Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand derived from cinnamaldehyde

Article abstract of DOI:10.1016/j.cbi.2016.01.015

In this study, we have used aldehyde function of cinnamaldehyde to synthesize N, N′-Bis (cinnamaldehyde) ethylenediimine [C₂₀H₂₀N₂] and Co(II) complex of the type [Co(C₄₀H₄₀N₄)Cl₂]. The structures of the synthesized compounds were determined on the basis of physiochemical analysis and spectroscopic data (¹H NMR, FTIR, UV-visible and mass spectra) along with molar conductivity measurements. Anticandidal activity of cinnamaldehyde its ligand [L] and Co(II) complex was investigated by determining MIC₈₀, time-kill kinetics, disc diffusion assay and ergosterol extraction and estimation assay. Ligand [L] and Co(II) complex are found to be 4.55 and 21.0 folds more efficient than cinnamaldehyde in a liquid medium. MIC₈₀ of Co(II) complex correlated well with ergosterol inhibition suggesting ergosterol biosynthesis to be the primary site of action. In comparison to fluconazole, the test compounds showed limited toxicity against H9c2 rat cardiac myoblasts. In confocal microscopy propidium iodide (PI) penetrates the yeast cells when treated with MIC of metal complex, indicating a disruption of cell membrane that results in imbibition of dye. TEM analysis of metal complex treated cells exhibited notable alterations or damage to the cell membrane and the cell wall. The structural disorganization within the cell cytoplasm was noted. It was concluded that fungicidal activity of Co(II) complex originated from loss of membrane integrity and a decrease in ergosterol content is only one consequence of this.

Full text of DOI:10.1016/j.cbi.2016.01.015

Accepted Manuscript
Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with
ligand derived from cinnamaldehyde
Dr. Sheikh Shreaz, Rayees A. Shiekh, Vaseem Raja, Waseem A. Wani, Jawad M.
Behbehani
PII:
S0009-2797(16)30015-1
10.1016/j.cbi.2016.01.015
CBI 7577
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 17 May 2015
Revised Date: 29 December 2015
Accepted Date: 20 January 2016
Please cite this article as: S. Shreaz, R.A. Shiekh, V. Raja, W.A. Wani, J.M. Behbehani, Impaired
ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand derived from
cinnamaldehyde, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.01.015.
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Graphical Abstract
Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand
derived from cinnamaldehyde

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Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II)
complex with ligand derived from cinnamaldehyde
Sheikh Shreaza*, Rayees A. Shiekhb, Vaseem Rajac ,
Waseem A. Wanid, Jawad M. Behbehania
aOral Microbiology Laboratory, Faculty of Dentistry, Health Sciences Center,
Kuwait University, P.O. Box 24923, Safat, 13110, Kuwait
bDepartment of Chemistry, Faculty of Science, Taibah University, P.O. Box 30002,
Al Madinah Al Munawarrah, Kingdom of Saudi Arabia
cDepartment of Applied Sciences & Humanities, Jamia Millia Islamia (A Central
University), P.O. Box 110025, New Delhi, India
dInstitute of Bioproduct Development, Universiti Teknologi Malaysia, 81310,
UTM, Skudai, Johor, Malaysia.
*
Corresponding author mailing address: Dr. Sheikh Shreaz, Department of Bioclinical Sciences,
Faculty of Dentistry, Kuwait University, P.O. Box. 24923, Safat, 13110 Kuwait. Tel. no.: (+965)
98017163, Fax no.: (+965) 25326049.
E-mail: sheikh@hsc.edu.kw

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A B S T R A C T
In this study, we have used aldehyde function of cinnamaldehyde to synthesize N, N'-Bis
(cinnamaldehyde) ethylenediimine [C20H20N2] and Co(II) complex of the type
[Co(C40H40N4)Cl2]. The structures of the synthesized compounds were determined on the
basis of physiochemical analysis and spectroscopic data (1H NMR, FTIR, UV-visible and
mass spectra) along with molar conductivity measurements. Anticandidal activity of
cinnamaldehyde its ligand [L] and Co(II) complex was investigated by determining MIC80,
time-kill kinetics, disc diffusion assay and ergosterol extraction and estimation assay.
Ligand [L] and Co(II) complex are found to be 4.55 and 21.0 folds more efficient than
cinnamaldehyde in a liquid medium. MIC80 of Co(II) complex correlated well with
ergosterol inhibition suggesting ergosterol biosynthesis to be the primary site of action. In
comparison to fluconazole, the test compounds showed limited toxicity against H9c2 rat
cardiac myoblasts. In confocal microscopy propidium iodide (PI) penetrates the yeast cells
when treated with MIC of metal complex, indicating a disruption of cell membrane that
results in imbibition of dye. TEM analysis of metal complex treated cells exhibited notable
alterations or damage to the cell membrane and the cell wall. The structural disorganization
within the cell cytoplasm was noted. It was concluded that fungicidal activity of Co(II)
complex originated from loss of membrane integrity and a decrease in ergosterol content is
only one consequence of this.
Key words: Cinnamaldehyde, Candida, Co(II) complex, Ergosterol biosynthesis,
Anticandidal activity.

