Antifungal activity of α-methyl trans cinnamaldehyde, its ligand and metal complexes: Promising growth and ergosterol inhibitors

Article abstract of DOI:10.1007/s10534-011-9447-0

Antifungal effectivity and utility of cinnamaldehyde is limited because of its high MIC and skin sensitivity. In this study, α-methyl trans cinnamaldehyde, a less irritating derivative, have been self coupled and complexed with Co(II) and Ni(II) to generate N, N′-Bis (α-methyl trans cinnamadehyde) ethylenediimine [C₂₂H₂₄N ₂], [Co(C₄₄H₄₈N₄)Cl₂] and [Ni(C₄₄H₄₈N₄)Cl₂]. Ligand and complexes were characterized on the basis of FTIR, ESI-MS, IR and ¹HNMR techniques. Synthesized ligand [L] and complexes were investigated for their MICs, inhibition of ergosterol biosynthesis and H ⁺ extrusion against three strains of Candida: C. albicans 44829, C. tropicalis 750 and C. krusei 6258. Average of three species MIC of methyl cinnamaldehyde is 317 μg/ml (2168 μM). Compared to methyl cinnamaldehyde ligand [L], Co(II) and Ni(II) complex are found to be 4.48, 17.78 and 21.46 times more effective in liquid medium and 2.73, 8.93 and 10.38 times more effective in solid medium. At their respective MIC₉₀ average inhibition of ergosterol biosynthesis caused by methyl cinnamaldehyde, ligand [L], Co(II) and Ni(II) complex, respectively was 80, 78, 90 and 93%. H ⁺ extrusion was also significantly inhibited but did not co-relate well with MIC₉₀. Results indicate ergosterol biosynthesis as site of action of α-methyl cinnamaldehyde, synthesized ligand and complexes. α-methyl cinnamaldehyde and ligand did not show any toxicity against H9c2 rat cardiac myoblast cell, whereas Co(II) and Ni(II) complexes on an average produced 19% cellular toxicity.

Full text of DOI:10.1007/s10534-011-9447-0

Biometals (2011) 24:923–933
DOI 10.1007/s10534-011-9447-0
Antifungal activity of a-methyl trans cinnamaldehyde,
its ligand and metal complexes: promising growth
and ergosterol inhibitors
•
Sheikh Shreaz Rayees A. Sheikh Rimple Bhatia
•
•
•
Khan Neelofar Sheikh Imran Athar A. Hashmi
•
•
•
Nikhat Manzoor Seemi F. Basir Luqman A. Khan
•
Received: 25 November 2010 / Accepted: 26 March 2011 / Published online: 8 April 2011
Ó Springer Science+Business Media, LLC. 2011
Abstract Antifungal effectivity and utility of cin-
namaldehyde is limited because of its high MIC and
skin sensitivity. In this study, a-methyl trans cinna-
maldehyde, a less irritating derivative, have been self
coupled and complexed with Co(II) and Ni(II) to
generate N, N0–Bis (a-methyl trans cinnamadehyde)
ethylenediimine [C22H24N2], [Co(C44H48N4)Cl2] and
[Ni(C44H48N4)Cl2]. Ligand and complexes were
characterized on the basis of FTIR, ESI–MS, IR and
1HNMR techniques. Synthesized ligand [L] and com-
plexes were investigated for their MICs, inhibition of
ergosterol biosynthesis and H? extrusion against three
strains of Candida: C. albicans 44829, C. tropicalis
750 and C. krusei 6258. Average of three species MIC
of methyl cinnamaldehyde is 317 lg/ml (2168 lM).
Compared to methyl cinnamaldehyde ligand [L],
Co(II) and Ni(II) complex are found to be 4.48,
17.78 and 21.46 times more effective in liquid
medium and 2.73, 8.93 and 10.38 times more effective
in solid medium. At their respective MIC90 average
inhibition of ergosterol biosynthesis caused by methyl
cinnamaldehyde, ligand [L], Co(II) and Ni(II) com-
plex, respectively was 80, 78, 90 and 93%. H?
extrusion was also significantly inhibited but did not
co-relate well with MIC90. Results indicate ergosterol
biosynthesis as site of action of a-methyl cinnamal-
dehyde, synthesized ligand and complexes. a-methyl
cinnamaldehyde and ligand did not show any toxicity
against H9c2 rat cardiac myoblast cell, whereas Co(II)
and Ni(II) complexes on an average produced 19%
cellular toxicity.
