Newly synthesized dopamine ester derivatives and assessment of their antioxidant, antimicrobial and hemolytic activities

Article abstract of DOI:10.1016/j.procbio.2013.07.022

Preparation of dopamine derivatives was carried out as a response to the increasing demand for new lipophilized antioxidants in food, cosmetic and pharmaceutical industries. A large series of dopamine esters (DA-C₃ to DA-C_18:1) with

Full text of DOI:10.1016/j.procbio.2013.07.022

Process Biochemistry 48 (2013) 1481–1487
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Newly synthesized dopamine ester derivatives and assessment of
their antioxidant, antimicrobial and hemolytic activities
a
b,c
a
d
a
Mohamed Sellami , Ali Châari , Imen Aissa , Mohamed Bouaziz , Youssef Gargouri ,
Nabil Miled
a
a,∗
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Université de Sfax, route de Soukra, BPW 1173-3038 Sfax, Tunisia
Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), UMR CNRS 7086, UFR de Chimie, Université Paris Diderot - Paris 7, Bat.
b
Lavoisier, 15 rue Jean Antoine de Baïf, 75205 Paris Cedex 13, France
c
Laboratoire de Génétique et Biologie Cellulaire Université de Versailles Saint Quentin en Yvelines, 45 avenue des Etats-Unis, 78000 Versailles cedex, France
Laboratoire des Bioprocédés, Centre de Biotechnologie de Sfax (CBS), Université de Sfax, BP 1177, 3018 Sfax, Tunisia
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 March 2013
Received in revised form 23 July 2013
Accepted 24 July 2013
Available online 1 August 2013
Preparation of dopamine derivatives was carried out as a response to the increasing demand for new
lipophilized antioxidants in food, cosmetic and pharmaceutical industries. A large series of dopamine
esters (DA-C3 to DA-C18:1) with increasing lipophilicity was synthesized using lipase from Candida
antarctica (Novozyme 435) as a biocatalyst. The highest conversion yield (52.75%) was reached when
caprylic acid (DA-C8) was used as acyl donor. Synthesized compounds were purified and evaluated for
their antioxidant activity using the DPPH and the ABTS tests. Results showed that esterification had little
effect on radical-scavenging capacity. However, long chain fatty acid esters displayed higher protective
Keywords:
Dopamine
Antioxidant
◦
effect of oil against oxidation at 70 C as compared to the parent dopamine or to the BHT. The hemolytic
activity of dopamine esters was studied. Middle chain length derivatives (DA-C8 and DA-C12) of dopamine
and oleic acid derivative (DA-C18:1) showed the highest hemolytic activity against human erythrocyte. The
antimicrobial activities of dopamine esters were also evaluated using well diffusion and minimal inhibi-
tion concentration methods. Among all the tested compounds, DA-C8 and DA-C12 exhibited the highest
antibacterial activities. These results open up potential applications by using dopamine derivatives as
antioxidants and antimicrobial compounds in cosmetic, food and pharmaceutical industries.
Antimicrobial activity
Lipophilicity
Hemolytic activity
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), propyl
gallate were extensively used with the intention to delay or pre-
vent the negative effects of lipid peroxidation by scavenging free
radicals or diminishing the formation of initiating lipid radicals.
However, they were not so well accepted by consumers as dietary
components, and there has been growing concerns about their
possible side-effects such as carcinogenicity [4]. Development of
new antioxidants with less toxicity is desirable for the prevention
and/or treatment of a number of diseases and for better food
preservation. Extensive work has been carried out over the last
decade to find novel naturally occurring compounds as alternatives
to the synthetic antioxidants [3,5]. Dopamine (DA), a phenolic com-
pound derived from tyrosine and belonging to the catecholamine
family, is a natural constituent of all aerobic organisms and is
widely used in medicine as well as in food [6]. DA has been shown
Fats, oils and lipid-based foods deteriorate through several
degradation reactions both on heating and on long-term storage
1]. The main deterioration processes were oxidation reactions
[
that result in decreased nutritional value and sensorial quality. The
spontaneous reaction of atmospheric oxygen with lipids, known as
auto-oxidation, is the most common process leading to oxidative
deterioration [2]. In addition to the prevention of oxygen access,
the use of low temperatures, the inactivation of enzymes causing
oxidation, or a suitable packaging, oxidation may be also inhibited
by the use of specific additives (antioxidants) that may vary in their
chemical structure and mode of action. Antioxidants have been
added to food for decades to control the oxidation process and they
are widely used today for better food preservation [3]. Synthetic
antioxidants such as butylated hydroxyanisole (BHA), butylated
•
to be an effective scavenger of superoxide (O 2) and hydroxyl
•
radicals (HO ) [6] since it is an uncomplexed catecholamine [7].