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1. Introduction
Fungi are neglected pathogens as indicated by the fact that amphotericin B discovered in
1956 is still the gold standard to treat fungal infections. The incidence of infections caused
by Candida species has increased dramatically worldwide due to increase in
immunocompromised patients [1-5]. The majority of commonly used antifungals have
serious drawbacks regarding toxicity to the host tissues, and their continuous deployment in
treating infections has led to the emergence of drug-resistant fungal strains [6,7].
Cinnamaldehyde either in pure form or a crude extract of cinnamon has displayed exciting
antifungal properties [8-10]. Na+/K+ ATPase and Cu2+-ATPase as representatives of P- and
CPx-type of ATPases have been established as possible intracellular targets for the action of
cinnamaldehyde [11]. Importantly, cinnamaldehyde causes damage to cell walls, cell
membranes, cytoplasmic contents and other membranous structures in a fungal cell [12,9].
Its antifungal action has been target specific and has demonstrated activities over a diversity
of sensitive and resistant fungal species [8,11,13,14]. Cinnamaldehyde occurs naturally in
the bark of cinnamon trees and in approved quantities it exhibits lower toxicities to host
cells [8,15].
Macrocycles represent a class of cyclic macromolecules with diverse chemistry, a wide
range of molecular topologies and sets of donor atoms [16]. Macrocyclic ligands and their
coordination complexes are of significant interest as antitumor, antibacterial and antifungal
agents [17,18]. The efficacies of such complexes can be easily optimized by complexing
with various metals ions [19]. Organocobalt complexes of octanedioic acid and nonanedioic
acid have shown high anticandidal activities [20]. Aside from the exciting biology of
macrocycles other interesting classes of chemical compounds are Schiff base ligands and
their complexes. Schiff bases and their complexes have shown significant antifungal
activity towards Candida albicans and other human pathogenic fungi [21]. Cobalt (II)

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complexes of ligands derived from reduced N, N’-o-Phenylenebis (Salicylideneimine) have
been reported as effective against all tested Candida species [22].
Earlier we have reported the synthesis and anticandidal activity of a cinnamaldehyde
derived ligand and its Ni(II) complex [23]. In the present study, Cobalt(II) has been
employed to complex synthesized ligand with an aim to have increased antifungal activity.
The initial antifungal screening was performed by determining minimum inhibitory
concentration (MIC80), disc diffusion assay and time-kill kinetics. Ergosterol biosynthesis is
also an important target pathway of existing antifungals like azoles, as also for the
development of novel antifungals. We have investigated the effect of cinnamaldehyde, its
ligand, and metal complex [CoLCl2] on total ergosterol content of yeast cells. To gain
insight into the mechanism of antifungal action of Co(II) complex, we have carried out
confocal scanning laser microscopy (CSLM) and transmission electron microscopy (TEM)
analysis. Co(II) complex displayed markedly improved anticandidal activity with limited
toxicity against H9c2 rat cardiac myoblast cells.
2. Materials and Methods
Cinnamaldehyde in oil form with 99% purity was obtained from Sigma-Aldrich, USA
(Catalog No. C80687). The solvents were purchased from Merck, Ltd., India and used as
received. The initial stock solution of cinnamaldehyde was 1g/ml, which was then dissolved
in 1% dimethyl sulfoxide (DMSO) (control solvent) to dilute it further for obtaining lower
concentrations as required to carry out different antifungal assays. Glass apparatus with
standard joints were used throughout experimental work and stringent precautions were
taken to exclude moisture. All the syntheses and handling were carried out under an
atmosphere of dry oxygen-free di-nitrogen, using standard Schlenk techniques. To carry out
microanalysis samples were vacuum dried to achieve a constant weight. Perkin Elmer 2400
CHNSO Elemental Analyser was used for elemental analysis. IR and far-IR spectra were

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recorded as KBr pellets and CsI pellets (region 650-100 cm-1), using Perkin Elmer 1620 FT
IR and JASCO FT IR spectrophotometers, respectively. Bruker AVANCE DPX-600
1
spectrometer was used to record H NMR spectra with TMS as internal standard and
DMSO-d6/CDCl3 was used as a solvent. The splitting patterns were shown as: s, singlet; d,
doublet; dd, doublet of doublets; t, triplet and m, multiplet. Values for chemical shifts were
reported in ppm. Bruker (esquire3000_00037) instrument was used to measure positive and
negative ESI mass spectra. Gouy method was used to carry out magnetic susceptibility
measurements. UV-Vis Dual Beam 8 auto cell UVS–2700 LABOMED, INC, US
spectrophotometer was used to record electronic spectra with DMSO as a solvent. Metrex
melting point apparatus was used to record melting points.
2.1. Synthesis of ligand (L)
For the synthesis of ligand, the same procedure was followed as reported by Khalaji and
Weil [24] and the yield obtained was 78%. The synthesis of ligand that involved the
condensation reaction between cinnamaldehyde and ethylene diamine is shown in Scheme
1.
2.2. Synthesis of cobalt(II) complex
The synthesis of the ligand, and its Co(II) complex was carried out according to the steps
shown in Scheme 1. A [2+1] condensation reaction of cinnamaldehyde and
ethylenediamine formed the ligand (L) in excellent yield, which was further cyclised with
cobalt metal chloride by mixing 2:1 ratio of the ligand and corresponding metal chloride.
The obtained complexes are microcrystalline solids that are stable in air, soluble in DMF,
DMSO, sparingly soluble in MeOH, EtOH and DCM but insoluble in water. The analytical
data of the complexes corresponded well with the general formula [M(L)X2] where L =
ligand; M = Co(II) and X = Cl-. The molar conductance values in DMSO (10-3 M) are too

