Substituted imino and amino derivatives of 4-hydroxycoumarins as novel antioxidant, antibacterial and antifungal agents: Synthesis and in vitro assessments

Article abstract of DOI:10.1016/j.foodchem.2009.11.040

A series of imino and amino derivatives of 4-hydroxycoumarins were synthesised and evaluated for antioxidant potential, through different in vitro models such as (DPPH) free radical-scavenging activity, linoleic acid emulsion model system, reducing power

Full text of DOI:10.1016/j.foodchem.2009.11.040

Food Chemistry 120 (2010) 1011–1018
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Substituted imino and amino derivatives of 4-hydroxycoumarins as novel
antioxidant, antibacterial and antifungal agents: Synthesis and in vitro assessments
*
Nenad Vukovic , Slobodan Sukdolak, Slavica Solujic, Neda Niciforovic
University of Kragujevac, Faculty of Science, Department of Chemistry, Laboratory for Biochemistry, P.O. Box 60, 34000 Kragujevac, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of imino and amino derivatives of 4-hydroxycoumarins were synthesised and evaluated for anti-
oxidant potential, through different in vitro models such as (DPPH) free radical-scavenging activity, lin-
oleic acid emulsion model system, reducing power assay and phosphomolybdenum method. Also,
antimicrobial activity of obtained coumarins was evaluated against 13 bacteria and eight fungi. All pre-
pared compounds possessed good antioxidant activity and among them a p-nitrophenol derivative with
Received 16 June 2009
Received in revised form 26 September
2009
Accepted 23 November 2009
IC50 at 25.9 lM possessed radical-scavenging activity which was comparable to BHT. Observed data for
antibacterial activity indicated strong activity of all tested amino derivatives, while imines showed better
antifungal properties.
Keywords:
4-Hydroxy coumarin
Imines
Ó 2009 Elsevier Ltd. All rights reserved.
Amines
Antioxidant activity
Antimicrobial activity
1. Introduction
breaks and are considered to be the first step in several human
degenerative diseases, cancer and ageing (Festa et al., 2001).
In vivo molecular oxygen is easily converted to reactive free rad-
icals such as superoxide anions (Oꢀ2 Å) and hydroxyl radicals (HOÅ),
which are highly reactive substances that react with lipids, pro-
teins and DNA, provoking irreversible changes of their biomolecu-
lar structure (Halliwell, 1990). Reactive oxygen species (ROS) are
continuously generated in very low amounts by the transfer of
one electron to an oxygen molecule during various physiological
processes, such as respiration chain, oxygenase and cellular immu-
nization reactions (Dröge, 2002; Filomeni, Rotilio, & Ciriolo, 2006).
They play an essential role in the control of cell functions. They are
intermediate metabolites in several enzymatic reactions, involved
in post-translational protein turnover and play a role in the control
of signal transduction. Many components of the vascular system,
such as leucocytes, monocytes and endothelial cells are able to re-
lease ROS upon appropriate stimulation. Thus, ROS are associated
with the incidence of various diseases, such as heart diseases,
thrombosis, hypertension, Alzheimer’s disease, Parkinson’s disease
and amyotrophic lateral sclerosis (Juliano, Colavita, Leo, Pratico, &
Violi, 1997; Lassegue & Griendling, 2004; McIntosh, Trush, &
Troncoso, 1997). Besides oxidative stress, reactive oxygen species
are associated with the induction of DNA single- and double-strand
Tissues with high oxygen consumption rate and the central ner-
vous system (CNS), in particular, are more easily susceptible to oxi-
dative damage under conditions of oxidative stress, due to the
presence of excitatory amino acids, such as glutamate, elevated
iron stores, cell membranes rich in polyunsaturated fatty acids
and low levels of the natural antioxidant glutathione in neurons
(Barnham, Masters, & Bush, 2004). Furthermore, the blood–brain
barrier reduces the permeability and the protective efficacy of
most antioxidants (Gilgun-Sherki, Melamed, & Offen, 2001; Gil-
gun-Sherki, Rosenbaum, Meland, & Offen, 2002).
The emergence of multi-drug resistant microorganisms has
made the treatment of infectious diseases difficult and has, over
the last decades, become a serious medical problem. As bacteria
and fungi continuously evolve mechanisms of resistance to cur-
rently used drugs, the discovery of novel and potent antimicrobial
agents is the best way to overcome bacterial and fungal resistance
and develop effective therapies.
The imino group (–C@N–), containing compounds typically
known as Schiff bases, form a significant class of compounds in
medicinal and pharmaceutical chemistry with several biological
applications that include antibacterial (Pannerselvam, Nair, Vijaya-
lakshmi, Subramanian, & Sridhar, 2005; Sithambaram Karthikeyan,
Jagadesh Prasad, Poojary, & Subramanya Bhat, 2006), antifungal
(Pandeya, Sriram, Nath, & Declercq, 1999) and antitumour activity
(Mladenova, Ignatova, Manolova, Petrova, & Rashkov, 2002).
* Corresponding author. Tel.: +381 34336223; fax: +381 34335085.
E-mail address: nvukovic@kg.ac.rs (N. Vukovic).
0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.11.040

1012
N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
From the fundamental point of view, reduction of imines to the
Sukdolak, Solujic, & Milosevic, 2008), we present here the prepara-
tion of some new imino and amino derivatives of 4-hydrox-
ycoumarins and the investigation of their antibacterial,
antifungal and antioxidative properties.
corresponding amines represents one of the most widely used and
valuable functional group transformations in synthetic organic
chemistry, since amines constitute important precursors to
compounds that are of much interest in the pharmaceutical and
agricultural industries (Moglie, Alonso, Vitale, Yusb, & Radivoy,
2006).
2. Materials and methods
Coumarin (1,2-benzopyrone) derivatives constitute one of the
most common families of green plant secondary metabolites, sev-
eral of them being reported to display multiple biological proper-
ties (Egan et al., 1990; Jurd, Corse, King, Bayne, & Mihara, 1971).
Many products which contain a coumarin subunit exhibit biologi-
cal activities, such as molluscicidal, anthelmintic, hypnotic, and
insecticidal activities (Schonberg & Latif, 1954). Also, the medicinal
properties of coumarins, include inhibition of platelet aggregation,
2.1. Chemicals
All applied chemicals and reagents were of the highest purity
available and purchased from the Sigma–Aldrich Chemical Com-
pany (St. Louis, MO), Difco and Merck Laboratory Supplies (Darms-
tadt, Germany).