The ability of the catechol ring to scavenge free radicals was
attributed to the ability of the structure to donate electrons [6]. DA
functions as a neurotransmitter. It is produced in several areas of
the brain, including the substantia nigra and the ventral tegmental
∗ Corresponding author. Tel.: +216 74675055; fax: +216 74675055.
E-mail address: nmiled@yahoo.com (N. Miled).
1
359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.07.022

1
482
M. Sellami et al. / Process Biochemistry 48 (2013) 1481–1487
area. DA plays a major role in the brain system that is responsible
for reward-driven learning and its absence in the organism causes
several neuro-degenerative diseases such as Parkinson [8]. DA pro-
vides an antioxidant defense in the brain against oxidant and free
radical induced damages [9]. It has been found to protect against
intestinal mucosal injury by modulating eicosanoid synthesis
which contributes to the anti-inflammation [10]. DA was identi-
fied at a high level ranging from 80 to 560 mg per 100 g in peel and
area of the ester product divided by the total area, multiplied by 100 as described
previously by Araujo et al. [19]. All experiments were performed in triplicates.
2
.3. HPLC analysis
The identification and the conversion yield of DA derivatives were carried
out by HPLC analysis. It was performed using a Dionex apparatus composed of
an LC-10ATvp pump and an SPD-10Avp detector. The column used was a C-18
◦
(
1
4.6 mm × 250 mm; Shimpack VP-ODS), maintained at 30 C. The flow rate used was
.0 mL/min and detection of DA and its derivatives were carried out by measuring
2
.5 to 10 mg in pulp of banana to shield them against peroxidizing
the absorbance at 280 nm. The mobile phase used was 0.1% acetic acid in water
(A) versus 0.1% acetic acid in acetonitrile (B) for a total running time of 25 min and
the following proportions of solvent B were used for the elution: 0–5 min: 10%;
factors since banana, a tropical fruits, grows under hot and intense
sunlight that facilitates lipid peroxidation [11]. Unfortunately, DA
exhibits a low solubility and stability in the lipophilic media which
can limit its efficient use in these conditions. Hence, lipophilic
derivatives of DA and, in particular, long acyl chain esters, would
exhibit a better affinity with lipophilic membrane constituents of
drug target cells [12]. This property would make from DA deriva-
tives good candidates to be used in pharmaceutical and cosmetic
lipophilic formulations [13]. Some DA derivatives have been found
in rat and bovine brains and their role as endogenous cannabi-
noid/vanilloid regulators has been speculated [14,15]. They could
be considered as a transport form of DA capable of crossing the
blood-brain barrier and producing the dopaminergic effect [12].
The interest in new compounds with antimicrobial properties
has been revived because of current problems associated with the
use of antibiotics [16]. Aissa et al. [5] have synthesized a large series
of tyrosyl esters with increasing lipophilicity using lipase of Can-
dida antarctica. Authors showed that the parent tyrosol does not
have any effect on various pathogenic bacteria. However, tyrosyl
esters and especially medium chain tyrosyl derivatives exhibited
an antimicrobial activity against staphylococcus strains.
5–10 min: 10–100%; 10–20 min: 100% and 20–25 min: 100–10%.
2
.4. LC/MS analysis
The LC/MS experiments were carried out with an Agilent 1100 LC system con-
sisting of degasser, binary pump, auto sampler, and column heater. The column
outlet was coupled to an Agilent MSD Ion Trap XCT mass spectrometer equipped
with an ESI ion source. Data acquisition and mass spectrometric evaluation were
carried out on a personal computer with Data Analysis software (Chemstations).