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low to account for any dissociation of the complexes. Therefore, the complexes were
considered to be non-electrolytes.
Yield: 65%; m.p.: > 300oC; UV-Vis (DMSO) nm: 520, 570, 682, 739, 824; IR (KBr, cm-1):
1
2977(C-H), 1420(C-N), 1043, 881, 748; Far IR (CsI, cm-1): 445(Co-N), 330(Co-Cl); H
NMR (300 MHz, δ ppm from TMS in DMSO-d6, 300 k): δ 7.40-7.58(20H, aromatic C-H), δ
3.41-3.87(8H, C=N-C-H2), δ 6.92 (4H, C=C-CH), δ 7.79 (4H, C-N=CH); ESI-MS (m/z):
707 [M]+, 709 [M+2]+; Molar conductance, Λm (ꢀ-1cm-1 mol-1, 10-3 DMSO, r.t.): 34; ꢁeff
(r.t., BM): 4.82; Elem. anal. calcd.: C 68.02, H 5.66, N 7.93%; found: C 68.04, H 5.69, N
7.94%.
2.3. Growth conditions and determination of MIC80
C. glabrata ATCC 90030, C. tropicalis ATCC 750 and C. krusei ATCC 6258 strains
involved in the present study were obtained from Regional Research Laboratory, Jammu
India. Clinical isolates of C. albicans STD number 1128 and C. guilliermondii STD number
1685 were obtained from Institute of Pathology, New Delhi, India, respectively. Species
identification was done by examining microscopic morphology on Corn Meal Agar and Hi
Chrome Agar. Germ tube test, nitrogen assimilation test, and ascospore production on malt-
extract agar was also done. The yeast strains were cultured in Yeast Extract, Peptone and
Dextrose (YEPD) broth as required, maintained on YEPD agar plates at 4ºC and restreaked
after every 4-6 weeks. The culture was initiated with a loop full of cells maintained on
YEPD slants into a 50 ml of appropriate medium (YEPD) and grown at 37ºC in a rotary
shaker (150-170 rpm). For long term storage cultures were stored at -20ºC with 1:1 glycerol
as glycerol stocks.
Minimum inhibitory concentration (MIC) was defined as the lowest concentration of test
entity that causes 80% decrease in absorbance (MIC80) compared with control. This
sensitivity testing procedure was carried out as per NCCLS document M27-A2, 2002 [25],

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in microtitre plates. Cultures were grown with/without test compounds in the media. Stock
solutions of the test compounds and standard drug were prepared in 1% DMSO. The cells
were grown in YEPD medium. The diluted cell suspensions were added to the wells of
round-bottomed 96 well microtitre plates (100µl/well) containing equal volumes of media
(100µl/well) and different concentrations of test compounds. A drug-free control was also
included. The plates were incubated at 35ºC for 24 h. The MIC test end point was also
evaluated both visually and by observing OD595 in a microplate reader (BIO-RAD, i Mark,
US).
2.4. Disc diffusion assay
Strains were inoculated into liquid YEPD medium and grown at 37ºC. Overnight grown
cells were then centrifuged and washed 2-3 times with distilled water. 105 cells/ml were
added to molten agar media at 40ºC and then cells together with media was poured into
100-mm diameter petri plates. 4-mm-diameter sterile filter discs were impregnated with
cinnamaldehyde, its ligand, and Co(II) complex, and then 2 mg/ml concentrations of the test
compounds (dissolved in 1% DMSO) were spotted on the disc in 10 µl volume. The
diameter of the zone of inhibition was recorded in millimeters after 48 h and was compared
with that of control [26]. Sensitivity Index (SI) was calculated for all the five Candida
isolates and was defined as:
Diameter of ZOI (mm)/ concentration of drug (mg/ml) = Clearing (mm/mg).
2.5. Time kill kinetics
Yeast isolates were sub cultured at least twice and grown for 24 h at 35ºC on YEPD plates,
and initial inoculum was adjusted to 4.5×105 c.f.u/ml as described earlier [8]. Final
concentrations of cinnamaldehyde, its ligand, and Co(II) complex were MIC, MIC/2, and
MIC/4 for each test sample. 5 ml final volume cultures were incubated at 35ºC with

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agitation at 200 rpm. At pre-determined time points (0, 2, 4, 8, 12 and 24 h), 100 ꢁl aliquots
were removed and transferred to eppendorf tubes, centrifuged (3,900g at 4ºC for 1 min) and
rinsed twice with 0.9 ml of sterile distilled water to obtain compound free cells. Pellets were
suspended in 100 ꢁl of sterile distilled water and were serially diluted as required. Diluted
culture (20 ꢁl) was spread onto YEPD plates and incubated at 35ºC for 48 h to determine
the numbers of c.f.u/ml.
2.6. Ergosterol extraction and estimation assay
Total intracellular sterols were extracted following the method of Brevik and Owades [27].
A single Candida colony from an overnight YEPD agar plate culture was used to inoculate
50 ml of YEPD broth for control and various concentrations of cinnamaldehyde, its ligand,
and Co(II) complex. The cultures were incubated for 16 h and harvested by centrifugation at
2,700 rpm for 5 min. The wet weight of the cell pellet was determined. 25% alcoholic
potassium hydroxide solution was added to each pellet and vortexed for 1 min. Sterol
extraction was carried out by the 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. After this, 20 µl aliquots
of the sterol extract was diluted fivefold in 100% ethanol and scanned
spectrophotometrically between 240 and 300 nm. The detailed procedure can be obtained
from the method by Brevik and Owades [27]. Ergosterol content is calculated as a
percentage of the wet weight of the cell by the following equations:
% ergosterol + % 24(28) DHE = [(A281.5/ 290) x F]/pellet weight,
% 24(28) DHE = [(A230/518) x F]/pellet weight,
% ergosterol = [% ergosterol + % 24(28) DHE] - % 24(28) DHE,
Where F is the factor for dilution in ethanol and 290 and 518 are the E values (in
percentages per centimeter) determined for crystalline ergosterol and 24(28) DHE,
respectively.