Keywords Candida ꢀ a-methyl trans
cinnamaldehyde ꢀ Metal complexes ꢀ PM-ATPase ꢀ
Ergosterol
S. Shreaz ꢀ R. Bhatia ꢀ K. Neelofar ꢀ N. Manzoor ꢀ
S. F. Basir ꢀ L. A. Khan (&)
Introduction
Department of Biosciences, Jamia Millia Islamia (Central
University), Enzyme Kinetics Laboratory, Room NO-106,
New Delhi 110025, India
Primary and opportunistic fungal infections continue
to increase rapidly because of prolonged use of
antibiotics, cancer chemotherapy regimes and HIV
infections. Candida albicans is causative agent of
most candidiasis; but other emerging Candida species,
including C. glabrata and C. krusei, are now posing
serious threat (Nguyen et al. 1996; Pfaller et al. 1998).
Existing classes of antifungals: azoles and polyenes
e-mail: profkhanluqman@gmail.com
R. A. Sheikh ꢀ A. A. Hashmi
Department of Chemistry, Jamia Millia Islamia (Central
University), New Delhi 110025, India
S. Imran
Department of Applied Sciences, Jamia Millia Islamia
(Central University), New Delhi 110025, India
123

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Biometals (2011) 24:923–933
have severe hepatotoxicity, nephrotoxicity and are
being increasingly resisted by infecting fungi (Odds
et al. 2003; Gupta and Thomas 2003). Natural
remedies may offer safe alternatives for less severe
infections.
acid (Scheme 1a). The ligand [L] was then refluxed at
room temperature with nickel chloride to form its
metal complex [NiLCl2] and cobalt chloride to form
its metal complex [CoLCl2] (Scheme 1b). Elemental
analyses were performed by a Perkin Elmer 2400
CHNSO Elemental Analyser. IR spectra were
recorded as KBr pellets using a Perkin Elmer 1620
FT IR spectrophotometer. Far IR spectra were
recorded as CsI pellets in the region 650–100 cm-1
Cinnamaldehyde is effective in inhibiting growth
of bacteria, yeast and filamentous molds and is
reported to interfere with F-type mitochondrial
ATPase and lowering of intracellular ATP (Gill and
Holley 2004). Antimicrobial activity of cinnamalde-
hyde is attributed to its membrane action (Bang et al.
2000). It has been tentatively established that aro-
matic ring and hydrocarbon chain affect antifungal
properties (Sheikh et al. 2010). Antifungal MICs of
cinnamaldehyde is high, its use is further limited by
its irritant effect on skin which is attributed to
aldehyde function (Wang et al. 2005). a-substituted
cinnamaldehyde are known to be less skin sensitizer
because they react slowly with amine (Vimala and
Raymonds 1997). a-methyl cinnamaldehyde is one
such derivative, which is also expected to be more
effective because of its increased hydrophobicity.
In this study we report synthesis of self coupled
ligand N, N0–Bis (a-methyl trans cinnamaldehyde)
ethylenediimine [C22H24N2] and its Ni(II) and Co(II)
complexes of the type [Co(C44H48N4)Cl2] and
[Ni(C44H48N4)Cl2]. Synthesized compound have been
characterized employing standard chemical tech-
niques. Antifungal efficacy of synthesized compounds
have been tested against three standard Candida
species: C. albicans ATCC 44829, C. tropicalis
ATCC 750 and C. krusei ATCC 6258 by determining
MICs, sensitivity and investigating membrane asso-
ciated properties of PM-ATPase function and ergos-
terol biosynthesis. a-methyl cinnamaldehyde and its
ligand and complexes display markedly improved
anticandidal activity, compared to cinnamaldehyde
and its complexes (Sheikh et al. 2010) and showed
very limited toxicity against H9c2 rat cardiac myo-
blast cells.
1
using a JASCO FT IR spectrophotometer. H NMR
spectra were recorded using a Bruker DPX-300 MHz
spectrophotometer operating at room temperature
with DMSO d6 as solvent. The chemical shift (d) is
reported in parts per million (ppm) using tetrameth-
ylsilane as internal standard. Positive and negative
ESI mass spectra were measured by Bruker (esquire
3000–00037) instrument. Magnetic susceptibility
measurements were carried out from a microanalysis
laboratory by Gouy method at room temperature.
Electronic spectra were recorded on a spectro-UV–
Vis Dual Beam 8 auto cell UVS-2700 LABOMED,
INC, US spectrophotometer using DMSO as solvent.