2.5. Purification and identification of dopamine esters
The reaction mixture resulting from the esterification of DA with various fatty
acids contains a mixture of DA ester and residual substrates. After removal of the
enzyme and the molecular sieves by centrifugation at 8000 rpm for 15 min, sol-
◦
vent (2-methylbutan-2-ol) was evaporated at 50 C under vacuum. DA esters except
DA propionate (DA-C3) were purified by liquid–liquid extraction according to the
method described by Ardhaoui et al. [20]. The residual DA was removed in the water
◦
phase at 50 C by addition of water/hexane mixture (2/3, v/v) under stirring during
2
0 min. DA esters were recovered in the acetonitrile phase by adding a mixture of
acetonitrile/hexane = 3/5 (v/v) at 50 ◦C. The purification of DA-C was achieved by
3
chromatography on a silica gel 60 column (Merck) (25 cm × 2 cm) according to Aissa
et al. [5]. Final purity of the products obtained was checked using LC/MS analysis.
A
great variety of chemical compounds possessing both
2
.6. DPPH radical scavenging assay
hydrophilic and lipophilic properties induce hemolysis at given
concentration, a consequence of their amphiphilic character
Free radical scavenging activity of DA derivatives was determined using the
[
17,18]. Although the molecular events in the hemolytic process are
DPPH method described by Brand-Williams et al. [21]. The changes in color (from
purple to a residual pale yellow color) were measured at 517 nm on an UV/visible
light spectrophotometer (Secomam, Uvi Light XT 5). An aliquot of ethanol absolute
solution (0.1 mL) containing different concentrations (1:2 serial dilutions from initial
sample) of DA and its esters were added to 3.9 mL of DPPH solution (0.06 mM in
ethanol) and vortexed. The absorbance of the remaining DPPH was determined after
not fully known, they are generally believed to consist of 5 steps:
adsorption of the surfactant to the cell surface, intercalation of the
surfactant into the membrane, induction of changes in the organi-
zation of the membrane, increase in permeability of the membrane,
and rupture of the erythrocyte [17]. Because of the extensive use
of surfactants studies on the hemolytic assay, it is of interest to
analyze the performance of new amphiphilic compounds with dif-
ferent fatty acid chain lengths to study the relationship between
their structure and their hemolysis potency.
We have synthesized a large series of DA fatty acid esters by
direct esterification of DA with various fatty acids using Novozyme
4
microbial activity against several pathogenic strains and hemolytic
activity against human erythrocytes.
60 min at 517 nm against ethanol blank. BHT was used as standard control. IC50
values denote the concentration of sample, which is required to scavenge 50% of
DPPH free radicals.
2.7. ABTS radical scavenging assay
The ABTS radical-scavenging activity was determined according to Re et al. [22].
The ABTS radical cation was prepared by reacting an aqueous solution of ABTS
(
7 mM) with potassium persulfate (2.45 mM, final concentration) which was kept
35 as a catalyst and evaluated their antioxidant potential, anti-
◦
in the dark at 25 C for 12–16 h. The obtained solution was diluted in ethanol to
an absorbance of 0.70 (± 0.020) at 734 nm before use. 10 ␮L of trolox or sample
in ethanol were mixed with 990 ␮L of this diluted solution and the absorbance
was determined at 734 nm after 6 min of initial mixing. The antioxidant activ-
ity was calculated relative to the equivalent trolox concentration. The activity of
each antioxidant was determined at three concentrations, within the range of the
dose–response curve of trolox and the radical-scavenging activity was expressed as
the trolox equivalent antioxidant capacity (TEAC), defined as mM of trolox per gram
of sample. BHT was used as reference compound.
2
. Materials and methods
2
.1. Materials
Dopamine, caprylic, lauric, myristic, palmitic, oleic, stearic acids and 2-
methylbutan-2-ol were purchased from Fluka (Germany). n-Hexane from Prolabo
Paris, France), acetonitrile and proprionic acid from Pharmacia (Uppsala, Sweden).
Lipase from Candida antarctica (Novozyme 435) was from Sigma Aldrich (Germany).
2.8. Lipophilicity evaluation
(
The lipophilic character of DA esters was evaluated as described by Viskupicova
et al. [23] by determining the partition coefficient (miLog P). miLog P values of DA
esters were calculated using Molinspiration software, which is based on fragmental
methods (available online at www.molinspiration.com).