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2.7. Confocal scanning laser microscopy (CSLM)
This microscopic experiment was used to evaluate the effect of Co(II) complex on the
architecture of yeast cells as done earlier [28]. Mid-log phase Candida cells (106 cells/ml)
were incubated without and with MIC of Co(II) complex. The samples were then incubated at
37 ºC for 30 min in the dark. After this, the suspensions were centrifuged, washed and
resuspended in PBS. To determine general cell morphology, one µg/ml of propidium iodide
(PI) was added to the cell suspensions. In case the test agent causes the damage to the cell, PI
present at the surface finds its way inside the cell, showing increased red fluorescence [29].
The cells were examined with Olympus Laser Confocal Scanning Microscope equipped with
green helium neon laser (543 nm) for PI.
2.8. Transmission electron microscopy (TEM)
Co(II) complex (MIC) treated cells (~1×106) were incubated for 12 h at 30 ºC and then fixed
with 2% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 20 ºC [30,31]. Cells were
washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed by 1% OSO4 in 0.1 M phosphate
buffer for 1 h at 4 ºC. For ultrastructure study, the samples were dehydrated with graded
acetone, cleared with toluene and infiltrated with toluene and araldite mixture at room
temperature, and then finally in pure araldite at 50 ºC and embedded in eppendoff tubes (1.5
ml) with pure araldite mixture at 60 ºC. Sectioning was done with Ultramicrotome (Lecia EM
UC6) and then observed under TEM. Other details can be obtained from the method by
Mares and Borgers et al., [30,32].
2.9. MTT cell viability assay
H9c2 rat cardiac myoblasts were cultured and maintained as monolayer in Dulbecco’s
modified Eagle’s medium (DMEM), high glucose, supplemented with 10% foetal bovine
serum (heat inactivated), 100 units/ml penicillin, 100 ꢁg/ml streptomycin, and 2.5 ꢁg/ml
amphotericin B, at 37 °C in humidified incubator with 5% CO2 [33]. For treatments, test

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compound stock solutions were prepared in dimethyl sulfoxide (DMSO) and added to wells
to give the indicated final concentrations. Final DMSO concentration was 0.2% in all wells
including the untreated cells (control) and fluconazole controls. Cells were incubated for 48
h at 37 ºC in 5% CO2 humidified incubator together with the untreated control sample. After
incubation, cells were washed with PBS and incubated with MTT solution for 45 min at 37
ºC. After discarding the supernatant, MTT crystals were dissolved in acid iso-propanol and
the absorbance was measured at 570 nm. Percent viability was defined as the relative
absorbance of treated versus untreated control cells.
2.10. Statistical analysis
The descriptive statistics have been presented as Mean ± SD. The mean values were
compared using t test between control and treated samples (MIC & sub-MIC values), and
one-way analysis of variance (ANOVA) was used to test the significance among the
different test agents. The two tailed probability value (p< 0.001) was considered statistically
significant. The data management and statistical analysis was conducted using Statistical
Package of Social Sciences, IBM SPSS, Version 22.0 (IBM Corp. in Armonk, NY, USA).
3. Results
The physical properties and analytical data of the Co(II) complex supported its
proposed structure. A facile [2+1] condensation reaction of cinnamaldehyde and
ethylenediamine produced ligand in excellent yield (78%). The complex was a
microcrystalline solid and stable in air, soluble in DMF, DMSO, sparingly soluble in
MeOH, EtOH and DCM; but insoluble in water The analytical data of complex
corresponded well with the composition as shown in Scheme 1. The molar conductivity of
Co(II) complex showed low value, which indicated that the complex was a non-electrolyte
1
[34]. The FTIR, H NMR, UV-Visible and ESI-MS results further confirmed the structure
of ligand and its complex. The geometry of the complex was well ascertained by the

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presence of characteristic UV-Visible absorption bands in the region of 520-824 nm and a
magnetic moment of 4.82 BM.
3.1. IR spectra of ligand and complex
IR spectrum of ligand did not exhibit any band corresponding to the free primary diamine
and carbonyl group [35,36]. A broad absorption band in the region of 3300-2800 cm-1
characteristic for hydrogen atom of (H-C=O), and the absence of absorption band in the
region of 1675-1775 cm-1 characteristic for carbonyl group (HC=O) in cinnamaldehyde,
indicated that the oxygen atom of cinnamaldehyde was detached from the HC=O group to
form a bond between aldehydic carbon atom and amino nitrogen of ethylenediamine. It also
suggested complete condensation of reactants and elimination of water molecule. This was
confirmed by the appearance of a strong signal at 1460 cm-1 that might be attributed to the
C=N bond [37].
This coordination behaviour shows how a ligand with multiple donor sites can
coordinate to obtain a simple but stable geometry. The amide groups that are more close to
the metal ion centre maintaining less metal-ligand bond distance, participate in the
complexation. On complexation, the shifting in the band of υ(C=N) (1420) towards the
lower wave number indicated that the coordination took place through the nitrogen of the
υ(C=N) group. This further implied that the ligand is bidentate. The appearance of new
vibrating signals at 445 and 330 cm-1 in the spectrum of metal complex gave clear proof for
the presence of metal-nitrogen (M-N) and metal-chlorine (M-Cl) bonds in Co(II) complex,
respectively [35,38].
3.2. 1HNMR spectra
A multiplet in the range 7.36-7.54 ppm corresponded to the phenyl ring protons (10H).
Another multiplet in the range 3.37-3.81 ppm has been ascribed to methylene protons
(C=N-CH2, 4H). The two sharp signals; one at the pseudo-aromatic region 6.85 ppm (2H,