Melting point was recorded on a Metrex melting
point apparatus. Synthesis of ligand N, N0–Bis
(a-methyl trans cinnamadehyde) ethylenediimine, its
Ni(II) and Co(II) complexes was carried out broadly
2HN
A
O
+
2
2HN
-2H2O
EtOH
Reflux
N
N
CH3
CH3
Ligand
B
N
MCl2 + 6 H 2
O
2
+
N
CH3
CH3
R.T
Reflux
MeOH
Materials and methods
Cl
M
H3
C
C
H3
N
N
Materials
N
N
CH3
CH 3
Cl
M(II) Complex
Where M = Ni and Co
The ligand was prepared by condensing the a-methyl
trans cinnamaldehyde and the diamine (ethylene
diamine) in presence of the few drops of hydrochloric
Scheme 1 a Synthesis of ligand [L] b Metal complexes
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Biometals (2011) 24:923–933
925
Filter disk assay
by procedure detailed in reference (Khalaji and Weil
2007; Sheikh et al. 2010). The compounds were
recovered in solid state and were recrystallized from
methanol. Yield 65% Ni (II) and 62% Co (II)
complexes.
Strains were inoculated into liquid YPD medium and
grown overnight at 37°C. The cells were then pelleted
and washed three times with distilled water. Approx-
imately 105 cells/ml were inoculated in molten agar
media at 40°C and poured into 100 mm diameter
petriplates. Filter discs were kept on solid agar and
test compound was spotted on the disc as described
previously (Ahmad et al. 2010). Test compounds
(ten-fold more than MIC) dissolved in 10% DMSO,
or solvent control (10% DMSO) were pipetted onto
4-mm-diameter filter disc. 200 lg/ml of fluconazole
was also on the discs to serve as positive control. The
diameter of zone of inhibition was recorded in
millimeters as difference in zone diameter from the
centre of disc for test compound with that of solvent
control, after 48 h and was compared with that of
control. The results are presented as Mean stan-
dard deviation of at least three replicate experiments
performed on separate days.
Growth conditions and media
C. albicans ATCC 44829, C. tropicalis ATCC 750
and C. krusei ATCC 6258 used in this study were
kind gifts from I.A Khan (Clinical microbiology unit,
IIIM Jammu, India). All of the strains were grown on
Yeast extract Peptone Dextrose (YPD) medium
containing 2% (w/v) glucose, 2% peptone, and 1%
yeast extract (Hi Media, India). YPD agar plates
containing 2.5% Agar (Hi Media) in addition were
used to maintain the culture. a-methyl trans cinna-
maldehyde was purchased from ALDRICH chemi-
cals (Germany), whereas all inorganic chemicals
were of analytical grade and procured from E. Merck
(India). Fluconazole was purchased from Hi Media,
India.
Ergosterol extraction and estimation assay
Total intracellular sterols were extracted after
(Arthington et al. 1999). Briefly, a single Candida
colony from an overnight Sabouraud dextrose agar
plate culture was used to inoculate 50 ml of Sabou-
raud dextrose broth (Hi Media) containing various
concentrations of the test compounds along with
positive control fluconazole. The cultures were incu-
bated for 16 h with shaking at 35°C. The stationary-
phase cells were harvested by centrifugation at
2,700 rpm (Sigma 3K30) for 5 min and washed once
with sterile distilled water. The net wet weight of the
cell pellet was determined. Three milliliters of 25%
alcoholic potassium hydroxide solution was added to
each pellet and vortex mixed for 1 min. Cell suspen-
sions were transferred to sterile borosilicate glass
screw-cap tubes and were incubated in an 85°C water
bath for 1 h. Following incubation, tubes were
allowed to cool to room temperature. Sterols were
then extracted by addition of a mixture of 1 ml of
sterile distilled water and 3 ml of n-heptane followed
by vigorous vortex mixing for 3 min. Prior to analysis,
a 1 ml aliquot of sterol extract was diluted fivefold in
100% ethanol and scanned spectrophotometrically
between 240 and 300 nm using LABOMED, INC
Spectrophotometer (USA). The presence of ergosterol
Methods
Determination of minimum inhibitory
concentration (MIC90)
Microtiter assay
Cells were grown for 48 h at 30°C to obtain single
colonies, which were resuspended in a 0.9% normal
saline solution to give an optical density at 600 nm
(OD600) of 0.1. The cells were then diluted 100-fold
in YNB medium containing 2% glucose and the
respective auxotrophic supplements. The diluted cell
suspensions were added to the wells of round-
bottomed 96-well microtiter plates (100 ll/well)
containing equal volumes of medium and different
concentrations of test agents (Kohli et al. 2002).
Fluconazole was included as positive control. In
addition to this a drug-free control was also included.
The plates were incubated at 30°C for 24 h. The MIC
test end point was evaluated both visually and by
observing the OD620 in a microplate reader (BIO-
RAD, iMark, US) and is defined as the lowest
compound concentration that gave C90% inhibition
of growth compared to the controls.