2
.2. Esterification reactions
DA lipophilic esters (DA-C3 to DA-C18:1) were prepared by direct esterification of
2.9. Measurement of conjugated diene (CD) and triene (CT)
DA with different fatty acids in screw-capped flasks. DA (10 mg/mL) was dissolved
in 2 mL of 2-methylbutan-2-ol. The fatty acid concentration was adjusted to obtain
an equimolar ratio. The mixture was stirred at 50 C in an orbital shaker at 200 rpm,
Conjugated diene (CD) and conjugated triene (CT) are suited for measuring the
oxidative state of oil [24]. DA and its derivatives were added to refined soya oil at
200 ␮g/g of oil and stored at 70 C. The reaction was monitored for 15 days and
◦
◦
in the presence of 10 mg/mL of Novozyme 435 and 100 mg/mL of molecular sieves
4
A˚ . Control reactions in the absence of lipase were also carried out. Aliquots from
the reaction mixture were withdrawn within 168 h of reaction and filtered to be
used for HPLC analysis. The conversion yield of DA derivatives was calculated as the
every day, 0.1 g of soya oil was withdrawn and dissolved in 25 mL of hexane and the
absorbance was measured at 232 nm (for CD) and 270 nm (for CT), using hexane as
a blank. All measurements were performed in duplicates.

M. Sellami et al. / Process Biochemistry 48 (2013) 1481–1487
1483
2
2
.10. Antibacterial activity
.10.1. Diffusion well method
The bactericidal effects of DA and its acyl esters were tested against several
Gram positive and Gram negative bacteria on Luria Broth (LB) medium agar plate by
agar spot assay [25]. Bacteria (previously pre-incubated 12 h in LB medium) were
◦
cultivated in LB medium at 37 C for 3 h. A basal layer of LB containing 16 g/l agar,
was cooled in Petri dishes. When plates were dried, 10 mL of soft LB (8 g/l agar)
containing 107 cells were overlayed. Wells were then punched in the agar plate
◦
and filled with 20 ␮l of each sample (3 mg/mL). After 24 h of incubation at 37 C, the
zone of growth inhibition was measured to determine the level of bactericidal effect.
Ampicillin (10 ␮g/well) was used as positive control. Several bacterial strains were
used: i.e., gram-positive bacteria: Bacillus cereus, Micrococcus luteus, Brevibacterium
flavum, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus xylosus,
Bacillus subtilis and Enterococcus faecium and gram-negative bacteria: Pseudomonas
aeruginosa, Salmonella, Klebsielle pneumoniae, Enterobacter cloacae and Escherichia
coli.
2.10.2. Determination of the minimum inhibitory concentration (MIC)
The minimum inhibitory concentrations (MIC) were determined for all strains
which showed significant zones of inhibition (more than 15 mm). The MIC was deter-
mined in LB broth using a macrobroth dilution method as described by the Clinical
and Laboratory Standards Institute (CLSI) [26]. The final concentration of bacteria in
6
each macrobroth dilution tube was approximately 6.8 × 10 CFU/mL of LB. The MIC
Fig. 1. Effect of fatty acid chain length on dopamine conversion yield estimated
was defined as the lowest concentration of the phenolic compound that resulted in
◦
by HPLC analysis. The biosynthesis reaction of dopamine with different fatty acids
no visible growth after 24 h of incubation at 37 C.
◦
(
equimolar ratio) was catalyzed by Novozyme 435 lipase at 50 C and 200 rpm in
2
-methylbutan-2-ol (168 h). Results are expressed as mean ± SD. Symbols DA; DA-
2.11. Hemolytic activity
C3–DA-C18:1 refers to DA derivatives as specified in Table 1.
Hemolytic activity of DA and its derivatives on human erythrocytes was mea-
sured by a modified method according to Malagoli [27]. Freshly collected human
blood samples were immediately mixed with anticoagulant, Alsever’s solution (pH
observation may be attributed to the structure of the acyl binding
site of C. antarctica lipase which can better accommodate medium
chain fatty acids [29]. These results are also in line with findings
of Aissa et al. [5] and Viskupicova et al. [23]. Likewise, Pedersen
et al. [29] and Vaysse et al. [30] reported that the conversion yield
increases with decreasing chain length of the acyl donor. These
results are, however in contradiction with those described by Ard-
haoui et al. [20] who reported no significant effect of the carbon
chain length on the conversion yield of rutin and esculin alkyl
esters when using the same lipase. These discrepancies might be
attributed to the change of the lipase selectivity depending on the
composition of the reaction medium (solvent, water content, sub-
strate chemistry) as reported previously [31–34].
