The inhibitory effect of the amino acid complexes of Zn(II) on the growth of Aspergillus flavus and aflatoxin B1 production

Article abstract of DOI:10.1007/s13738-018-01581-3

The zinc(II) complexes with amino acids as the ligands, Zn(Gln)₂ (1), [Zn(Arg)₂(OAc)]OAc·3H₂O (2), Zn(His)₂ (3), Zn(Gly)₂ (4), Zn(Met)₂ (5), and Zn(Cys)₂ (6) (Gln = glutamine, Arg = arginine, His = histidine, Gly = glycine, Met = methionine, and Cys = cysteine) have been synthesized in aqueous solutions and characterized by elemental analysis and spectroscopic methods. In addition, the solid-state structure of 1 and 2 has been determined by single-crystal X-ray crystallography. X-ray analysis revealed that the central Zn(II) atom is six- and five- coordinated in 1 and 2, respectively. Furthermore, the antifungal activity of the synthesized complexes was investigated against the Aspergillus flavus fungus. The aqueous solutions of the zinc(II) amino acid complexes at various concentrations were added to the potato dextrose agar (PDA) medium containing spores of Aspergillus flavus, and the mixtures were then incubated at 25–30?°C for 6 days. The aflatoxin B₁ produced in vitro was measured using enzyme-linked immunosorbent assay (ELISA). The increasing concentration of these complexes decreased the growth of Aspergillus flavus and aflatoxin B₁ production, consequently. Furthermore, the results showed that the synthesized Zn(II) amino acid complexes are more antifungal active than the zinc(II) ion.

Full text of DOI:10.1007/s13738-018-01581-3

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-01581-3
ORIGINAL PAPER
The inhibitory effect of the amino acid complexes of Zn(II)
on the growth of Aspergillus flavus and aflatoxin B1 production
Marzieh Daryanavard1 · Nafiseh Jarrah2
Received: 6 June 2018 / Accepted: 24 December 2018
© Iranian Chemical Society 2019
Abstract
The zinc(II) complexes with amino acids as the ligands, Zn(Gln)2 (1), [Zn(Arg)2(OAc)]OAc·3H2O (2), Zn(His)2 (3), Zn(Gly)2
(4), Zn(Met)2 (5), and Zn(Cys)2 (6) (Gln=glutamine, Arg=arginine, His=histidine, Gly=glycine, Met=methionine, and
Cys = cysteine) have been synthesized in aqueous solutions and characterized by elemental analysis and spectroscopic
methods. In addition, the solid-state structure of 1 and 2 has been determined by single-crystal X-ray crystallography. X-ray
analysis revealed that the central Zn(II) atom is six- and five- coordinated in 1 and 2, respectively. Furthermore, the antifungal
activity of the synthesized complexes was investigated against the Aspergillus flavus fungus. The aqueous solutions of the
zinc(II) amino acid complexes at various concentrations were added to the potato dextrose agar (PDA) medium contain-
ing spores of Aspergillus flavus, and the mixtures were then incubated at 25–30 °C for 6 days. The aflatoxin B1 produced
in vitro was measured using enzyme-linked immunosorbent assay (ELISA). The increasing concentration of these complexes
decreased the growth of Aspergillus flavus and aflatoxin B1 production, consequently. Furthermore, the results showed that
the synthesized Zn(II) amino acid complexes are more antifungal active than the zinc(II) ion.
Keywords Zn(II) complexes · Amino acids · Aspergillus flavus · Aflatoxin · Potato dextrose agar (PDA) · Antifungal
activity
Introduction
Mycotoxins are the toxic secondary chemical metabolites,
which are produced by the filamentous fungi. Mycotoxins do
not belong to a single class of the chemical compounds, and
they have different toxicological effects [10, 11]. Among the
various dietary approaches to reduce the effects of mycotox-
ins, the addition of mycotoxin binders to contaminated diets
has drawn much attention from researchers [12]. Therefore,
many reviews about the mycotoxin binders have been pub-
lished [13–15]. The binder significantly prevents the animal
toxicity, so that counteracts mycotoxins in the feedstuff by
binding them strongly enough to avoid the toxic interactions
with the consuming animal.
The transition metals complexes have been widely studied
for their antimicrobial and antifungal properties [1, 2]. They
have also attracted considerable attention in modern medi-
cine [3–5]. A literature survey indicates that the zinc(II)
complexes of aminobenzolamide derivatives have shown
notable antifungal activities [6]. Other antifungal studies
have been done on the amino acid-derived compounds [7],
zinc(II) carboxylates [8], and zinc complexes of sulfadrugs
such as sulfadiazines and sulfamerazines [9].