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Biometals (2011) 24:923–933
and the late sterol intermediate 24(28) DHE in the
extracted sample resulted in a characteristic four-
peaked curve. The absence of detectable ergosterol in
extracts was indicated by a flat line. Ergosterol
content was calculated as a percentage of the wet
weight of the cell.
Direct reduction or interaction of MTT observed with
ligand and its complexes varied from 5 to 20%
depending upon concentration used in the study.
Given the low magnitude of direct reduction involved,
any reduction by compounds accumulated by cells
would be very minimal. No correction has been made
for this reduction.
Proton efflux measurements
Mid-log phase cells harvested from YEPD medium
were washed twice with distilled water and routinely
100 mg cells were suspended in 5 ml solution
containing 0.1 M KCl and 0.1 mM CaCl2. Suspen-
sion was kept in a double-jacketed glass container
with constant stirring. Test compounds were added to
achieve the desired concentration in this 5 ml solu-
tion. For glucose stimulation experiments, 100 ll of
glucose was added to achieve a final concentration of
5 mM. The container was connected to a water
circulator at 25°C. H? extrusion rate was calculated
from the volume of 0.01 N NaOH consumed as
reported earlier (Rashid et al. 2004).
Results
Physical properties and IR Spectra
Physical properties and analytical data of the ligand
and its metal complexes support its proposed struc-
ture. The molar conductivity (^m) of the Ni(II) and
Co(II) complexes measured in 1 9 10-3 mol l-1
DMSO at room temperature shows values of 38,
32 X-1 cm2 mol-1 respectively. Elemental analysis
results show that the composition of the complexes is
in agreement with its formulation given in (Sche-
me 1a, b). No band corresponding to free primary
diamine and hydroxyl group was detected similarly in
the IR spectrum of ligand absence of a broad
absorption band in the region 1660–1765 cm-1
characteristic for carbonyl group of (HC=O) in
a-methyl trans cinnamaldehyde, indicates that the
Oxygen was detached from the HC=O. Appearance
of a strong signal at 1445 cm-1 may be attributed to
the C–N bond (Nakamoto 1970). The shifting in the
band of t(C–N) towards the lower wave number in
the metal complex indicated that the coordination
takes place through the nitrogen of the t(C–NH)
group, hence implying that the ligand [L] is tetra-
dentate. This was finally established through far IR
spectra by the appearance of new signals seen at 450,
455, 340, and 345 cm-1 in the spectra of metal
complexes.
MTT cell viability assay
H9c2 rat cardiac myoblasts were cultured and main-
tained as monolayer in Dulbecco’s modified Eagle’s
medium (DMEM), high glucose, supplemented with
10% fetal bovine serum (heat inactivated), 100 units/ml
penicillin, 100 lg/ml streptomycin, and 2.5 lg/ml
Amphotericin B, at 37°C in humidified incubator with
5% CO2 (Gupta et al. 2006). For treatments, com-
pound stock solutions were prepared in dimethyl
sulfoxide (DMSO) and added to wells to give the
indicated final concentrations. Final DMSO concen-
tration was 0.2% in all wells including the untreated
cells (control) and fluconazole controls. Cells were
incubated for 48 h at 37°C in 5% CO2 humidified
incubator together with untreated control sample.
After incubation cells were washed in PBS and
incubated with MTT solution for 45 min at 37°C.
After discarding the supernatant MTT crystals were
dissolved with acid isopropanol and the absorbance
was measured at 570 nm. All assays were performed
in triplicate. Percent viability was defined as the
relative absorbance of treated versus untreated control
cells. Plates were analysed in an ELISA plate reader
(Labsystems Multiskan RC, Helsinki, Finland) at
570 nm with a reference wavelength of 655 nm.
1H NMR Spectra
1H NMR also supports the formation of ligand by
showing the absence of an aldehydic peak (H–C=O) at
10 ppm (Fig. 1). The aromatic region of the ligand
shows two doublet signals in the range 7.271–
7.301 ppm (Ar–CH, 4H), 7.412–7.450 ppm (Ar–CH,
4H) and one sharp singlet at 7.18 ppm (Ar–CH, 2H),
because these aromatic protons are in different chem-
ical environment. Another sharp singlet at 2.46 ppm
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Biometals (2011) 24:923–933
927
1
Fig. 1 A region of the H
NMR spectrum of the
ligand [L]
(C=N–CH2, 4H) has been ascribed to methylene
protons. Two more sharp signals one in the pseudo
aromatic region at 6.85 ppm (2H, C=C–CH) and
another at 7.71 ppm (2H, C–N=C–H) have been
ascribed to methene protons. The signals of
C–N = C–H has been shifted to down field due to
the deshielding effect of nitrogen group attached to it.