7
.4) to prevent blood coagulation. To obtain a pure suspension of erythrocytes, blood
sample was washed three times in 9 volumes of sterile 0.9% NaCl saline solution.
◦
After each washing, cells were pelleted by centrifugation at 72 × g for 15 min at 4 C.
The supernatant was then removed by gentle aspiration. Erythrocytes were finally
resuspended in PBS to make 1% solution for hemolytic assay. Various ethanolic con-
centrations of pure DA derivatives (1–1000 ␮g/mL) were added to the suspension
◦
of red blood cells. The DA esters mixed with erythrocytes were incubated at 37
C
◦
for 30 min in water bath and then centrifuged at 72 g for 5 min at 4 C. Supernatants
were transferred to 96-well microplates and the absorbance at 405 nm was deter-
mined by using a spectrophotometric microplate reader (BioTek Instruments) to
measure the extent of red blood cell lysis. Positive control (100% hemolysis) and
negative control (0% hemolysis) were also run by incubating erythrocytes with 1%
Triton X-100 in PBS and 2.5% ethanol in PBS, respectively.
HC50, the concentration where 50% of erythrocytes are lysed, was determined for
each surfactant. The hemolysis percentage was calculated by the equation: Hemoly-
ꢀ
ꢀ
sis (%) = ((Abs − Abs0)/(Abs100 − Abs0)) × 100 where ‘Abs’, ‘Abs0 , and ‘Abs100 were
sample absorbance, control with PBS and control in the presence of hemolytic dose
of triton X-100, respectively.
3.2. Purification and identification of dopamine fatty acid esters
Biocatalyst and molecular sieves were discarded as a pellet
3
. Results and discussion
after centrifugation. The solvent was removed from the super-
natant upon rotary evaporation. The reaction mixture was then
subjected to liquid extraction using a water/hexane mixture (2/3,
v/v), whereby residual DA was discarded in the water phase. This
selective extraction phenomenon was presumed to be related to
a lower water solubility of the DA esters, as well as subsequent
differences in partitioning behavior of the compounds between
the extraction phases. The organic phase containing DA esters and
residual fatty acids was then subjected to a second liquid extrac-
tion using acetonitrile. DA esters were successfully extracted into
the acetonitrile phase. HPLC and LC/MS analysis confirmed that
the second step of this extraction process was sufficiently selec-
tive to separate DA esters from residual substrates without the
need of further chromatographic separation (Fig. 2A and B). The
same procedure was also used by Ardhaoui et al. [20] and Lue
et al. [35] to recover esculin and rutin fatty acid esters. Many
of the esters could be identified tentatively by expected elution
order in UV spectra from the individual reactions, but there are
several overlapping species that required MS analysis for unequiv-
ocal identification. The purified reaction product was analyzed by
HPLC (Fig. 2A) and identified by LC/MS. Fig. 2B shows the spec-
troscopic analysis of DA-C16 as a typical example of dopamine
derivatives. LC/MS analysis in negative mode of DA-C16 exhibited
3.1. Enzymatic synthesis of dopamine fatty acid esters
In order to synthesize DA esters (DA-C3 to DA-C18:1), sev-
eral solvents such as hexane, chloroform, acetone and toluene
were checked as reaction media. However, DA was only sol-
uble in relatively polar solvents like tertiary alcohols such as
2
-methylbutan-2-ol which was used as a reaction medium. This
reaction medium effectively solubilized all reactants as well as final
products. Lipase from C. antarctica has been used as a catalyst for the
esterification reactions. This lipase was known for its high tolerance
toward polar solvents [28].
Molecular sieves are usually made of synthetic zeolites. They
were employed for water removal in order to drive the reac-
tion toward biosynthesis. A molecular sieves concentration of
1
00 mg/mL was used. Such a concentration was described to
enhance synthesis reactions [20].