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C=C-CH) and another at 7.71 ppm (2H, C-N=C-H) have been ascribed to methine protons.
The signal of C-N=C-H has been shifted downfield due to the deshielding effect of the
nitrogen group attached to it. These aromatic and aliphatic proton signals undergo
downfield shifting in Co(II) complex of the ligand due to the paramagnetic effect of Co(II)
ion and hence support the coordination of ligand towards Co(II) ion [39,40].
3.3. Electro spray ionization mass spectra (ESI MS)
The ESI-MS of the ligand and complex were studied in DMSO solution. A negative ion ESI
mass spectrum of ligand confirmed the proposed formula by showing a peak at m/z 289
corresponding to the moiety [(C20H20N2) of atomic mass m/z 288.21. The series of peaks at
m/z 102.9, 192.9, 244.7, etc may be assigned to various fragments. All these suggested the
2+1 condensation of cinnamaldehyde and ethylenediamine, respectively. Similarly negative
ion ESI-MS of Co(II) complex showed a peak at m/z 707 corresponding to the moiety
[(CoC40H40N4Cl2) of atomic mass m/z 706.25] (Fig. 1), which is consistent with the
molecular ion fragment, and it supports the proposed structure of the complex. [M+2]+
fragments were also observed in the metal complex. This may be due to the low quantities
of isotopic chlorine. Molecular ion peak was also associated with water molecules, solvent
and some adduct ions from the mobile phase solution in some cases [41-43].
3.4. Electronic spectra and magnetic measurements of Co (II) complex
The electronic spectrum of Co(II) complex recorded in DMSO supported the geometry
around the respective metal atom. The presence of electronic transition bands at 520 and
4
565 nm; assigned to the transition T1g→4T1g(P), 682 nm; assigned to the transition
4T1g→2T1g, and 739 and 824 nm; assigned to the transition 4T1g→4T2g (Fig. 2) suggested an
octahedral geometry around the Co(II) ion in the complex [44].
The Co(II) complex has an effective magnetic moment value (ꢁeff) of 4.82 BM. This value
is indicative of an octahedral geometry around the Co(II) ion. The magnetic properties of

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4
octahedral Co(II) complexes are governed by the orbitally degenerate ground term T1g.
This provides an orbital contribution to the magnetic moment so that the room temperature
moments are experimentally found to be in the range 4.7-5.2 B.M [41,45,46].
3.5. Minimum inhibitory concentration (MIC80) and disc diffusion assay
Table 1 summarizes the minimum inhibitory concentration (MIC80) values in microgram/ml
(µg/ml) and micromolar (µM) of cinnamaldehyde, ligand and Co(II) complex against five
different Candida species. On the concentration basis, average MIC of cinnamaldehyde
was 3022 µM. The average MIC of ligand and Co(II) complex is 664 and 144 µM,
respectively. Compared to cinnamaldehyde, ligand [L] is 4.55 times more efficient whereas
Co(II) complex is 21.0 times more efficient. Anticandidal MIC of fluconazole varies
between 6.5 µm (2 µg/ml) and 104.5 µM (32 µg/ml). Thus the synthesized complex is less
effective than fluconazole by a factor ranging from 4.5 to 29.
Disc diffusion is one of the standard methods of measuring the antifungal activity of
molecules against different Candida strains grown in agar culture plate. The test compounds
used in this study showed varying zones of clearance at a particular concentration. Discs
impregnated with the vehicle, 1% DMSO (negative control) showed no zone of inhibition.
Our results showed that growth was inhibited by cinnamaldehyde its ligand and Co(II)
complex. The halo was completely clear, which indicated potential fungicidal activity.
Yeasts were found to be more sensitive to Co(II) complex as compared to ligand and
cinnamaldehyde. Diameter of the inhibition zone (mm) by cinnamaldehyde, against C.
glabrata, C. tropicalis, C. krusei, C. albicans and C. guilliermondii at 2 mg/ml
concentration was 5 (± 0.3), 4 (± 0.5), 3 (± 0.2), 5 (± 0.5) and 3 (± 0.3), respectively. The
average of five species being 4.0 mm. For ligand, these values increased to 9 (± 0.4), 7 (±
0.4), 6 (± 0.2), 11 (± 0.3) and 6 (± 0.2), respectively; with an average of 7.8 mm. For Co(II)
complex the values were 12 (± 0.4), 11 (± 0.5), 9 (± 0.5), 14 (± 0.5) and 11 (± 0.4), ),

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respectively; with average being 11.4 mm. Sensitivity index (S.I) was calculated as the ratio
of total clearing by test compounds (in mm) and the total amount of test compound (in mg).
Results were calculated for cinnamaldehyde, its ligand and Co(II) complex for five sensitive
isolates used in this study. Sensitivity index for all five strains was found to be the highest in
the case of Co(II) complex with 5.7 mm/mg followed by ligand [L] with 3.9 mm/mg and
then cinnamaldehyde with 2.0 mm/mg. On the concentration basis, ligand and Co(II)
complex were more effective compared to cinnamaldehyde, but the results obtained implied
that diffusion in solid medium restricts effectiveness of Co(II) complex to an extent.
3.6. Time kill kinetics
Fig. 3 shows the killing activity of Co(II) complex against C. glabrata ATCC 90030, C.
tropicalis ATCC 750, C. krusei ATCC 6258, C. albicans STD 1128 and C. guilliermondii
STD 1685, respectively. At its respective MIC and MIC/2 values the Co(II) complex
showed potential killing activity against all the tested Candida isolates. A significant
decrease in the number of cfu per ml was observed in both standard and clinical
(susceptible) Candida strains. The complete fungicidal endpoint was reached in an average
time of 12-14 h following incubation with MIC of the test agent. No systematic difference
was observed between standard and clinical isolates. Results obtained demonstrated that the
ability to kill Candida species is concentration as well as time dependent. The increase in
compound concentrations leads to a significant decrease in cfu/ml. This decrease in cfu/ml
was also obtained for cinnamaldehyde and ligand used in this study (data not shown). After
24 h incubation and at half MIC of the test compounds the average reduction in c.f.u/ml
with respect to control for all the five Candida isolates was 43% in the presence of
cinnamaldehyde, 59% in the presence of ligand, and 84% in the presence of Co(II) complex
respectively. The aim was to examine the killing activity in presence of Co(II) complex
therefore we have also given the decrease in cfu/ml after 24 h incubation when different