2.2. Instrumentation and apparatus
cytochrome P450 and steroid 5a-reductase (Hoult & Paya, 1996;
Kostova, 2005). However, the best-known compounds in this series
are some 4-hydroxycoumarins, such as the drugs warfarin and ace-
nocoumarol, which have been widely used in anticoagulation ther-
apy for over 20 years (Hirsh et al., 2001). A number of coumarins
were found to affect the formation and scavenging of ROS, exhibit-
ing tissue-protective antioxidant properties, which may include
numerous different molecular mechanisms and are probably re-
lated to their structural analogy with flavonoids and benzophenon-
es (Beillerot, Rodríguez Domínguez, Kirsch, & Bagrel, 2008). Indeed,
this structure type can bind Fe(III) and thus inhibit hydroxyl radical
and hydrogen peroxide formation produced by Fenton’s reactions.
The hydroxyl groups of some hydroxycoumarins are potent HÅ do-
nors for free radical acceptors, due to electron delocalization across
the molecule (Sharma, Rajor, Chopra, & Sharma, 2005). Also, some
simple hydroxylated coumarin derivatives have been reported to
inhibit xanthine oxidase (Ferrari, Sgobba, Gamberini, & Rastelli,
2007).
The microwave-assisted reactions were carried out in
a
MICROSYNTH Microwave Synthesis System manufactured by Mile-
stone Inc. (Shelton, CT). Products were identified by determination
of melting points (Kofler-hot stage apparatus), using elemental
analysis (Carlo Erba 1106 microanalyser), IR (Perkin-Elmer Grating
Spectrophotometers Model 137 and Model 337, KBr disc,
m in
cmꢀ1), 1H-NMR (Varian, Palo Alto, CA; Gemini 200 spectrometer,
CDCl3, d in ppm) and GC/MS (Agilent, Santa Clara, CA) techniques
(HP 5MS capillary column, 30 m ꢁ 0.25 mm i.d., film thickness
0.25 lm; injector and transfer line temperatures of 250 and
280 °C, respectively; oven temperature programmed at 150 °C with
an increase of 10 °C/min to 300 °C (isothermal for 10 min); carrier
gas, helium; MS quadruple temperature 150 °C; mass scan range,
35–500 amu at 70 eV). Analytical TLC was performed on Merck Sil-
ica gel 60 F254 TLC plates (layer thickness, 0.25 mm), and moni-
tored by UV light (254 and 365 nm) or iodine vapours. Column
chromatography was carried out using Merck 7734 (60 200
mesh) silica gel and monitored by TLC.
Prompted by the above-mentioned biological properties of
coumarin derivatives and in continuation of our previous work
´
´
´
´
(Sukdolak, Vukovic, Solujic, Manojlovic, & Krstic, 2004; Vukovic,
2.3. Synthesis of imino and amino derivatives of 4-hydroxycoumarins
2.3.1. Condensation of 3-acetyl-4-hydroxy-chromene-2-one 1 with
amines 2a–9a
Table 1
Substituents and yields of obtained compounds 2b–8b.
2.3.1.1. Method A – conventional method. A mixture of 3-acetyl-4-
hydroxy-chromene-2-one 1 (0.01 mol), amine 2a–9a (0.01 mol)
and catalytic amount of p-toluenesulphonic acid (10ꢀ5 mol) in
anhydrous toluene (50 ml) was heated with azeotropic removal
of water over a period of 10–12 h. Progress of reaction was moni-
tored by TLC (toluene:acetone = 7:3). At the end of reaction, the
solvent was evaporated to one quarter of its volume; then the ob-
tained solid products were filtered, dried, purified via column chro-
matography (benzene:acetone = 8:2) to give compounds 2b–9b.
No.
R
Conventional
method A
Microwave assisted
method B
Time of
synthesisa
(h)
Yieldb
(%)
Irradiation
time
Yieldb
(%)
(min)
2b
3b
4b
5b
6b
7b
8b
9b
Ph
p-tolyl
m-tolyl
o-tolyl
p-NO2-phenyl
m-NO2-phenyl
Benzyl
9
9.5
9
9.5
13.5
12.5
10
75
73
84
73
51
62
75
42
3
3
3
3
3
3
3
3
95
97
94
94
92
97
97
87
2.3.1.2. Method B – microwave method. Catalytic amount of p-tolu-
enesulfonic acid (10ꢀ5 mol) was added to 50 ml toluene solution of
equimolar amounts (0.01 mol) of 3-acetyl-4-hydroxy-chromene-2-
one 1 and amines 2a–9a. The mixture was heated under micro-
wave for 3 min. After cooling, the solvent was evaporated to one
x
-C4H8COOH
10
a
Reflux in heating equipments.
Percentage of yield calculated from practical and theoretical yields.
B
Fig. 1. Synthesis of imino and amino derivatives of 4-hydroxy coumarins.

N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
1013
quarter of its volume, then the obtained solid was filtered and
recrystallised from methanol (Table 1, Fig. 1).
chloroacetic acid. The blank sample contained 300
water, 300 l of phosphate buffer, 300
anide and 300 l of 10% trichloroacetic acid. Ascorbic acid and
l
l distilled
l
ll of 1% potassium ferricy-
l
BHT were used as positive controls. The reducing power of samples
was calculated by the following formula:
2.3.2. Reduction of imino derivatives of 4-hydroxy-chromene-2-one
2b–9b to amines 2c–9c
Sodium borohydride (0.129 g, 0.0034 mol) was slowly added in
several portions to a solution of 0.0034 mol of the imine 2b–9b in
50 ml MeOH/THF (8:2). The reaction mixture was stirred at room
temperature for 4 h and monitored periodically by TLC. Upon com-
pletion, the solvent was evaporated and the residue was treated
with hydrochloric acid (10 w/v). Then the reaction mixture was
made basic with a saturated aqueous NaHCO3 solution and the or-
ganic products were extracted with dichloromethane (3 ꢁ 50 ml).
The organic layer was dried over anhydrous sodium sulphate and
was filtered. After evaporation of the solvent, the obtained residue
was purified via column chromatography (benzene:acetone = 8:2)
to give amines 2c–9c.
RPð%Þ ¼ ðAB ꢀ AAÞ ꢁ 100;
where: RP is reducing power; AB is absorbance of control sample
(100%); AA is the absorbance of the tested sample. Percentage of
reducing power RP (%) was plotted against concentration, and the
equation for the line was used to obtain the RP50 value. All determi-
nations were carried out in triplicate. A lower RP50 value indicates
greater reducing power ability.