At 1.95 ppm (C–CH3, 6H) sharp singlet has been
ascribed to methyl protons. These proton signals
undergo down field shifting in the metal complexes
of the ligand, because of the paramagnetic effect of
metal (II) ion, hence it supports the coordination of the
ligand towards the metal ion (Chaudhary and Singh
2004; Kemp 1975).
Electro spray ionization mass spectra (ESI MS)
The electro spray ionization mass spectrum (ESI MS)
of the ligand was studied in DMSO solution (Fig. 2a).
A negative ion ESI mass spectrum of ligand confirms
the proposed formula by showing a peak at m/z 317
corresponding to the moiety [(C22H24N2) atomic
mass m/z 316.23]. The series of peaks in the range
m/z 120.7, 164.6, 254.7 etc., may be assigned to
various fragments. This data suggests the 2 ? 1
condensation of a-methyl trans cinnamaldehyde and
ethylenediamine. Their intensity gives an idea of
stability of fragments. Similarly negative ion ESI–
MS of the nickel and cobalt complexes shows a peak
at m/z 763 (Fig. 2b) and m/z 763.5 respectively,
which is consistent with the molecular ion fragment
and it supports the proposed structure of the complex.
Fig. 2 Electro spray ionization mass spectrum a ligand [L],
b Ni(II) complex
Electronic spectra and magnetic measurements of
Cobalt (II) and Nickel (II) complex of a-methyl trans
cinnamaldehyde.
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Biometals (2011) 24:923–933
magnetic moment at leff = 3.29 BM, which is in
agreement with an octahedral geometry around the
Ni(II) ion.
Minimum inhibitory concentration (MIC90)
and sensitivity assay
Table 1 summarizes the minimum inhibitory concen-
tration (MIC90) values, in microgram/ml (lg/ml) and
microMolar (lM) of methyl cinnamaldehyde, ligand
[L], Co(II) and Ni(II) complex against three different
Candida species. On concentration basis average
MIC90 of methyl cinnamaldehyde was 2168 lM.
Average MIC90 of ligand [L], Co(II) and Ni(II)
complex is 483.8, 122 and 101 lM, respectively.
Fluconazole had a mean MIC90 of 120 lM. Com-
pared to methyl cinnamaldehyde, thus ligand [L] is
4.48 times more effective whereas Co(II) and Ni(II)
complex is 17.78 and 21.46 times more effective.
Antifungal activity on solid media examined by disc
diffusion assay yields results along similar lines.
Yeasts were found to be more sensitive to Ni(II) and
Co(II) complex, compared to ligand [L] and original
compound (Table 2). Average value of zone of
inhibition of three species at 3 mg/ml methyl cinna-
maldehyde, ligand [L], [Co(L)Cl2] and [Ni(L)Cl2]
was 8.3, 10.6, 14.3 and 16.6 mm. For fluconazole
(200 lg/ml) the average value was 15.3 mm. On
concentration basis thus ligand [L] was 2.73 times
more effective compared to methyl cinnamaldehyde.
Average fold effectiveness of [Co(L)Cl2], [Ni(L)Cl2]
and fluconazole was determined to be 8.93, 10.38 and
57.58 times compared to methyl cinnamaldehyde.
300
400
500
600
700
800
900
wavelength (nm)
Fig. 3 Electronic spectrum of the Co(II) complex
The electronic spectra of the Co(II) complex
(Fig. 3) consist of transition bands at 520, 565 nm
4
which are assigned to the transition T1 g
?