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Candida isolates were grown in MIC and sub-MIC concentrations of Co(II) complex. The p
values were calculated between control and treated samples (MIC & sub-MIC values) for each of
the five strains (Table 2).
3.7. Ergosterol extraction and estimation assay
Ergosterol is an important component of cell membranes of yeasts, which differentiates
them from animal and plant cells. A characteristic four peaked curve is obtained due to
presence of ergosterol and the late sterol intermediate 24 (28) dihydroergosterol (DHE) in
the extracted sample. As the concentration of test molecules increases, suppression of four
peaks is observed. Thus, we can clearly distinguish the inhibitory effect of various
concentrations of test molecules on the sterol profile of Candida strains. Table 3
summarizes the effect of cinnamaldehyde, its ligand, and Co(II) complex on ergosterol
biosynthesis in five Candida isolates. The total ergosterol content was determined for each
isolate grown in varying concentrations of test compounds. A dose-dependent decrease in
ergosterol production was observed when isolates were grown in the presence of
cinnamaldehyde, its ligand, and Co(II) complex. As shown in Table 3, the average decrease
in total cellular ergosterol content for different Candida isolates after exposure to their
respective MIC80 of cinnamaldehyde was 62%. The average decrease in total cellular
ergosterol after exposure to MIC80 of ligand and Co(II) complex was 76 and 88%,
respectively. Respective decrease at MIC80 of fluconazole was 92.73%. From the results, it
is clear that the increase in the concentration of test molecules decreases ergosterol content.
Finally, at MIC value, near absence of ergosterol occurs in the sample. MIC80 values of
Co(II) complex co-relates very well with percent inhibition of sterol biosynthesis. It would
thus appear that major mode of action of the complex is through inhibition of ergosterol
biosynthesis.
3.8. Confocal scanning laser microscopy (CSLM)

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It is a technique used for obtaining high-resolution optical images with depth selectivity.
Studies were performed to validate the discrimination of live and dead Candida albicans
STD 1128 cells due to MIC of Co(II) complex in the presence of PI. PI is used as a DNA
stain for both flow cytometry and microscopy to evaluate cell viability, and to visualize the
nucleus and other DNA containing organelles. It is membrane impermeant and generally
excluded from viable cells. Therefore, it is used for identifying dead cells in a population. It
can be used to differentiate necrotic, apoptotic and normal cells. Cells with severe
membrane lesions leading to the inherent loss of viability will internalize PI, resulting in an
increase in red fluorescence [29]. The laser confocal images of stained C. albicans STD
1128 cells exposed to MIC value of Co(II) complex are shown in Fig 4. The left panel
shows the laser confocal images of stained Candida cells without any treatment, and both
the images in the right panel show stained cells exposed to MIC of Co(II) complex,
respectively. The right panel shows disruption in cell membranes of cells treated with Co(II)
complex, which causes the imbibition of dye. More the damage to cell population more is
the number of cells which turn red. Our results confirmed that PI penetrates in Co(II)
complex treated cells implying that the structure of cell membrane was disrupted by the test
agent. It was worth noticing that in the case of treated cells entire yeast cell appeared red
(maximum entry of the dye inside the cell), which confirmed both cell wall and plasma
membrane damage. These results formed firm basis to carry out further microscopic
analysis of the treated cells by employing transmission electron microscopy.
3.9. Transmission electron microscopy (TEM)
Transmission electron microscopy was used to study the ultrastructural changes caused by
Co(II) complex in C. albicans STD 1128 cells after 12 h exposure. Fig 5. A, at a direct
magnification of 25000 X shows (untreated) cells of C. albicans STD 1128. Control cells
showed the typical normal morphology of Candida with a uniform central density and

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endomembrane system enveloped by a regular, intact cell wall and membrane. Fig. 5 B
shows the Co(II) complex (MIC) treated cells at 20000 X magnification. The treated cells
exhibited notable alterations or damage in the cell membrane and cell wall. The structural
disorganization within the cell cytoplasm and leakage of intracellular content has also been
noted (Fig. 5 B).
3.10. Toxicity profile
To evaluate the toxicity of synthesized compounds they were tested against H9c2 rat
cardiac myoblasts. Sub-confluent populations of H9c2 cells were treated with increasing
concentrations of these compounds, and the number of viable cells was measured after 48 h
by MTT cell viability assay. Table 4 shows that cinnamaldehyde and its ligand showed
almost 100% viability at the concentration range of 6.25 µg/ml, while as at the same
concentration, Co(II) complex and reference drug fluconazole showed 98% and 90%
viability, respectively. It was observed that 100 µg/ml of cinnamaldehyde, its ligand, and
Co(II) complex offered a remarkable viability of 94, 90 and 84%, respectively. At 200
µg/ml concentration, cinnamaldehyde, ligand, and Co(II) complex showed 8, 11 and 20%
toxicity, respectively. Toxicity of reference drug fluconazole at 200 µg/ml was 55%.
4. Discussion
Schiff base ligands and their transition metal complexes with macrocyclic
architectures are being preferred for the development of drugs and drug candidates with
exciting biological properties [47]. The ligand reported in the present study was prepared by
a facile condensation involving the two primary amine groups of one ethylenediamine
molecule and two aldehydic functional groups of two cinnamaldehyde molecules. The
potentially bidentate ligand coordinated with Co(II) ions and formed a 1:2 metal to ligand
complex with octahedral geometry. The structure of the complex was fully ascertained from
1
the microanalysis, UV-Vis, FTIR, H NMR, and ESI-MS spectral date. The low molar