2.4.3. Inhibition (%) of lipid peroxidation in a linoleic acid emulsion
The total antioxidant activity of synthesised compounds was
carried out by use of a linoleic acid system (Masude, Isibe, Jitoe,
& Naramati, 1992). The linoleic acid emulsion was prepared by
mixing 0.2804 g of linoleic acid, 0.2804 g of Tween 20 as emulsifier
and 50 ml of phosphate buffer (0.2 M, pH 7.0), and then the mix-
ture was homogenised. A 0.5-ml ethanol solution of different con-
2.4. Measurement of antioxidant activity
2.4.1. DPPH assay
centrations of imines and amines (1000, 500, 250 and 125 lM) was
The method used by Takao, Watanabe, Yagi, and Sakata, (1994),
was adopted with suitable modifications. DPPH (8 mg) was dis-
solved in MeOH (100 ml) to obtain a concentration of 80 lg/ml. Se-
mixed with a linoleic acid emulsion (2.5 ml, 0.02 M, pH 7.0) and
phosphate buffer (2 ml, 0.2 M, pH 7.0). The reaction mixture was
incubated at 37 °C in the dark to accelerate the peroxidation pro-
cess. Aliquots of 100 ll were taken at different intervals during
incubation. The degree of oxidation was measured by sequentially
adding ethanol (4.7 ml, 75%), an ammonium thiocyanate sample
solution (100 ll, 30%) and ferrous chloride (100 ll, 0.02 M in
3.5% HCl). After 3 min, the absorbance at 500 nm was read. Ascor-
bic acid and BHT were used as reference compounds. A control was
performed with linoleic acid but without the tested compounds.
All data reported are the average of triplicate analyses. Percentage
inhibition of lipid peroxide generation was calculated using the
formula:
rial dilutions were carried out with stock solutions (4 mM) of the
compounds (2b–9b; 2c–9c) in methanol to obtain concentrations
of 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, 0.0078 and
0.0039 mM. Diluted solutions (2 ml each) were mixed with DPPH
(2 ml) and allowed to stand for 30 and 60 min for any reaction to
occur. The absorbance was recorded at 517 nm using a Perkin–El-
mer Lambda 25 UV/Vis spectrophotometer. The experiment was
performed in triplicate and the average absorbance was noted for
each concentration. The IC50 value, which is the concentration of
the test compound that reduces 50% of the initial free radical con-
centration, was calculated in lM. Ascorbic acid and BHT were used
as reference standards, at the same concentrations in methanol as
were used for the tested compounds. Control sample was prepared
containing the same volume without test compounds and refer-
ence compounds. The radical-scavenging activity of the tested
samples, expressed as percentage inhibition of DPPH, was calcu-
lated according to the formula:
%Inhibition ¼ ½ðA0 ꢀ AtÞ=A0ꢂ ꢁ 100;
where At is the absorbance value of the tested sample and A0 is the
absorbance value of the control sample.
2.4.4. Determination of total antioxidant capacity
The antioxidant activity of the tested compounds was evaluated
by the phosphomolybdenum method according to the procedure of
Prieto, Pineda, and Aguilar, (1999), with some modification. The as-
say is based on the reduction of Mo(VI) to Mo(V) by the test com-
pounds and subsequent formation of a green phosphate/Mo(V)
complex at acid pH. One hundred microlitres of the solution of
ICð%Þ ¼ ½ðA0 ꢀ AtÞ n A0ꢂ ꢁ 100;
where At is the absorbance value of the tested sample and A0 is the
absorbance value of blank sample, at a particular time. Percentage
inhibition after 30 and 60 min was plotted against concentration,
and the equation for the line was used to obtain the IC50 value. A
lower IC50 value indicates greater antioxidant activity.
tested compounds (15.6–250 lg/ml) was combined with 1 ml of
reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate
and 4 mM ammonium molybdate). The tubes containing the reac-
tion solution were incubated at 95 °C for 90 min. Then the absor-
bance of the solution was measured at 695 nm against a blank
2.4.2. Reducing power assay
The reducing power test is based on reduction of ferric to fer-
rous by the potent antioxidant. In the presence of cyanide ions,
and adding a new amount of Fe3+, blue colour of Fe4[Fe(CN)6]3
develops. The reducing activity of prepared compounds was mea-
sured according to the method described by Oyaizu (1986). Sample
after cooling to room temperature. Ethanol (100 ll) in the place
of the solution of the tested compounds was used as the blank.
The antioxidant activity is expressed as the number of equivalents
of ascorbic acid. Determinations of total antioxidant capacity were
carried out in triplicate.
of 300
solutions) was mixed with 300
6.6) and 300 l of 1% potassium ferricyanide. The mixtures were
incubated at 50 °C for 20 min. After incubation 300 l of 10% tri-
ll of various dilutions (from 2 to 0.0039 mM, methanol
l
l of phosphate buffer (0.2 M, pH
l
l
2.5. Determination of antibacterial and antifungal activity
chloroacetic acid were added to the mixture, which was then cen-
trifuged at 3000 rpm for 10 min. The upper layer (0.6 ml) of
2.5.1. Culture of microorganisms
solution was mixed with 0.6 ml of distilled water and 120
0.1% FeCl3 and the absorbance was measured at 700 nm. The con-
trol sample contained 300 l distilled water, 300 l of phosphate
buffer, 300 l of 1% potassium ferrocyanide and 300 l of 10% tri-
l
l of
Test bacteria which were used in this experiment are: (a) Gram-
positive bacteria: Bacillus mycoides (FSB 1), Bacillus subtilis (FSB 2),
Micrococcus lysodeikticus (ATCC 4698), Staphylococcus aureus (ATCC
25923) and Staphylococcus aureus (FSB 30); (b) Gram-negative
l
l
l
l

1014
N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
bacteria: Azotobacter chroococcum (FSB 14), Enterobacter cloaceae
(FSB 22), Erwinia carotovora (FSB 31), Escherichia coli (ATCC
25922), Klebsiella pneumoniae (FSB 26), Pseudomonas fluorescens
(FSB 28), Pseudomonas glycinea (FSB 40) and Pseudomonas phaseoli-
cola (FSB 29). All tested bacteria are from the Faculty of Agriculture,
Beograd, University of Beograd, Serbia. The Laboratory for Microbi-
ology, Department of Biology, Faculty of Science, Kragujevac, Uni-
versity of Kragujevac, Serbia confirmed identification of all tested
bacteria.
values were determined visually after 48 h of incubation, as the
lowest concentration of drug that caused no detectable growth.