Aflatoxins are the highly toxic secondary metabolites
and the main naturally occurring mycotoxins in agricultural
products, which are produced by a complex biosynthetic
pathway [16]. First time, they discovered and characterized
in 1960s, when more than 100,000 turkey poults in Eng-
land died of evident poisoning from contaminated peanut
meal [17, 18]. Aflatoxins are produced by several species
of Aspergillus genus, especially Aspergillus flavus, Asper-
gillus parasiticus, and Aspergillus nomius [19, 20]. They
contaminate many commodities used for human food and
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-018-01581-3) contains
supplementary material, which is available to authorized users.
* Marzieh Daryanavard
m.daryanavard@ch.iut.ac.ir
1
Department of Chemistry, Estahban Higher Education
Center, Estahban 74519-44655, Iran
2
Department of Chemistry, Isfahan University of Technology,
Isfahan 84156-83111, Iran
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
animal feed, which leads to serious economic and health
problems worldwide. There are four most common aflatox-
ins including B1, B2, G1, and G2, which have been named
based on their fluorescence color under ultraviolet light (blue
or green) and their relative mobility on a thin-layer chroma-
tographic plate. In addition, there are less common aflatoxins
including M1, B2a, G2a, P1, and Q1, which have been defined
as the mammalian biotransformation products of the main
metabolites [21]. Aflatoxin B1 (Fig. 1) is the most potent
liver toxin [22], which is classified by the International
Agency of Research on Cancer as the Group 1 carcinogen
[23]. Aflatoxin B1 exhibits an intense blue fluorescence
visible at 450 nm, when excites with the long-wavelength
ultraviolet light (365 nm) as the excitation wavelength [24].
Aspergillus flavus produces only B aflatoxins, while Asper-
gillus parasiticus and Aspergillus nomius produce both B
and G aflatoxins [25]. The fungi are more difficult to quan-
tify than bacteria, because they usually grow slowly and
often in multicellular forms. Therefore, the complicated
experiments are required to evaluate the in vitro or in vivo
properties of a promising antifungal agent [26].
the commercial availability of ELISA test kits for most
of the mycotoxins, ELISA is used for the routine analysis
of mycotoxins in food and feed [34].
Herein, we report the synthesis and characterization of
the Zn(II) amino acid complexes including Zn(Gln)2 (1),
[Zn(Arg)2(OAc)]OAc∙3H2O (2), Zn(His)2 (3), Zn(Gly)2 (4),
Zn(Met)2 (5), and Zn(Cys)2 (6) (Gln=glutamine, Arg=argi-
nine, His=histidine, Gly=glycine, Met=methionine, and
Cys=cysteine). Furthermore, the antifungal activity of these
complexes has been studied on growth of the Aspergillus
flavus in PDA culture medium along with determination of
amount of aflatoxin B1 produced, using the ELISA method.
Experimental
Materials and methods
All chemicals and solvents were commercially available in
analytical grade and were used without further purification.
Pure Aspergillus flavus was received from the Department
of Microbiology, Isfahan University of Technology. Zinc(II)
acetate dihydrate, Zn(OAc)2·2H2O, amino acid ligands, and
PDA culture medium were purchased from Merck Com-
pany. ELISA kits Ridascreen aflatoxin B1 (R-Biopharm
AG, Germany) were used according to the manufacturer’s
instructions. Double-distilled water was used in all experi-
ments. Elemental analyses were performed using a Leco
CHNS-932 elemental analyzer. Infrared spectra were taken
in the 4000–400 cm−1 region as KBr pellets using an FT-IR
JASCO 680-PLUS spectrophotometer. Electronic absorption
spectra were recorded on a JASCO 7580 UV–Vis double-
beam spectrophotometer. The cultivation processes were
performed in an IKA KS 4000i control shaking incubator.
1H NMR spectra were recorded on a Bruker-400 MHz spec-
trometer at room temperature in D2O.
The different countries have developed the regulations
for aflatoxins in food and feed materials because of the
negative effect of aflatoxins on human health. It has been
reported that the maximum admissible levels of aflatox-
ins in food are 10 and 5 µg kg−1 for total aflatoxins and
aflatoxin B1, respectively, in more than 75 countries [11].