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conductance value of the complex in DMSO indicated its non-electrolytic nature, and thus it
might be inferred that the chlorido ligands do not dissociate in the solution phase. The two
chlorido ligands, therefore, satisfy both primary and secondary valencies of the Co(II) ion in
this complex [48]. The absence of bands due to free primary amine and carbonyl groups in
the FTIR spectrum of ligand, and the presence of bands due to C=N bond confirmed the
condensation of cinnamaldehyde and ethylenediamine to form ligand. The coordination of
ligand with Co(II) ion was ascertained by the shifting of υ(C=N) band towards the lower
wave number. Besides, the appearance of vibrating frequencies of Co-N and Co-Cl further
confirmed the complex formation. 1H NMR spectral data was highly useful for the structure
determination. The characteristic signals for phenyl ring, methylene, pseudo-aromatic and
methine protons established the formation of ligand. The metal coordination of the ligand
was ensured due to the downfield shifting of the signals of aromatic and aliphatic protons in
the 1H NMR spectrum of Co(II) complex. ESI-MS data further revealed the composition of
the ligand and complex. The ESI-MS spectrum of the Co(II) complex (Fig. 1) confirmed the
molecular composition of the complex as, [(CoC40H40N4Cl2) with molecular mass m/z
706.25. Finally, the geometry of the complex was ascertained from the results of electronic
spectral and the magnetic moment data of the Co(II) complex. The presence of electronic
4
4
4
transitions such as T1g→4T1g(P), T1g→2T1g and T1g→4T2g (Fig. 2) in the electronic
spectrum of complex suggested an octahedral geometry around the cobalt(II) ion in the
complex, which was further confirmed from the magnetic moment value of the complex
(4.82 BM).
Drug resistance has become an important problem in a variety of infectious
diseases, including human immunodeficiency virus (HIV) infection, tuberculosis, and the
majority of fungal infections [49]. The rise in the incidence of fungal infections has
exacerbated the need for the next generation of antifungal agents since many of the

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currently available antifungal drugs like polyenes and azoles have undesirable side effects,
are ineffective against new or re-emerging fungi, or lead to the rapid development of
resistance [49-52]. Alternative forms of treatment may also address the growing problems
of drug resistance in microbes. In this study, cobalt(II) has been employed to complex
cinnamaldehyde based ligand with an aim to have increased antifungal activity. On the basis
of spectroscopic analysis, it appears that ligand is formed through condensation of two
molecules of cinnamaldehyde and one molecule of ethylene diamine. Cobalt(II) coordinated
with four nitrogen atoms of two ligands as shown in Scheme 1.
Cinnamic aldehyde has antifungal properties against fungi involved in respiratory
tract mycoses [53]. The precise mechanism by which cinnamaldehyde exerts its
antimicrobial action is not clear. Being lipophilic, it permeates the membrane and hence,
becomes accessible to various intracellular sites [54]. Antifungal effect of cinnamaldehyde
may also originate from inhibition of fungal cell wall synthesizing enzymes, beta-(1,3)-
glucan synthase and chitin synthase [55]. Structural component of cinnamaldehyde, which
may contribute to its antimicrobial effects are aromatic ring, hydrocarbon chain outside the
ring and aldehyde function. Most of the irritant effects of cinnamaldehyde especially at
higher concentrations are attributed to aldehyde function [56]. Skin sensitization of
cinnamaldehyde is probably initiated by the reaction of cinnamaldehyde with ε-amino
groups on protein side chains. We used aldehyde function of cinnamaldehyde to form a
ligand employing ethylenediamine (Scheme 1). The ligand formation led to increase in
lipophilicity and extension of the hydrocarbon chain outside the ring. The ligand with
structure twice that of cinnamaldehyde was further complexed employing cobalt as a central
metal ion with no aldehyde function, and thus complex structure four times that of
cinnamaldehyde was obtained. Chelation reduces the polarity of the metal ion because of
partial sharing of its positive charge with the donor group within the whole chelate ring

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system [57]. This process of chelation thus, increases the lipophilic nature of central metal
ion, which in turn, favours its permeation through the lipid layer of membrane thus, causing
the metal complex to cross the membrane more effectively.
Initial screening for antifungal activity of cinnamaldehyde, its ligand, and Co(II)
complex was carried out by evaluating MIC80 against five Candida species of the
fluconazole-sensitive category. MIC80 results obtained in this study showed that the test
compounds exhibited varying degrees of antifungal activity. The use of total mean MICs
obtained gave a good indication of the overall antimicrobial effectiveness of each test
compound. MIC values as determined by NCCLS method decreased almost
stoichiometrically with the multiplication of structure for all five species of Candida.
Average MIC of five species for cinnamaldehyde is 3022 µM. Anticandidal MIC decreased
tremendously for ligand as 664 µM, and complex as 144 µM, representing an increase in
effectivity by 4.55 times and 21 times compared to cinnamaldehyde. Anticandidal MIC of
fluconazole varies between 6.5 µm (2 µg/ml) and 104.5 µM (32 µg/ml), thus the
synthesized complex could be less effective as compared to fluconazole by a factor ranging
from 4.5 to 29. Results obtained in time-kill assays demonstrated that the increase in the
concentration of test compounds leads to significant killing activity. Full fungicidal
endpoint was reached in an average time of 12-14 h following incubation with MIC of
cinnamaldehyde, its ligand and Co(II) complex (Fig. 3). No systematic difference was
observed between standard and clinical isolates. The average reduction in c.f.u/ml with
respect to control was maximum in the presence of Co(II) complex (84%) followed by
ligand (59%) and cinnamaldehyde (43%). Effectiveness of test molecules was evaluated on
solid media to check whether the different state of media alters their antifungal approach.
This was evaluated by disc diffusion assay in which filter paper disc impregnated with
various concentrations of test molecules were put on agar plates. In the case of disc