4T1 g(P), 682 nm which is assigned to the transition
4T1 g ? T1 g, and 739, 824 nm which are assigned
2
4
4
to the transition T1 g ? T2 g. The Co(II) complex
has an effective magnetic moment value (leff) of 4.82
BM. These values are indicative of an octahedral
geometry around the Co(II) ion. Electronic spectra of
the Ni(II) complex showed transition bands at 415,
530 nm which are assigned to the transition
3A2 g ? T1 g(F), 690, 750 and 860 nm which are
3
3
3
assigned to the transition A2 g ? T2 g. An indica-
tion of the most probable geometric configuration of
the synthesized metal complex is their magnetic
moment values. The Ni(II) complex possesses
Table 1 Minimum inhibitory concentrations (MIC90) of four bioactive compounds and standard drug tested against different
Candida spp
Bioactive compound
MIC90 (lg/ml)
C. albicans
ATCC 44829
C. tropicalis
ATCC 750
C. krusei
ATCC 6258
Methyl trans cinnamaldehyde
250
110
60
300
160
100
80
400
190
120
100
64
Ligand [L]
[Co(L)Cl2]
[Ni(L)Cl2]
Fluconazolea
a
50
16
32
Positive control L- [C22H24N2]. Average of three species: methyl trans cinnamaldehyde; 2168 lM, ligand [L]; 483.8 lM,
[Co(L)Cl2]; 122 lM, [Ni(L)Cl2]; 101 lM, fluconazole; 120 lM
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Biometals (2011) 24:923–933
929
Table 2 Disc diffusion assay of methyl cinnamaldehyde its ligand [L], Co(II) and Ni(II) complexes against different Candida spp
Zone of inhibition (mm)
Methyl cinnamaldehyde
Ligand [L]
Co(II) complex
Ni(II) complex
Fluconazolea
17 ( 0.5)
Candida albicans
3 mg/ml
11 ( 0.9)
02 ( 0.5)
14 ( 1.0)
06 ( 1.5)
17 ( 0.9)
09 ( 0.5)
19 ( 0.0)
10 ( 0.3)
1 mg/ml
Candida tropicalis
3 mg/ml
08 ( 0.4)
02 ( 0.5)
10 ( 1.0)
05 ( 1.5)
14 ( 1.0)
09 ( 0.5)
16 ( 0.5)
11 ( 0.8)
15 ( 1.0)
14 ( 0.5)
1 mg/ml
Candida krusei
3 mg/ml
06 ( 1.5)
01 ( 0.8)
08 ( 0.6)
04 ( 0.5)
12 ( 1.0)
07 ( 1.0)
15 ( 0.5)
08 ( 0.6)
1 mg/ml
The data represents (Mean S.D) of three experiments
a
Positive control (200 lg/ml). Average value of three species at 3 mg/ml: methyl trans cinnamaldehyde; 8.3 mm, ligand [L];
10.6 mm, [Co(L)Cl2]; 14.3, [Ni(L)Cl2]; 16.6 and fluconazole (200 lg/ml); 15.3 mm
Ergosterol extraction and estimation assay
glucose (Serrano 1980; Ben-Josef et al. 2000; Elias
et al. 2001). Proton- pumping ability of fungi
mediated by the Pma1p at the expense of energy is
crucial for the regulation of internal pH and growth
regulation of fungal cell. Table 4 gives rate of H?
extrusion by Candida species in presence and absence
of glucose. No significant variation was observed
between various organisms. Untreated Candida cells
(control) in presence of 0.1 mM CaCl2 and 100 mM
KCl showed acidification starting from pH 7.0.
Acidification decreased in the presence of methyl
cinnamaldehyde its ligand [L], Co(II) and Ni(II)
complex. In the absence of glucose, average reduc-
tion in H? extrusion at MIC90 by methyl cinnamal-
dehyde was 62.11%. Ligand [L], Co(II) and Ni(II)
complex caused average inhibition of 70.21, 82.89
and 90.96% respectively. Presence of glucose
(5 mM) stimulated H?-efflux by 3.88, 5.22 and
3.86 fold with respect to control in C. albicans,
C. tropicalis and C. krusei respectively. Average
inhibition of glucose stimulated H? efflux was 30.09,
44.32, 50.32 and 55.75% for methyl cinnamaldehyde
its ligand [L], Co(II) and Ni(II) complex respectively.
H? efflux in the presence of glucose therefore did not
correspond with MIC90 of methyl cinnamaldehdye or
synthesized ligand and complexes.
Table 3 summarizes the effect of a -methyl cinna-
maldehyde its ligand [L], Co(II) and Ni(II) complex
on ergosterol biosynthesis in three Candida strains.
The total ergosterol content was determined for each
isolate grown in presence of varying concentrations
of test compounds. As shown in (Table 3), the
average decrease in total cellular ergosterol content
for different Candida isolates after exposure to their
respective MIC90 of methyl cinnamaldehyde was
80%. Average decrease in total cellular ergosterol
after exposure to MIC90 of ligand [L], Co(II) and
Ni(II) complex was 78, 90 and 93%, respectively.
Inhibition at MIC90 of fluconazole was 91%. From
the results, it is clear that with increase in test
compound concentrations, %ergosterol inhibition
increases and finally at MIC90 value close to 90%
reduction in ergosterol content is obtained. MIC90
values of methyl cinnamaldehyde its ligand [L],
Co(II) complex, Ni(II) complex and fluconazole
corresponds very well with percent inhibition of
sterol biosynthesis. It would thus appear that major
mode of action of methyl cinnamaldehyde, its ligand
and complexes is through inhibition of ergosterol
biosynthesis.