The average of 3 values was calculated to give the MIC for the
tested compounds and standard drug.
3. Results and discussion
3.1. Synthesis of imino and amino derivatives 4-hydroxycoumarins
Fungi which were used in this experiment are: Aspergillus glau-
cus (FSB 32), Aspergillus niger (FSB 31), Candida albicans (ATCC
10259), Fusarium oxysporum (FSB 91), Penicillium verrucosum (FSB
21), Trichoderma longibrachiatum (FSB 13), Trichoderma harzianum
(FSB 12) and Trichoderma viride (FSB 11). All tested fungi are from
the Laboratory for Micology, Department of Biology, Faculty of Sci-
ence, University of Kragujevac.
Bacteria were cultured for 24 h at 37 °C in Mueller–Hinton
broth. Suspensions of fungal spores were prepared from two-day
old cultures which were grown on SDA plates at 30 °C. The final
inoculum size was 106 CFU/ml (according to 0.5 McFarland stan-
dard; turbidimetric method was applied) for the antibacterial as-
say and 104 CFU/ml for the antifungal assay.
For the synthesis of imino derivatives of 4-hydroxy-chromene-
2-one (2b–9b), the requisite starting material, 3-acetyl-4-hydroxy-
chromene-2-one (1), was prepared from commercially available
4-hydroxy-chromene-2-one by the previously reported procedure
(Sukdolak et al., 2004). Preparation of imino derivatives 2b–9b
by conventional condensation method with azeotropic removal
of water was characterised with lower yields of desired
compounds (not more than 84%) followed by time-consuming
purifications by column chromatography. Thus, we reported
improvements in the synthesis of these compounds using a micro-
wave-promoted reaction. Homogenous heating under microwave
exposure resulted in increasing yields of desired compounds 9b–
16b (up to 87%), decrease in time of reaction and use of the purifi-
cation procedure only from the point of recrystallisation from
appropriate solvents. As presented in Table 1, the yield has been
significantly increased using reaction under microwave heating
conditions, particularly in the case of imino derivatives with p-
nitrophenyl (92%), m-nitrophenyl (97%) and pentanoic acid substit-
uents (87%). Their further transformation to the corresponding
amines (2c–9c) was performed by reduction with sodium borohy-
dride in a methanol:tetrahydrofuran mixture (8:2). After reduc-
tion, the solvent mixture was removed on a rotary evaporator at
40 °C and the residue was treated with 10% HCl for a period of
15 min. Then a solution of NaHCO3 was added to pH 7 and an
extraction with methylene chloride was performed; the organic
layer was separated, dried under anhydrous sodium sulphate and
removed under reduced pressure to form amines 2c–9c.
The IR spectral data of the compounds 2b–9b confirmed the
presence of coumarine –OH (at 3406–3467), lactone –C@O (at
1697–1712) and –C@N– group (at 1606–1614). Similarly NMR
broadened singlets in the range of 15.8–16.15 ppm also confirmed
the presence of coumarine –OH, while multiplets in the range of
7.3–7.7 ppm indicated the presence of protons from the benzenoid
part of the molecule. Observed singlets at 2.61–2.81 ppm revealed
the presence of methyl protons from imino groups. The imino
derivatives 3b, 4b and 5b showed additional singlets (2.30–
2.42 ppm) from methyl protons attached at the aromatic part,
while compound 8b possesses a singlet at 3.25 ppm from –C@N–
CH2 group. In contrast to other prepared imino derivatives, com-
pound 9b revealed triplets from two methylene protons at 2.46
and 2.70 ppm, a multiplet at 1.82 ppm from two protons at C00-2
and a broadened singlet from COOH group at 11.3 ppm, which
indicated presence of the n-pentanoic acid part of molecule.
In general, the IR spectral data of the amines 2c–9c revealed
bands at 3429–3455 cmꢀ1 (coumarine –OH), 3179–3193 cmꢀ1
(–NH group) and 1664–1681 cmꢀ1 (lactone –C@O). Additional
bands for compound 9c from –COOH group were observed in a re-
gion of 2590–3611 cmꢀ1 (–OH) and at 1710 cmꢀ1 (–C@O). In the
1H-NMR spectral data, all the compounds showed a quartet of
one proton at around 1.33 ppm and a doublet of three protons at
around 3.78 ppm, accounted for by the –CH–CH3 group. Broadened
singlets in a region at 3.87–4.14 ppm were assigned to NH groups,
while singlets at low field (16.55–17.19 ppm) confirmed presence
of C-4–OH protons. Characteristic resonances from methylene
groups at 1.82, 2.46 and 3.60 ppm, were observed for compound
9c, and indicated C4 fragment of molecule.
2.5.2. Assay for in vitro antibacterial activity
The minimal inhibitory concentration (MIC) of the prepared
compounds against tested bacteria was determined based on a mic-
rodilution method in 96 multi-well microtitre plates (Sarker, Nahar,
& Kumarasamy, 2007). The dissolved compounds were first diluted
to the highest concentration to be tested (500 mg/ml); 50
Mueller–Hinton broth was distributed from the 2nd to the 12th
well, a volume of 50 l from each of the compounds initially pre-
pared was pipetted into the 1st test wells of each microtitre line,
and then 50 l of scalar dilution were transferred from the 2nd to
the 12th well. To each well 10 l of resazurin indicator solution
(prepared by dissolving a 270-mg tablet in 40 ml of sterile distilled
water) and 30 l of Mueller–Hinton broth were added. Finally, 10
ll of
l
l
l
l
ll
of bacterial suspension (106 CFU/ml) were added to each well, to
achieve a concentration of 105 CFU/ml. The final concentrations of
the compounds adopted to evaluate antibacterial activity ranged
from 0.244 to 500 mg/ml. Two columns in each plate were used
as controls: one column with a broad-spectrum antibiotic as a po-
sitive control (streptomycin in a serial dilution of 0.244–500 mg/
ml) and one column containing the solvent ethanol as negative con-
trol. Plates were wrapped loosely with cling film to ensure that bac-
teria did not become dehydrated and prepared in triplicate, and
then they were placed in an incubator at 37 °C for 18–24 h. Colour
change was then assessed visually. Any colour change from purple
to pink or colourless was recorded as positive. The lowest concen-
tration at which colour change occurred was taken as the MIC value.