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diffusion, sensitivity index was calculated on the basis of zone of inhibition. Like MIC
studies in liquid media, no systematic or significant difference was seen between various
strains for all the three tested molecules. Accordingly, sensitivity index was calculated for
all five tested isolates. Cinnamaldehyde showed the mean sensitivity index of 2.0 mm/mg;
ligand showed 3.9 mm/mg and Co(II) complex showed 5.7 mm/mg of clearance. Therefore,
Co(II) complex showed 2.85 folds and ligand showed 1.95 folds sensitivity index as
compared to cinnamaldehyde. In comparison to the liquid medium, the anticandidal effect
of synthesized compounds on solid medium showed that diffusion could be a constraint for
the complex.
Ergosterol is important constituent of yeast cell membrane that distinguishes them
from host cell membranes. Ergosterol biosynthesis is thus an important target pathway of
existing antifungals like azoles, as also for the development of new antifungals. Conditions
that affect membrane also play a role in regulating ergosterol biosynthesis. Decreased
biosynthesis of ergosterol leads to decreased viability of yeasts. Effect of promising
antifungals can, therefore, be explained by estimating ergosterol content in their presence.
Present study, therefore, explored the possible target sites for the antifungal actions of
cinnamaldehyde, its ligand, and Co(II) complex. Earlier studies have shown that a major
site of action of cinnamaldehyde is cell membrane and ergosterol biosynthesis [9]. One
membrane parameter viz. ergosterol content was measured in this study. Inhibition of
ergosterol biosynthesis correlates well with MIC80 values. Ergosterol reduction of 62, 76
and 88% was estimated in the presence of MIC80 of cinnamaldehyde, ligand, and Co(II)
complex. This co-relation suggests that ergosterol is the primary site of action of
cinnamaldehyde, its ligand, and Co(II) complex. Although cinnamaldehyde acts by
disrupting the microbial cell membranes, however, specific mechanisms involved in its
mode of antifungal action remain poorly characterized. Ergosterol is unique to fungi, and

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the potential fungicidal effect of the compounds used in this study hints affinity for this
specific target, and hence its biosynthesis pathway. In this study, an attempt has been made
to compare the antifungal potential of cinnamaldehyde, its ligand, and Co(II) complex by
performing some initial screening experiments and then studying the effect on the
morphological aspects of membrane integrity and sterol biosynthesis. In case of Co (II)
complex, ergosterol content co-related well with MIC80 suggesting that inhibition of
ergosterol biosynthesis could be a leading/primary cause of antifungal action. With these
studies, it was not possible to figure out whether Co(II) complex causes direct inhibition of
ergosterol biosynthesis or decrease in ergosterol is a consequence of disruption of
membrane integrity. Co-relation of MIC80 with ergosterol decrease was found in case of
Co(II) complex but was far off in case of cinnamaldehyde. If the mechanism of action of
cinnamaldehyde, its ligand, and Co(II) complex is taken as same, it would indicate that
killing originates from loss of membrane integrity, and decrease in ergosterol content is
only one consequence of this. Other factors appear to have an equal contribution in
cinnamaldehyde promoted killing.
To demonstrate the effect of Co(II) complex on yeast cells, microscopy studies
were carried out against cells exposed to MIC concentration of Co(II) complex. It was
observed that treated cells showed less viability as indicated by increased absorption of
propidium iodide visualized by confocal scanning electron microscopy. The effect on
membrane integrity, thus, appears to originate from inhibition of ergosterol biosynthesis as
visible from confocal microscopy results (Fig. 4). TEM studies with Co(II) complex
showed disruption of outer morphology and internal cellular structure to a greater extent.
Cell wall breakage and leakage of intracellular content could be seen from TEM
micrographs (Fig. 5). Hence, it can be concluded that fungicidal activity of Co(II) originates

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from loss of membrane integrity, and a decrease in ergosterol content is only one
consequence of this.
5. Conclusion
In summary, Schiff base ligand was synthesized from cinnamaldehyde in a good
yield via facile condensation reaction. The synthesized ligand coordinated with Co(II) ions
in a bidentate fashion. The basic approach behind the synthesis of ligand and its complex
was to increase their size and so the lipophilicity. Ligand and Co(II) complex attained
structures two times and four times, respectively the size of cinnamaldehyde. Both the
compounds were markedly active against different Candida strains in liquid as well as solid
media. Interestingly, the compounds also showed insignificant toxicity at MIC80. The
mechanistic studies revealed ergosterol biosynthesis as the primary site of action of Co(II)
complex. Overall, the excellence of the anticandidal properties in addition to the
insignificant toxicity of Co(II) complex; demands more insightful studies into all its
possible mechanisms of action.
Acknowledgements
The authors are thankful to Research Sector, Office of the Vice President for Research,
Kuwait University, Kuwait for providing Postdoctoral Research Fellowship to Dr. Sheikh
Shreaz (SRUL01/14). Authors are also thankful for the laboratory research facilities
provided by the Office of the Vice President for Research, Kuwait University (Project No.
DRO4/14), and Kuwait Foundation for the Advancement of Sciences (Project No: 2011-
5502-01).
Conflict of Interest
The authors declare no conflict of interest.

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