The effect of fatty acid chain length (C –C18) on DA esters syn-
3
thesis yield was investigated (Fig. 1). The highest conversion yield
(
(
52.75%) was reached for the medium chain length caprylic acid
C8). With increasing the fatty acid chain length (from C12 to C18),
the synthesis yields of esters gradually decreased (Fig. 1). This

1
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M. Sellami et al. / Process Biochemistry 48 (2013) 1481–1487
Fig. 2. (A) HPLC profile of dopamine palmitate (DA-C16) following biosynthesis and purification. The biosynthesis reaction of dopamine with palmitic acid (equimolar ratio)
◦
was catalyzed by Novozyme 435 lipase at 50 C and 200 rpm in 2-methylbutan-2-ol (168 h). HPLC analysis was carried out on a C-18 column by gradient elution using acidified
water and acetonitrile at a flow rate of 1 mL/min. Symbol DA-C16 refers to dopamine palmitate as specified in Table 1 and (B) LC/MS spectra of purified dopamine palmitate
(
DA-C16).
a molecular ion at m/z = 390.40 [M−H]-attributed to the molecular
weight of a monoester (Fig. 2B). The peaks corresponding to ions at
m/z = 436.37 [M + 46]- and m/z = 781.82 [2M]- are attributed respec-
tively to ester linked to a formic acid molecule and a duplicate mass
of the native ester.
the total amount of an antioxidant standard consumed (Trolox),
while the DPPH method measures the speed of radical consump-
tion. Table 1 shows the scavenging activity of DA and its derivatives
on DPPH and ABTS radicals. Results were expressed by means
of the IC50 (␮g/mL) and mM Trolox equivalent (mM TEAC) for
DPPH and ABTS methods, respectively. A higher DPPH and ABTS
radical-scavenging activity were associated with lower IC50 val-
ues and higher TEAC ones, respectively. DA and their fatty acid
esters were shown to be strong DPPH and ABTS radical scavengers.
Esterification of DA seems not to significantly affect its DPPH
radical-scavenging activity. DA and its esters have two hydroxyl
groups in their structures, which have been recognized to func-
tion as an electron or hydrogen donors to quench electron mobility
with interruption of the free radical chain reaction [38]. DA and its
derivatives were three to four times more efficient antioxidants
than the synthetic antioxidant (BHT), most commonly applied
in food industries. Dopamine oleate (DA-C18:1) exhibited a high
antioxidant capacity as compared to the parent dopamine and
to the other esters when tested by the ABTS assay. Our find-
ings are consistent with those of Mateos et al. [39] who have
synthesized a large series of hydroxytyrosyl esters and evaluated
their antioxidant capacity via ABTS assay. Authors showed that
hydroxytyrosyl esters were more effective than that of the par-
ent hydroxytyrosol especially hydroxytyrosyl oleate and the order
of the scavenging activities toward the ABTS radical was hydrox-
ytyrosyl esters > alphatocopherol > hydroxytyrosol > BHT. Likewise,
The liquid extraction procedure followed above was not suitable
for the purification of DA-C . In fact, DA, propionic acid and its ester
3
(
DA-C ) were all soluble in the water phase. For this reason, we have
3
achieved the purification of DA-C3 by chromatography on a silica
gel 60 column according to Aissa et al. [5] and the final purity of the
product was checked with HPLC analysis (Fig. 2A).
3.3. Antioxidant activity
There is evidence that acylation of phenolic compounds pro-
vides a useful tool for the improvement of the antioxidant capacity
5,36,37]. For an efficient application of acylated DA into lipophilic
[
formulations, it was necessary not only to increase their hydropho-
bicity but also to maintain the antioxidant capacity. The potential
effect of selective DA modification on antioxidant efficiency of
lipophilized DA derivatives was checked using DPPH and ABTS
scavenging tests. DPPH and ABTS stable free radical methods are
rapid and sensitive widely used tests for the determination of
antioxidant activity of a specific compound [21,22]. These two
methods were applicable to the study of both water-soluble and
lipid-soluble antioxidants. The determination of ABTS measures

M. Sellami et al. / Process Biochemistry 48 (2013) 1481–1487
1485
Table 1
Physico-chemical parameters of dopamine and its esters related to lipophilicity and its antioxidant activities determined by DPPH (IC 50) and ABTS (TEAC) method as well
as hemolytic activities estimated by the HC50 values (concentration for lyses of 50% of cells).