H? extrusion studies
Toxicity profile
Primary determinant or source of acidification in
yeast is Pma1p, both in absence and presence of
Synthesized compounds were tested against H9c2
cardiac myoblasts for toxicity. Subconfluent populations
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Biometals (2011) 24:923–933
Table 3 % Decrease in ergosterol content of control cells and treated samples
C.albicans ATCC 44829
C.tropicalis ATCC 750
C.krusei ATCC 6258
Sample
% Decrease in
Ergosterol content
Sample
(lg/ml)
% Decrease in
Ergosterol content
Sample
(lg/ml)
%Decrease in
Ergosterol content
Methyl cinnamaldehyde
Control
0
Control
100
0
Control
100
0
100 lg/ml
200 lg/ml
250 lg/ml
Ligand [L]
Control
21.13 0.297
41.12 0.189
55.01 0.104
24.09 1.423
63.22 1.234
94.08 0.213
21.11 0.678
57.23 2.213
91.09 0.189
200
300
300
400
0
Control
50
0
Control
50
0
40 lg/ml
18.13 0.799
39.04 0.436
66.00 1.234
29.08 1.324
59.00 0.876
86.00 0.289
20.00 0.231
51.09 0.098
81.12 0.678
80 lg/ml
100
100
110 lg/ml
Co(II) complex
Control
160
190
0
Control
30
0
Control
40
0
20 lg/ml
23.17 1.530
43.12 0.209
77.00 0.085
34.00 2.123
66.08 0.456
97.19 1.324
29.00 0.987
62.21 1.103
96.18 0.099
40 lg/ml
60
80
60 lg/ml
100
120
Ni(II) complex
Control
0
Control
25
0
Control
30
0
20 lg/ml
28.24 1.423
59.20 0.111
80.21 0.324
40.00 0.987
71.08 0.056
100 0.178
37.09 0.719
69.00 1.170
100 1.167
40 lg/ml
50
60
50 lg/ml
80
100
Fluconazolea
Control
0
Control
0
Control
0
16 lg/ml (MIC)
81.00 1.423
32 (MIC)
93.12 1.423
64 (MIC)
100 1.423
The data represents (Mean S.D) of three experiments
a
Positive control. Average ergosterol inhibition of three species at their respective MIC90: methyl trans cinnamaldehyde; 80%,
ligand [L]; 78%, [Co(L)Cl2]; 90%, [Ni(L)Cl2]; 93%, Fluconazole; 91%
of H9c2 cells were treated with increasing concen-
trations of these compounds and the measure of
viable cells was obtained after 48 h by measuring
optical density. Figure 4 depicts that all the com-
pounds including the reference compound fluconaz-
ole showed a viability of 100% at the concentration
range of 3.13 lg/ml. At 100 lg/ml methyl cinnamal-
dehyde, its ligand [L], Co(II) and Ni(II) complex
offered a remarkable viability of 95, 90, 86 and 85%.
At 200 lg/ml methyl cinnamaldehyde, ligand [L],
Co(II) and Ni(II) complex showed 7, 11, 18 and 20%
toxicity, respectively. Toxicity of reference drug
fluconazole at 200 lg/ml was 55%. It was noticed
that MIC90 concentration of synthesized compounds,
which had profound effect on growth, ergosterol
biosynthesis and H? ATPase activity of Candida,
showed significant measure of viable cells. This
indicates that the synthesized compounds have low
cytotoxic activity.
Discussion
Results derived from structure-antifungal activity
relationship of cinnamaldehyde suggest that aromatic
ring, length of hydrocarbon chain outside the ring and
lipophilicity affect antifungal properties of cinnamal-
dehyde (Ooi et al. 2006; Cheng et al. 2008). We have
previously reported increase in antifungal activity of
cinnamaldehyde following its coupling and metal
123

Biometals (2011) 24:923–933
931
Table 4 Effect of test compounds on rate of H?- efflux in different Candida spp at pH 7.0: Control stands for cells without any
compound, incubated in 0.1 mM CaCl2 and 0.1 M KCl at 25°C
Incubation with
Relative H?- efflux rate (n mol/mg yeast cells/min)
C. albicans
ATCC 44829
C. tropicalis
ATCC 750
C. krusei
ATCC 6258
Average
inhibition
Control
1a
1a
1a
–
Control ? methyl cinnamaldehyde (MIC90
)
0.35
0. 24
0.11
0.05
3.88
2.31
2.09
1.83
1.61
0.38
0.28
0.18
0.08
5.22
3.20
2.82
2.48
2.18
0.40
0.37
0.23
0.13
3.86
2.50
2.28
2.10
1.91
62.11
70.21
82.39
90.96
–
Control ? ligand [L] (MIC90
Control ? [Co[L] Cl2] (MIC90
)
)
Control ? [Ni[L] Cl2] (MIC90
Control ? Glucose (5 mM)
)
Control ? methyl cinnamaldehyde ? Glucose
Control ? ligand [L] ? Glucose
Control ? [Co(L)Cl2] ? Glucose
Control ? [Ni(L)Cl2] ? Glucose
a
The control had an average proton efflux rate (n moles/mg yeast cells/min) of 5.4, 5.0 and 5.9 respectively for C. albicans ATCC