The average of 3 values was calculated to give the MIC for the tested
compounds and standard drug.
2.5.3. Assay for in vitro antifungal activity
Broth microdilution assays were performed in accordance with
the guidelines in CLSI document M27-A2 (National Committee for
Clinical Laboratory Standards, 2002). Stock solutions were pre-
pared in water for ketoconazole and ethanol for the compounds
in the present study. A final dilution was carried out in an RPMI
1640 medium, buffered to pH 7.0 with 0.165 M morpholenepro-
panesulphonic acid buffer. Each microdilution well containing
100
standard drug ketoconazole (0.244–500 mg/ml) was inoculated
with 100 l of the diluted twofold inoculum suspension (the final
volume in each well was 200
l and inoculum size of 103 CFU/
ll of the twofold concentrations of prepared compounds or
l
l
ml). Growth (drug free) and sterility controls were also included.
Microdilution trays were incubated in ambient air at 35 °C. MIC

N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
1015
3.2. Free radical-scavenging activity on 2,2-diphenyl-1-picrylhydrazyl
radical
30 min, DPPH antiradical scavenging activity was also time-
dependent.
This assay is based on the measurements of the scavenging
ability of compounds towards the stable 2,2-diphenyl-1-pic-
rylhydrazyl radical (DPPHÅ).The disappearance of this commer-
cially available radical is measured spectrophotometrically at
517 nm in a methanolic solution. The antioxidant activity was ex-
pressed as the 50% inhibitory concentration (IC50) based on the
amount of compound required for a 50% decrease of the initial
DPPH radical concentration.
As presented in Table 2, the imines 2b–9b with IC50 values in
the range of 304.1–446.9 lM showed lower radical-scavenging
activities in comparison to butylated hydroxytoluene (BHT) and
ascorbic acid. Among them, compounds 5b and 4b with o-tolyl
and m-tolyl substituents at imino nitrogen showed highest hydro-
gen donor ability to DPPH radical (IC50 values were 304.1 and
3.3. Evaluation of reducing power
The reducing power of prepared coumarin derivatives, which
may serve as a significant reflection of the antioxidant activity,
was determined using the iron(III) to iron(II) reduction assay. In
this assay, the yellow colour of the test solution changes to various
shades of green and blue, depending on the reducing power of
compounds. The presence of reductants in the solution causes
the reduction of the Fe3+/ferricyanide complex to the ferrous form.
Therefore, the Fe2+ ion can be monitored by measurement of the
formation of Perl’s Prussian blue at 700 nm. Table 2 showed the
reducing power of imino and amino derivatives of 4-hydroxy-chro-
mene-2-one compared to BHT and ascorbic acid. All tested com-
pounds showed some degree of reducing power; however, as
anticipated, their reducing power was inferior to ascorbic acid,
which is known to be a strong reducing agent. Assayed compounds
were able to reduce the ferric ions to corresponding ferrous ions,
reaching 50% of reduction, with RP50 values ranking from 278.8
334.6
2c–9c indicated more effective scavenging activity (IC50 values
were in the range of 25.9–138 M) related to imines 2b–9b, which
lM, respectively). Observed data for prepared compounds
l
was attributed to presence of hydroxyl and amino groups, as well
as formation of hydrogen bonds between them as the main factor
of increased acidity of protons from both groups or their ability to
be scavenged by DPPH. Compound 6c as p-nitrophenyl derivative
with IC50 at 25.9 lM possesses highest radical-scavenging activity,
which is comparable to BHT. On the other hand, better antiradical
to 350.3 lM for imines 2b–9b and 255.6 to 435.7 lM for amines
2c–9c. In the series of imines, compounds 9b as n-pentanoic acid
derivative and 4b with m-tolyl group attached to imino nitrogen
showed the best reducing power (RP50 values were 278.8 and
279.7
cases of compounds 3b, 5b and 8b (RP50 values were 350.3, 371.2
and 324.4 M, respectively). Comparison of these results with re-
lM, respectively). Low reducing power was gained in the
activity was observed in the case of phenyl derivative 2c and m-
nitrophenyl derivative 7c (IC50 values were 34.1 and 40.9
respectively) in comparison to ascorbic acid (IC50 42.4 M). In a
series of compounds with tolyl fragment, compound 5c as o-
methyl isomer with IC50 72.3 M showed best antiradical activity,
while m-tolyl derivative 4c with IC50 131.8 M showed least activ-
lM,
l
l
sults obtained in DPPH assay clearly indicated an opposite effect
of substituent bonded to imino nitrogen and position of methyl
or nitro group bonded to phenyl ring to reducing power. In the ser-
ies of tolyl and phenyl derivatives the reducing power followed the
following order: ascorbic acid > m-tolyl > phenyl > p-tolyl > o-to-
lyl > BHT. Obviously, presence of a methyl group on the phenyl ring
and type of substitution affected decrease in reducing power. Also,
from the point of substitution, similar results were gained in cases
of nitrophenyl derivatives 6b as para and 7b as meta isomer (RP50
l
l
ity. This indicated the influence of methyl substituent as an elec-
tron donor group at positions C00-2 and C00-6 of phenyl ring to
increase the capability of NH protons to scavenge the DPPH radi-
cals. Almost equal antiradical potency of compounds 8c and 9c
(IC50 were 62.6 and 63.4 lM, respectively) could be attributed to
similar electron donating natures of C6H5CH2 and C4COOH substit-
uents bonded to the NH group. Since the corresponding IC50 values
for all synthesised compounds after 60 min were lower than after
values were 307.3 and 296.0
amines, benzyl amino derivative 8c with RP50 value of 255.6
showed the best reducing power, while phenyl derivative 2c and
lM, respectively). In the group of
l
M
Table 2
Antioxidant activity of synthesised imino and amino derivatives of 4-hydroxy coumarins.
a
Comp.