Compound
Abbreviation
MW (g/mol)a
miLog Pa
Rt (min)b
IC 50 (␮g/mL)c
TEAC (mM)d
HC50 (␮g/mL)e
Dopamine
DA
153.18
209.24
279.38
335.48
363.54
391.59
419.65
417.63
–
−0.05
1.13
3.71
5.73
6.74
7.75
8.56
8.24
–
2.54
5.26
9.00
11.74
13.04
14.37
16.42
14.47
–
1.32 ± 0.20
1.46 ± 0.24
1.49 ± 0.19
1.48 ± 0.30
1.60 ± 0.27
1.57 ± 0.14
1.62 ± 0.18
1.68 ± 0.24
5.40 ± 0.86
0.64 ± 0.01
0.67 ± 0.04
0.83 ± 0.06
0.82 ± 0.08
0.82 ± 0.08
0.85 ± 0.00
0.82 ± 0.08
1.01 ± 0.06
0.27 ± 0.10
>1000
>1000
80.00
32.07
300.23
930.34
>1000
2.89
Dopamine propionate
Dopamine caprylate
Dopamine laurate
Dopamine myristate
Dopamine palmitate
Dopamine stearate
Dopamine oleate
DA-C3
DA-C8
DA-C12
DA-C14
DA-C16
DA-C18
DA-C18:1
BHT
Butylated hydroxytoluene
-
a
miLog P and MW values calculated using Molinspiration program.
Retention time from HPLC.
Antioxidant activity of dopamine and their fatty acid esters determined by DPPH method.
Antioxidant activity of dopamine and their fatty acid esters determined by ABTS method.
Hemolytic assay determined on human erythrocyte.
b
c
d
e
Bernini et al. [40] have synthesized a large panel of catechol esters
who showed that only medium chain tyrosyl esters had an antibac-
terial activity against staphylococcus strains. The authors suggested
that this fact might be due to the surfactant effectiveness of these
derivatives.
(
structural analogs of hydroxytyrosol) via chemical esterification.
The antioxidant capacity of lipophilic catechol esters were eval-
uated by ABTS assay. All tested esters showed good antioxidant
capacity, comparable to or higher than that of the parent cat-
echol. Furthermore, esters of unsaturated fatty acids display a
better radical-scavenging activity than the corresponding satu-
rated esters. The higher antioxidant capacity measured for oleic
and linoleic esters can derive from lowering of the acyl chain con-
formational freedom as a consequence of the double-bond rigidity.
To determine the effect of lipophilic DA derivatives on the oxida-
tive stability of oils, measurements of conjugated diene (CD) at
Minimum inhibitory concentrations (MIC) of the most effec-
tive antibacterial compounds (DA-C and DA-C12) were determined
8
(Table 3). DA did not show any inhibitory effect on the tested bacte-
ria, up to a concentration of 3 mg/mL. The DA-C8 and DA-C12 were
found to be equally effective against Staphylococcus strain with a
MIC value equal to 15 ␮g/mL. Moreover, DA-C12 displayed a high
antibacterial activity on B. flavum and P. aeruginosa. K. pneumonia
2
32 nm and triene (CT) at 270 nm were carried out. DA and its
esters were added to refined soy oil which CD and CT indexes were
◦
recorded upon 15 days storage at 70 C (Fig. 3A and B). The CD mea-
sured at 232 nm, determines the primary products of oil oxidation
whereas CT measured at 270 nm, evaluates the secondary oxidation
products such as aldehydes and ketones which are final products of
oil peroxidation. A significant increase was observed in CD and CT
of refined soya oil. The addition of DA derivatives as antioxidants
decreased the oil oxidation and exhibited prominent antioxidant
activity. Results indicated clearly that the DA and its derivatives
showed a high antioxidant activity. Interestingly, the protective
effect against oxidation by DA derivatives was higher than that of
DA or BHT and increased with the fatty acid chain length. DA and
DA-C3 derivative displayed the lowest protective effect of soya oil
against oxidation. Based on the increasing trend of miLog P values
(Table 1), it seems that DA esters having a high lipophilicity (miLog P
higher than 3.7) display the best antioxidant capacities.