44829, C. tropicalis ATCC 750 and C. kruesi ATCC 6258. The data represents mean of at-least three experiments (variation was less
than 5%)
38.09
44.32
50.32
55.75
120
100
80
60
40
20
0
3.13
6.25
12.5
25
50
100
200
Concentration (µg/ml)
Control
FCZ
Ni(II)
Co(II)
ligand
MCD
Fig. 4 Percentage of viable cells after 48 h pre-treatment of H9c2 myoblasts with fluconazole, Ni(II) complex, Co(II) complex,
ligand [L] and methyl cinnamaldehyde evaluated by MTT assay
complexing (Sheikh et al. 2010). As compared to
cinnamaldehyde, a-methyl trans cinnamaldehyde
with an additional carbon in chain shows increased
MIC90 of 250, 300 and 400 lg/ml for C. albicans,
C. tropicalis and C. krusei, respectively. Self cou-
pling of methyl cinnamaldehyde and further com-
plexing with Co(II) and Ni(II) increases lipophilicity,
which in turn may increase their permeation through
lipid membranes (Chohan et al. 2004). This is
reflected in significant drop in MIC90 of ligand
[L] as 110, 160 and 190 lg/ml, respectively, for three
Candida species. Complexation of two molecules of
ligand [L] employing Co(II) and Ni(II) ions leading
to further multiplication of methyl cinnamalde-
hyde structure and increased lipophilicity leading to
fold effectiveness of 17.78 and 21.46, respectively.
123

932
Biometals (2011) 24:923–933
Compared to methyl cinnamaldehyde fold effective-
ness of fluconazole was 18.06 which is in the same
range as of synthesized complex. Antifungal activity
on solid media examined by filter disc assay reinforce
MIC90 results that coupling and complexation leads
to increase in anticandidal effectivity of methyl
cinnamaldehyde against all the three strains. On solid
media, however, fold effectiveness of synthesized
ligand and complexes was 2.73, 8.93 and 10.38 as
compared to original molecule. Less fold effective-
ness of synthesized ligand and complexes on solid
media as compared to liquid medium indicates that
increase in structure leads to restricted diffusion on
solid media.
complexes. Sub confluent population of H9c2 rat
cardiac myoblast cells was employed for in vitro
MTT cell viability. No toxicity was displayed by
methyl cinnamaldehyde and ligand [L]. Co(II) and
Ni(II) complexes showed an average 19% cellular
toxicity.
To conclude a-methyl trans cinnamaldehyde, a
less skin sensitive derivative of cinnamaldehyde was
self coupled and complexed with Co(II) and Ni(II).
Synthesized ligand and complexes showed increased
anticandidal activity and have a very limited toxicity
towards vertebrate cells.
Acknowledgments Sheikh Shreaz greatly acknowledges the
financial support by ICMR (India) grant 45/93/09-Pha/BMS,
(S.R.F). The authors are thankful to Dr. Inshad A. Khan,
Scientist E1, BSL-3 Lab, IꢀI.I.M Canal road Jammu, India, for
providing standard Candida species. Authors are also indebted
to Shageer Ahmad from Department of Biosciences J.M.I
(India) for providing valuable technical assistance.
Mechanism of antimicrobial activity of cinnamal-
dehyde is not well established, available literature
supports both membrane action and specific cellular
processes (Kwon et al. 2003; Walsh et al. 2003). Two
key membrane associated properties have been
explored: PM-ATPase activity and ergosterol biosyn-
thesis. In the absence of glucose methyl cinnamalde-
hyde, its ligand [L], Co(II) and Ni(II) complex cause
mean inhibition in H?-extrusion of 62, 70, 82 and
91%. Presence of 5 mM glucose activated H?-
extrusion. Glucose stimulated H?-extrusion however,
was inhibited to a significantly lesser extent by all
tested compounds. Mean inhibition of three species
was only 38, 44, 50 and 56% by methyl cinnamal-
dehyde, ligand, Co(II) and Ni(II) complex at their
respective MIC90 values. Glucose modified form of
H?ATPase thus appears to be less sensitive. Inhibi-
tion values do not correspond well with MIC90 values
suggesting that inhibition of PM-ATPase may not be
the primary effect of methyl cinnamaldehyde and its
complexes.
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