DPPH assay
IC50 M)
Reducing power assay
bRP50
Inhibition (%) of lipid peroxidation in a linoleic acid emulsionc
(
l
30 min
60 min
(l
M)
1000
lM
500
lM
250
l
M
125 lM
2b
3b
4b
5b
6b
7b
8b
9b
2c
3c
4c
5c
6c
446.9 12.1
352.6 22.2
334.6 21.9
304.1 13.2
371.6 12.7
498.7 13.2
424.9 14.3
394.2 21.8
34.1 13.4
93.9 13.3
131.8 21.6
72.3 13.4
25.9 11.8
40.9 13.9
62.6 14.2
63.4 12.7
25.4 22.7
42.4 22.7
281.5 9.1
266.6 12.2
170.1 8.7
237.8 18.3
152.1 17.4
441.5 7.6
402.8 11.2
377.4 24.6
33.1 11.3
58.8 11.5
56.0 15.3
45.0 19.8
25.0 14.6
37.2 7.7
285.3 2.3
350.3 3.3
279.7 2.1
371.2 4.4
307.3 3.5
296.0 4.3
324.4 3.2
278.8 2.6
435.7 4.7
365.1 2.3
428.1 3.3
293.4 4.3
338.2 4.4
310.5 3.3
255.6 3.3
279.5 2.2
447.1 4.4
142.3 4.5
74.5 1.1
94.4 1.3
88.6 2.1
78.5 1.9
81.3 1.5
76.4 1.6
63.2 1.4
62.6 2.4
57.6 2.2
57.7 2.0
59.4 2.5
58.9 1.6
67.8 1.7
58.3 1.1
60.3 1.1
52.9 1.4
90.5 1.3
27.8 1.4
69.2 1.9
50.5 1.8
77.4 1.6
75.4 1.4
66.5 1.2
73.4 1.1
72.8 1.4
52.9 1.5
51.8 1.7
34.9 1.7
37.6 1.8
31.5 1.5
35.6 1.9
47.5 2.1
37.8 2.2
48.7 2.0
41.7 2.3
90.1 2.1
20.5 2.5
67.3 2.9
38.5 2.1
60.4 2.3
46.2 2.1
35.9 2.9
55.8 3.5
49.2 1.1
39.3 1.4
39.0 1.8
25.6 2.7
29.5 2.4
21.5 2.3
27.6 2.1
29.7 1.5
27.6 1.5
27.2 1.3
22.8 1.2
89.7 1.1
16.0 1.7
63.6 1.8
36.6 2.3
53.9 2.1
29.9 2.4
25.4 2.5
43.6 2.5
31.8 2.7
30.8 3.5
15.8 3.1
17.9 1.5
22.4 2.7
15.9 2.4
15.0 2.6
20.9 1.7
15.6 1.8
18.1 2.0
16.8 1.7
89.6 1.3
8.8 1.5
7c
8c
9c
BHT
Asc
56.9 21.2
58.4 14.5
12.78 7.6
27.22 14.3
a-Toc
60.2 2.1
a
b
c
IC50 values were determined by linear regression analysis. Results are mean values SD from at least three experiments.
RP50 values were determined by linear regression analysis. Results are mean values SD from at least three experiments.
Data of percentage are reported as means SD of three measurements.

1016
N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
Table 3
m-tolyl derivative 4c with RP50 values of 435.7 and 428.1 lM,
The total antioxidant activity expressed as equivalents of ascorbic acid (lg/ml).
respectively, possess low reducing power. Also, RP50 values are
influenced by the substituent bonded to amino group, as well as
the type of substitution of phenyl moiety. The reducing power of
tolyl and phenyl derivatives was decreased in order: o > p > m >
phenyl, which clearly demonstrated influence of position and
electron donor nature of methyl group attached to phenyl ring,
as well as low reducing power of phenyl derivative 2c. Also,
m-nitro compound 6c showed better activity than the correspond-
Comp. 250
l
g/ml
125
lg/ml
62.50
g/ml
31.25
g/ml
15.60
g/ml
l
l
l
Equivalents of ascorbic acid (
l
g/ml)a
2b
3b
4b
5b
6b
7b
8b
9b
2c
3c
4c
5c
6c
7c
8c
9c
12.03 0.25
11.70 0.54
11.83 0.13
11.71 0.24
12.58 0.29
12.33 0.27
12.11 0.57
12.18 0.65
8.64 0.29
9.25 0.32
9.47 0.38
9.37 0.27
8.84 0.41
8.82 0.47
9.18 0.39
9.22 0.36
5.29 0.31 3.18 0.27 2.29 0.17
6.14 0.32 3.96 0.35 2.92 0.19
5.99 0.58 3.44 0.47 2.18 0.45
5.18 0.24 2.92 0.44 1.92 0.11
4.77 0.22 3.18 0.41 2.70 0.26
4.68 0.35 3.25 0.38 1.73 0.11
5.92 0.41 4.10 0.33 1.95 0.38
6.22 0.11 3.73 0.35 3.14 0.24
7.96 0.14 4.25 0.51 0.55 0.25
ing p-nitro derivative 7c (RP50 values were 338.2 and 310.5 lM,
respectively).
45.50 0.68 20.30 0.34
46.98 0.29 23.15 0.37 12.77 0.25 4.77 0.23 1.77 0.28
47.09 0.35 20.89 0.38 11.14 0.31 4.55 0.21 0.95 0.21
3.4. Inhibition (%) of lipid peroxidation in a linoleic acid emulsion
47.53 0.31 20.63 0.25
8.07 0.24 3.51 0.18 0.84 0.17
In the present study, the antioxidant activities of imines and
amines of 4-hydroxychromene-2-one, determined by peroxidation
of linoleic acid using the thiocyanate method at 37 °C, after addi-
tion of different concentration of prepared compounds, were deter-
mined. During the linoleic acid peroxidation, peroxides are formed
and these compounds oxidise Fe2+ to Fe3+, the latter Fe3+ ion forms
a complex with SCNꢀ, which has a maximum absorbance at
500 nm. High absorbance (or low value of% of inhibition) is an indi-
cator of high concentration of peroxide formed during the emul-
sion incubation. As presented in Table 2, the antioxidant activity
of synthesised imines and amines exhibited an amount-dependent
manner. In the control sample without tested compounds, the
absorbance at 500 nm increased up to the maximal value in 72 h,
and then it decreased. The reason for this was that linoleic acid
hydroperoxides, generated from the peroxidation of linoleic acid,
decomposed to many secondary oxidation products, or the inter-
mediate products may be converted to stable end-products and
the substrate was exhausted. All tested concentrations added to
linoleic acid emulsion were able to reduce the formation of hydro-
peroxide. Generally, imines 2b–9b showed better ability to inhibit
lipid peroxidation than the corresponding prepared amines 2c–9c.
54.57 0.13 27.26 0.27 11.83 0.36 7.63 0.25 4.66 0.29
45.90 0.11 24.26 0.28 11.29 0.38 8.96 0.38 5.40 0.33
49.16 0.25 23.56 0.21 11.59 0.13 5.25 0.24 3.51 0.39
49.35 0.12 18.81 0.51 10.94 0.17 4.62 0.35 3.70 0.34
a
Data of percentage are reported as means SD of three measurements.