Therefore, several qualitative and quantitative analytical
methods for aflatoxin have been extensively developed,
which include thin-layer chromatography (TLC) [27],
high-performance liquid chromatography (HPLC) [28],
enzyme-linked immunosorbent assay (ELISA) [29],
immunoaffinity with fluorescence [30], and liquid chro-
matography (LC) coupled to tandem mass spectrometry
(LC–MS/MS) [31]. As compared to the TLC and HPLC
techniques, ELISA method can be used for screening
procedures due to its advantages, including high speed,
high sensitivity, ease of operation, low-sample-volume
requirement, and high-sample throughput with minimum
sample clean-up [32]. ELISA is a well-known immuno-
assay method, which appeared in 1960 [33]. Because of
The white single crystals of (1) and (2) were chosen for
the single-crystal X-ray diffraction study. Data were col-
lected on a Bruker APEX II CCD diffractometer using
graphite-monochromated Mo-Kα radiation (λ=0.71073 Å)
at 296(2) K. Data collection, cell refinement, data reduc-
tion, and absorption correction were performed using mul-
tiscan method with Bruker software [35]. The structures
were solved by direct method using SIR2004 [36]. The
non-hydrogen atoms were refined anisotropically by the
full-matrix least-squares method on F2 using SHELXL [37].
All the hydrogen atoms of the ligands were placed at the
calculated positions and constrained to ride on their parent
atoms. Details of the data collection and refinement param-
eters for Zn(Gln)2 are reported in Table 1, and the selected
bond lengths and angles of its molecular structure are listed
in Table 2. Crystal data of Zn(Gln)2 and [Zn(Arg)2(OAc)]
Fig. 1 Molecular structure of aflatoxin B1
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Table 1 Crystallographic data and structure refinement for Zn(Gln)2
General procedure for synthesis of zinc(II) amino
acid complexes
Chemical formula
C10H18N4O6Zn
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
355.65
296 (2)
Zn(OAc)2·2H2O (438.9 mg, 2 mmol) was dissolved in
5 mL of water, and the obtained colorless solution was
added slowly to an aqueous solution of amino acid ligand
(4 mmol of Gln, Arg, His, Gly, Met, or Cys). The reac-
tion mixture was then vigorously stirred and heated at
40–50 °C for 2 h. The solution was evaporated at room
temperature to dryness. The white precipitate of Zn(II)
amino acid complex was then washed with acetone and
dried at room temperature under vacuum. The complexes
(1–6) were recrystallized by slow diffusion of ethanol into
the saturated aqueous solutions of each complex. After 3
weeks at room temperature, single crystals of Zn(Gln)2
(1) and [Zn(Arg)2(OAc)]OAc∙3H2O (2), suitable for X-ray
crystallography, were formed in a yield of 81% and 87%,
respectively. In addition, the crystals of other Zn(II) amino
acid complexes were separated and washed with ethanol
and vacuum dried. However, they were unsuitable for
X-ray crystallography.
Monoclinic
P21
9.4226 (4)
5.0517 (2)
14.5028 (6)
90.00
105.253 (2)
90.00
666.02 (5)
2
1.773
0.13×0.07×0.03
368
2.24–25.00
9721
2321 (R(int)=0.0348)
2321/2/202
1.042
β (°)
γ (°)
V (Å3)
Z
Calculated density (Mg m−3
Crystal size (mm)
F(000)
)
θ range (°)
Reflections collected
Independent reflections (Rint)
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices
Zn(Gln)2: Anal. calc. for C10H18ZnN4O6
(355.66 g mol−1): C, 33.96; H, 4.52; N, 15.83. Found: C,
33.83; H, 4.49; N, 15.97%. UV–Vis (H2O) λmax/nm (ε/
M−1 cm−1): 203 (1567). IR (KBr pellet), υ/cm−1: 3408, 3213
(N–H); 1686 (C=O); 1667 (COO); 1162 (C–N).
R1 =0.0257, wR2 =0.0538
R1 =0.0317, wR2 =0.0700
−9/9, −20/20, −13/13
R indices (all data)
Range of h, k, l
[Zn(Arg)2(OAc)]OAc·3H2O: Anal. Calc. C16H40ZnN8O11
(585.93 g mol−1): C, 32.79; H, 6.82; N, 19.11. Found: C,
33.08; H, 6.86; N, 19.31%. UV–Vis (H2O) λmax/nm (ε/
M−1 cm−1): 209 (2350). IR (KBr pellet), υ/cm−1: 3346
(O–H); 3273 (N–H); 1677 (C=O); 1578 (COO).
Table 2 Selected bond lengths (Å) and bond angles (°) for Zn(Gln)2
Bond lengths (Å)
Zn(1)–O(1)
Zn(1)–O(3)
Zn(1)–N(1)
Zn(1)–N(2)
Zn(1)–O(4)
Zn(1)–O(6)
2.0520 (2)
2.0444 (2)
2.0520 (3)
2.0800 (2)
2.5210 (2)
2.3036 (2)
Zn(His)2: Anal. calc. for C12H16ZnN6O4 (373.68 g mol−1):
C, 38.77; H, 3.76; N, 22.60. Found: C, 38.61; H, 3.78; N,
22.76%. UV–Vis (H2O) λmax/nm (ε/M−1 cm−1): 226 (4830).