3.4. Antibacterial activity
The well diffusion method was performed in order to test the
antibacterial activity of DA and its fatty acid esters (Table 2). DA
and its derivatives were active against both Gram (+) and Gram (−)
bacteria. DA was active solely against Klebsielle pneumoniae species
and no activity was observed against the other strains. Interest-
ingly, medium chain DA derivatives (DA-C , DA-C12) exhibited high
8
antibacterial activities against both Gram (+) and Gram (−) strains.
DA-C derivative showed the highest inhibitory activity against B.
12
flavum, K. pneumoniae and P. aeruginosa and a moderate antibacte-
rial effect against S. aureus, S. xylosus, B. cereus and S. epidermidis.
Meanwhile, DA-C8 showed a high antibacterial activity against K.
pneumoniae then P. aeruginosa, S. xylosus and S. aureus. Among the
long chain DA derivatives, only DA-C14 displayed a low antimi-
crobial effect against K. pneumonia, P. aeruginosa, S. xylosus and
S. aureus strains since the diameter of inhibition was lower than
Fig. 3. Antioxidant activities of dopamine (closed square) and its derivatives (DA-
C3, solid triangle; DA-C8, open triangle; DA-C12, solid circle; DA-C14, open circle;
DA-C16, open square; DA-C18, open diamond; DA-C18:1, dashed line) tested by the
measurement of conjugated dienes (A) and conjugated trienes (B) of refined soya oil
◦
during storage at 70 C. For each test, negative (solid diamond) and positive controls
1
5 mm. These findings are in agreement with those of Aissa et al. [5]
using BHT (crosses) were run in parallel.

1
486
M. Sellami et al. / Process Biochemistry 48 (2013) 1481–1487
Table 2
Inhibitory spectrum of dopamine and its derivatives on several Gram-positive and Gram-negative bacteria. Symbols DA; DA-C3–DA-C18:1 refers to DA derivatives as specified
in Table 1.
Strain
Gram
Sensibility
DA
DA-C3
DA-C8
DA-C12
DA-C14
DA-C16
DA-C18
DA-C18:1
Ampicillin
Staphylococcus aureus
Staphylococcus xylosus
Brevibacterium flavum
Bacillus cereus
Staphylococcus epidermidis
Bacillus subtilis
Micrococcus luteus
Enterococcus faecalis
Pseudomonas aeruginosa
Klebsielle pneumoniae
Enterobacter cloacae
Escherchia coli
+
+
+
+
+
+
+
+
−
−
−
−
−
a
a
a
a
a
a
a
a
a
b
a
a
a
a
a
a
a
a
a
a
a
a
b
a
a
a
c
c
b
b
a
a
a
a
c
d
a
a
a
c
c
b
b
a
a
a
a
a
a
b
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
c
c
c
d
b
b
a
a
a
d
d
a
a
a
d
d
d
c
d
c
d
c
d
d
Salmonella
The bactericidal level was estimated by measuring the size of inhibition zone of the indicator strain.
a
Insensitivity.
Low sensitivity (diameter of inhibition < 15 mm).
High sensitivity (diameter of inhibition between 15 and 20 mm).
Very high sensitivity (diameter of inhibition > 20 mm), ampicillin (10 ␮g/well) was used as positive control.
b
c
d
Table 3
◦
Minimum inhibitory concentrations (MIC) of dopamine esters (DA-C8 and DA-C12) for five microbial strains after 24 h of incubation at 37 C.
Compounds
MIC (␮g/mL)
S. aureus
S. xylosus
B. flavum
P. aeruginosa
K. pneumoniae
DA-C8
DA-C12
15
15
15
15
30
7.5
15
7.5
3.75
3.75
was found to be the most sensitive strain to DA-C8 and DA-C12
esters with a MIC value equal to 3.75 ␮g/mL. Differences in MIC
values of bacteria may be related to the differential susceptibility
of bacterial cell wall, which is the functional barrier to minor differ-
ences present in the outer membrane in the cell wall composition
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This work represents a part of thesis of Mr Mohamed Sellami. It
received financial support from the Ministry of Higher Education,
Scientific Research in Tunisia. The authors would like to thank Pr.
Sofiane Bezzine (ENIS, Sfax-Tunisia) for generous gift of bacterial
strains. We are grateful to Dr. Christine Fahy (ITODYS, Paris-France)
for technical assistance. We thank Dr. Mohamed Rholam (ITODYS,
Paris-France) for his fruitful discussion and help during the prepa-
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