12.33
derivatives 3b, 4b and 5b were the lowest values measured
(11.70, 11.83 and 11.71 g ascorbic acid/ml, respectively). On the
lg ascorbic acid/ml, respectively), while in the case of tolyl
l
other hand, comparing the antioxidant capacity within the range
of all tested concentrations, best results were observed for com-
pounds 9b, 8b and 2b. In the group of amines 2c–9c, the highest
antioxidant capacity was observed for p-nitrophenyl derivative
6c. Also, good reducing activities for all tested concentrations were
found for compounds 8c and 9c, while phenyl derivative 2c, as well
as, o-tolyl compound 5c showed the lowest reducing activity.
3.6. Antimicrobial activity of tested compounds
The newly prepared coumarin derivatives 2b–9b and 2c–9c, to-
gether with streptomycin and ketoconazole as references, were
tested against a panel of Gram-positive and Gram-negative bacte-
ria and fungi (Tables 4 and 5).
Compound 3b at a concentration of 1000 lM showed better anti-
oxidant activity than BHT, while in cases of other compounds
and other tested concentrations BHT was superior in the inhibition
of peroxidation. All imines possess better antioxidant activity than
ascorbic acid, while at the concentration of 1000 and 500
pounds 3b–7b showed high level inhibitor properties to lipid per-
oxidation in contrast to -tocopherol. Other two tested imines 8b
as benzyl imino and 9b as n-pentanoic acid derivatives showed
lower activities than -tocopherol, which clearly indicated that
lM, com-
3.6.1. Antibacterial activity of tested compounds
a
As shown in Table 4, the amino compounds 2c–9c were more
potent than the corresponding imino compounds 2b–9b. In the
series of imino derivatives, high antibacterial activities (MIC values
a
the phenyl moiety attached to imino nitrogen has influence on for-
mation of stable end-products during the process of lipid peroxida-
tion. Among the amines 2c–9c, only p-nitrophenyl derivative 6c at
were 31.25 lg/ml) were observed for compounds with n-pentanoic
acid (9b) and benzyl fragments (8b) bonded to imino nitrogen
against seven of 13 test bacteria. Also, the compound 6b as p-
NO2-phenyl derivate of 4-hydroxycoumarin showed activity, with
a concentration of 1000
lM showed 67.8% inhibition, an antioxi-
dant activity a few percentage lower than that of 1000
lM
a
-
MIC values of 15.6–31.25 lg/ml against six test bacteria. On the
tocopherol (74.8%). Inhibitor activities of other amines are similar.
All abovementioned confirmed that the reduction of imino group
led to decrease of antioxidant activity, as well as that, in a series
of tested amines, only benzopyran moiety was responsible for
the inhibition of lipid peroxidation.
other hand, bacteria K. pneumoniae and P. glycinea were most sen-
sitive against tested imino derivatives (MIC values were in the
range of 7.8–31.25
lg/ml, except in the case of compound 4b, since
the observed MIC values were 31.25 and 62.5
lg/ml, respectively),
while S. aureus (ATCC 25923) and E. cloaceae were the most resis-
tant tested bacteria with observed MIC values in the range of 62.5–
3.5. Total antioxidant activity
125
lent antibacterial activities, especially n-pentanoic acid derivative
9c with observed MIC values in the range of 3.9–15.6 g/ml, which
are for bacteria S. aureus (FSB 30), E. coli and P. fluorescens are two
times lower and for S. aureus (ATCC 25923) four times lower than
the MIC values of standard drug streptomycin. Also, compounds
with phenyl (2c) and p-tolyl (3c) residues at NH group inhibited
lg/ml. Corresponding amino compounds 2c–9c showed excel-
The phosphomolybdenum method is based on the reduction of
Mo(VI) to Mo(V) by the antioxidant compounds and the formation
of green Mo(V) complexes with a maximal absorption at 695 nm.
Using this method the results from Table 3 indicate that the pre-
pared amines 2c–9c have better antioxidant capacity than the cor-
responding imines 2b–9b. In a series of imino derivatives of 4-
hydroxycoumarins, and at the highest tested concentration
l
the growth of all bacteria at the concentrations of 3.9–7.8
(except in a case P. phaseolicola, MIC values were 62.5 and
31.25 g/ml, respectively), which indicated their antibacterial po-
tency to be similar to streptomycin. The introduction of o-tolyl
lg/ml
(250
lg/ml), the best reducing activities were found for com-
l
pounds with nitro-phenyl substituents 6b and 7b (12.58 and

N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
1017
Table 4
Antibacterial activity of tested imino and amino derivatives of 4-hydroxycoumarins.
Comp.
Bacteria
Gram-positive
Gram negative
B.m.
B.s.
M.l.
S.a.
S.a.(i)
A.c.
En.cl.
Er.ca.
Es.co.
K.p.
P.fl.
P.gl.
P.ph.
