycerols of sunflower oil
(TGSO) at 100 ꢁC. After processing, all the kinetic curves
123

Med Chem Res
50
40
30
20
10
0
Amides with aromatic amino acid esters
Amides with aromatic amines
Reference standarts
SA FA
CafA
FA-DA
SA-DA
CA-DA
SA-Tryp
CafA-TA
CafA-Y-OMe
CafA-DA
CafA-PEA
CafA-F-OMe
CA-DOPA-OMe
CafA-Tryp
p-CoumA-DA
SA-W-OMe
CafA-W-OMe
FA-DOPA-OSMAe-DOPA-OMe
CafA-DOPA-OMe
-CoumA-DOPA-OMe
p
Fig. 2 Stabilization factor of hydroxycinnamoylamides and reference
compounds during oxidation of TGSO. Concentration for all
compounds 1 9 10-3 M. Data for hydroxycinnamoylamides with
aromatic amines were reported previously (Georgiev et al., 2010) and
stand to their corresponding precursor amides of aromatic amino
acids in this figure
N-sinapoyl-tryptophan methyl ester [ sinapic acid [ feru-
lic acid, during oxidation of TGSO. Data obtained show
that three of N-hydroxycinnamoyl amino acid amides had
better or equal activity when compared with the referent
antioxidant—caffeic acid, whereas all amides were more
potent from the ferulic and sinapic acids.
Discussion
Recently, tyrosinase inhibitors have received a special
attention because of suppressing of hyperpigmentation,
undesirable browning of mushrooms, vegetables, fruits and
etc. Therefore, anti-tyrosinase agents are connected with a
different range of applications: in medicine, cosmetic and
food industries (Mosher et al., 1983; Friedman, 1996;
Parvez et al., 2007).
Their stabilization factors and radical scavenging
activities were shown in Fig. 2 and Table 3.
The structural resemblance of hydroxycinnamic acids
(p-coumaric-, caffeic-, ferulic- and sinapic acids) to
L-DOPA or L-tyrosine appears to be an important element
for tyrosinase inhibitory activity (Kim et al., 2005; Mat-
suda et al., 1995). Recently, the increasing potential of
tyrosinase inhibition was observed after connection of
aromatic amino acids to hydroxyphenolic acids or kojic
acid (Noh and Lee, 2011; Noh et al., 2009). This was the
reason for several cinnamoyl- and hydroxycinnamoyla-
mides of aromatic amino acids (H-Y-OMe, H-W-OMe,
H-F-OMe and H-DOPA-OMe) to be obtained and their
tyrosinase inhibitory activity to be compared to that of the
corresponding amides of aromatic amines.
Antimicrobial activity
On the basis of our previous investigations on cinnamoyl
amides of biogenic monoamines (Georgiev et al., 2012),
we chose five N-hydroxycinnamoylamides of their amino
acid precursors to examine their antimicrobial activity
against two Gram-positive strains: Staphylococcus aureus,
Streptococcus pyogenes. The commercial available antibi-
otic tobramycin was used as a positive control. The
examination of the data (Table 2) revealed that two of N-
feruloyl amino acid amides (FA-Y-OMe and FA-DOPA-
OMe) and N-sinapoyl-tyrosine methyl ester showed MIC
values of 125 lg/ml against S. aureus. All tested amino
acid amides showed activity of 250 lg/ml against
S. pyogenes, whereas the free acids showed activity of
125 lg/ml against both pathogens.
Amongst the 34 cinnamic- and hydroxycinnamic acid
amides, the amides of p-coumaric acid demonstrated the
highest tyrosinase inhibitory activities, but remained less
potent than the used standard-hydroquinone. This study
123

Med Chem Res
indicated that para-hydroxylated phenyl ring is important
for tyrosinase inhibitory activities, whereas addition of
second hydroxy or two methoxy groups ortho- to the para-
hydroxy group in the same ring decreases the anti-tyrosi-
nase activity. Our observation confirmed the previously
found correlation between p-coumaroyl structure and anti-
tyrosinase activities by other research groups (Qiu et al.,
2009). On the other hand, we concluded that the extension
of SA-W-OMe or Caf-W-OMe with one more tryptophanyl
moiety did not lead to any significant difference in the anti-
tyrosinase activity.
might come to a conclusion that all tested compounds are
promising antioxidants; moreover, the amides with aro-
matic amines have values for stabilization factor almost
twice higher. For evaluation of contribution of indole ring
in the synthetic amides, we chose to test and compare the
compound with weaker activity (N-sinapoyltryptamine and
N-sinapoyl-tryptophan methyl ester), so that the influence
will be demarked better and not be covered by influence
from the catechol-containing moiety. The results obtained
showed the positive influence of indole moiety on the
activity, which is clearly expressed in comparison with the
sinapic acid. N-sinapoyl-tryptophan methyl ester and
N-sinapoyltryptamine acted as inhibitors of lipid peroxi-
dation, respectively two and three times better than the free
acid.
Cinnamic acid esters and amides have been reported to
be good antimicrobials against Gram-positive, Gram-neg-
ative and fungal strains (Narsimhan et al., 2004; Fu et al.,
2010). The results obtained for antimicrobial activity of the
studied cinnamoyl amino acid amides (Table 2) showed
that the compounds exhibit activity, but slower compared
with the referent antibiotic. In comparison of these results
to those obtained for cinnamoylamides of biogenic amines
against the same bacteria, it is evident that the presence of
ester group in hydroxycinnamoyl amino acid amides cau-
ses two times decrease in the growth inhibitory effect than
their analogues.
The in vitro results obtained, indicating the role of
indole nucleus on the antioxidant activity, have been in line
´
with the finding of Stetinova et al. (2002), who have
established that some indole-containing compounds could
stimulate antioxidative enzyme systems.
This fact should be examined further in the design of
new antioxidants.
Acknowledgments This work was supported by the Bulgarian
Science Fund (contracts DMU03/2) and by South-West University
‘‘Neofit Rilski’’, Bulgaria (contract SRP A4/12). The NMR spec-
trometer is part of the National NMR Network in Portugal and was
purchased under contract REDE/1517/RMN/2005, with funds from
The prevention of diseases by antioxidants is an area of
intense current interest. Antioxidants also play an impor-
tant role in the protection of processed foods from oxida-
tion. This is desirable not only in relation to the storage
properties of food, but in preventing the accumulation of
cytotoxic lipid peroxidation products.
ˆ
POCI 2010 (FEDER) and Fundac¸a˜o para a Ciencia e a Tecnologia
(FCT).
It is evident from the RSA results presented in Table 3
that the most active compounds contain catechol moiety.
The conjugation of catechol type compounds to not so
active cinnamic and p-coumaric acids leads to an increase
in their radical scavenging activity. These results confirmed
the radical scavenger effect against peroxynitrite of cate-
chol-containing antioxidants previously described in the
literature (Heijnen et al., 2001). The RSA of all compounds
was dose-dependent and correlated positively with the
concentrations (the only exception from the last conclusion
was N-caffeoyl-DOPA-OMe). These findings are in very
good correlation with our previously obtained results on
the RSA of hydroxycinnamoylamides of biogenic amines
(Georgiev et al., 2012). According to the results of DPPH
test, it was evident that catechol moiety played a sub-
stantial role in the examined activity, whereas the presence
of methoxy groups decreased the activity (compared with
catechol containing compounds), which suppose another
mechanism of action of the formed radicals. However, the
activity of the last compounds is still greater than the acids
itself, which shows importance of the amide bond.
Conflict of interest None.
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