MIC values of tested compounds (
lg/ml)
2b
3b
4b
5b
6b
7b
8b
9b
2c
3c
4c
5c
6c
7c
8c
9c
31.25
125
62.5
125
62.5
125
62.5
62.5
125
62.5
62.5
31.25
3.9
3.9
3.9
3.9
3.9
62.5
125
62.5
62.5
62.5
125
125
125
3.9
3.9
7.8
15.6
15.6
31.25
3.9
62.5
62.5
31.25
125
31.25
62.5
31.25
62.5
7.8
7.8
7.8
7.8
15.6
15.6
3.9
3.9
7.8
125
62.5
62.5
62.5
31.25
62.5
62.5
62.5
62.5
125
62.5
62.5
125
125
3.9
3.9
3.9
3.9
7.8
7.8
7.8
7.8
1.95
125
125
62.5
62.5
31.25
62.5
31.25
62.5
7.8
7.8
3.9
3.9
7.8
62.5
62.5
62.5
62.5
125
15.6
31.25
31.25
15.6
15.6
7.8
15.6
31.25
7.8
7.8
7.8
15.6
7.8
15.6
7.8
62.5
62.5
31.25
62.5
31.25
31.25
31.25
15.6
62.5
7.8
7.8
7.8
7.8
7.8
62.5
62.5
62.5
62.5
125
125
31.25
31.25
62.5
31.25
31.25
62.5
15.6
7.8
62.5
62.5
62.5
62.5
31.25
62.5
62.5
62.5
62.5
31.25
15.6
7.8
15.6
62.5
15.6
15.6
15.6
7.8
62.5
31.25
15.6
62.5
31.25
31.25
7.8
125
125
125
31.25
31.25
7.8
7.8
7.8
7.8
7.8
15.6
31.25
15.6
31.25
62.5
62.5
3.9
3.9
3.9
3.9
3.9
7.8
3.9
3.9
7.8
31.25
3.9
3.9
7.8
7.8
3.9
3.9
7.8
7.8
7.8
7.8
15.6
62.5
31.25
7.8
31.25
7.8
3.9
3.9
7.8
1.95
7.8
7.8
7.8
7.8
15.6
7.8
7.8
15.6
7.8
3.9
3.9
15.6
7.8
1.95
*
S
7.8
7.8
7.8
*
S
Streptomycin; B.m. – Bacillus mycoides (FSB 1); B.s. – Bacillus subtilis (FSB 2); M.l. – Micrococcus lysodeikticus (ATCC 4698); S.a. – Staphylococcus aureus (ATCC 25923); S.a.(i) –
Staphylococcus aureus (FSB 30); A.c. – Azotobacter chroococcum (FSB 14); En.cl – Enterobacter cloaceae (FSB 22); Er.ca – Erwinia carotovora (FSB 31); Es-co – Escherichia coli (ATCC
25922); K.p. – Klebsiella pneumoniae (FSB 26); P.fl. – Pseudomonas fluorescens (FSB 28); P.gl – Pseudomonas glycinea (FSB 40); P.ph. – Pseudomonas phaseolicola (FSB 29).
Table 5
Antifungal activity of tested imino and amino derivatives of 4-hydroxycoumarins.
Comp.
Fungi
A.gl.
A.n.
C.a.
F.o.
P.ve.
T.l.
T.h.
T.v.
MIC values of tested compounds (lg/ml)
2b
3b
4b
5b
6b
7b
8b
9b
2c
3c
4c
5c
6c
7c
8c
9c
62.5
62.5
62.5
125
31.25
62.5
31.25
31.25
62.5
125
62.5
125
62.5
62.5
62.5
62.5
7.8
125
125
125
31.25
31.25
31.25
125
62.5
125
125
125
250
31.25
31.25
62.5
62.5
125
125
125
62.5
62.5
125
62.5
62.5
125
62.5
125
125
250
62.5
31.25
31.25
125
62.5
31.25
62.5
62.5
62.5
31.25
125
31.25
31.25
62.5
31.25
31.25
125
125
125
62.5
62.5
62.5
125
31.25
31.25
31.25
125
125
31.25
31.25
125
125
125
250
62.5
62.5
62.5
62.5
62.5
31.25
62.5
31.25
62.5
62.5
31.25
31.25
31.25
62.5
62.5
31.25
31.25
62.5
62.5
62.5
62.5
125
125
250
250
125
125
62.5
62.5
62.5
62.5
125
125
125
62.5
125
62.5
125
3.9
62.5
125
125
250
250
125
125
125
62.5
62.5
7.8
125
1.95
*
K
7.8
3.9
7.8
7.8
*
K
Ketoconazole; A.gl – Aspergillus glaucus (FSB 32); A.n – Aspergillus niger (FSB 31); C.a. – Candida albicans (ATCC 10259); F.o. – Fusarium oxysporum (FSB 91); P.ve – Penicillium
verrucosum (FSB 21); T.l. – Trichoderma longibrachiatum (FSB 13); Trichoderma harzianum (FSB 12); T.v. – Trichoderma viride (FSB 11).
fragment at NH group (compound 5c) decreased the potency
against B. mycoides, B. subtilis and P. phaseolicola (MIC values were
fragment at the imino nitrogen and at the concentration of
31.25 g/ml inhibited growth of five test fungi, while at the same
l
62.5
l
g/ml, respectively). The most sensitive bacteria against syn-
MIC value benzyl imino compound 8b showed inhibitor activity
against four tested fungi. The most sensitive fungi were C. albicans
and T. longibrachiatum and their growth was inhibited by com-
pounds 2b–5b, 8b and 9b at 31.25 lg/ml. Based on Table 5, pre-
pared amines 2c–9c, in contrast to imines 2b–9b, showed lower
thesised amino compounds 2c–9c were S. aureus (ATCC 25923), A.
chroococcum, E. carotovora, E. coli, P. fluorescens and P. glycinea,
while the resistant bacteria against tested compounds were B.
mycoides, B. subtilis, M. lysodeikticus and P. phaseolicola.
potency against tested fungal strains, with MIC values in the range
3.6.2. Antifungal activity of tested compounds
of 62.5–250
concentration of 62.5
fungi. The most resistant fungus was A. niger, whose growth was
inhibited by compounds 5c, 8c and 9c at 250 g/ml, which is not
comparable to the antifungal potency of the standard drug
ketoconazole.
l
g/ml. Among them, p-nitrophenyl derivative 6c at a
Antifungal activity of prepared imines 2b–9b was in the range
lg/ml showed potency against six of eight
of 31.25–125
as o-tolyl imino derivative against six of eight fungi (MIC values
were 31.25 g/ml, respectively). Also, good antifungal activity
was observed for compound 2b, which possesses phenyl
lg/ml, with high potency noted for compound 5b
l
l
a

1018
N. Vukovic et al. / Food Chemistry 120 (2010) 1011–1018
Jurd, L., Corse, J., King, A. D., Bayne, H., & Mihara, K. (1971). Antimicrobial properties
4. Conclusions
of 6,7-dihydroxy-, 7,8-dihydroxy, 6-hydroxy- and 8-hydroxycoumarins.
Phytochemistry, 10, 2971–2974.
In this study we presented the antioxidant and antimicrobial
activities of imino and amino derivatives of 4-hydroxycoumarins.
The synthesised compounds scavenged the DPPH radical, reduced
Fe3+ cations and inhibited lipid peroxidation in a concentration
and time-dependent manner. Also, prepared compounds showed
excellent antimicrobial activity against all test microorganisms.
These experiments show that introduction of new pharmaco-
phores on the 4-hydroxycoumarin moiety has a significant influ-
ence on its antimicrobial properties, resulting in potential
antimicrobial agents. On the other hand, their antioxidative activ-
ity and the fact that they are structurally modified derivatives of
naturally-occurring compounds make them potential dietary
antioxidants.
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