IR (KBr pellet), υ/cm−1: 3393 (N–H); 3134 (C–H); 1650
1
(C=N); 1550 (C=C). H NMR (D2O, δ (ppm)): 7.947 (s,
2H), 7.060 (s, 2H), 3.903 (t, 2H), 3.204–3.079 (dd, 4H).
Zn(Gly)2: Anal. Calc. for C4H8ZnN2O4 (213.51 g mol−1):
C, 22.71; H, 2.83; N, 13.24. Found: C, 22.90; H, 2.88; N,
13.31%. UV–Vis (H2O) λmax/nm (ε/M−1 cm−1): 196 (1223).
IR (KBr pellet), υ/cm−1: 3262 (N–H); 1654 (C=O); 1137
(C–N). 1H NMR (D2O, δ (ppm)): 3.404 (s, 4H).
Bond angles (°)
O(1)–Zn(1)–O(3)
N(1)–Zn(1)–N(2)
O(1)–Zn(1)–N(1)
O(3)–Zn(1)–N(2)
O(3)–Zn(1)–N(1)
O(1)–Zn(1)–N(2)
O(3)–Zn(1)–O(6)
O(1)–Zn(1)–O(6)
N(1)–Zn(1)–O(6)
N(2)–Zn(1)–O(6)
176.98 (9)
169.80 (11)
82.18 (9)
81.02 (9)
97.50 (9)
98.76 (9)
90.67 (8)
92.34 (7)
91.85 (9)
98.25 (8)
Zn(Met)2: Anal. calc. for C10H20ZnN2O4S2
(361.77 g mol−1): C, 33.38; H, 5.00; N, 7.78. Found: C,
33.46; H, 4.97; N, 7.69%. UV–Vis (DMF) λmax/nm (ε/
M−1 cm−1): 271 (1010). IR (KBr pellet), υ/cm−1: 3245
1
(N–H); 1607 (COO); 697 (C–S–C). H NMR (D2O, δ
(ppm)): 3.740 (s, 2H), 2.617–2.573 (dt, 4H), 2.200–2.014
(m, 4H), 2.080(s, 6H).
Zn(Cys)2: Anal. calc. for C6H10ZnN2O4S2
(305.69 g mol−1): C, 23.73; H, 3.29; N, 9.22. Found: C,
23.89; H, 3.33; N, 9.30%. UV–Vis (DMF) λmax/nm (ε/
M−1 cm−1): 275 (230). IR (KBr pellet), υ/cm−1: 3411, 3080
OAc∙3H2O have been deposited at the Cambridge Crystal-
lographic Data Center (CCDC nos. 1556782 and 1558139,
respectively).
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Journal of the Iranian Chemical Society
1
(N–H); 1629 (COO). H NMR (D2O, δ (ppm)): 3.864 (t,
treated with the inhibitors to calculate the normal growth of
2H), 2.693–2.576 (dd, 4H).
Aspergillus flavus fungus.
In vitro antifungal activity and minimum inhibition
concentration (MIC) of the zinc(II) complexes
Results and discussion
Antifungal activity of the Zn(II) ion as Zn(OAc)2∙2H2O and
its complexes with amino acid ligands was tested against the
Aspergillus flavus. The lowest concentration that prevents
the growth of fungus is considered as minimum inhibitory
concentration (MIC). The MIC of the compounds was evalu-
ated against the Aspergillus flavus using a dilution method.
In this method, the solutions of Zn(II) compounds at dif-
ferent concentrations (5–50 µg mL−1) were prepared in the
sterile test tubes by the serial dilution method. The culture
medium of PDA was prepared according to the manufac-
turer’s guideline. PDA (39 g) was dissolved in 1 L of water,
and the obtained solution was then autoclaved at 120 °C for
15 min. 20 mL of PDA solution was added to each petri dish.
The homogeneous suspension of Aspergillus flavus fungus
(1 mL), and the solutions of Zn(II) complexes (10 mL) were
also added immediately to each petri dish. The plates were
incubated at 25–30 °C for 6 days. Four replicates for each
concentration of the complexes were investigated simultane-
ously. Blank assays were done for cultures, which were not
Synthesis of the zinc(II) amino acid complexes
All zinc(II) amino acid complexes were synthesized in
good yields through the reaction of Zn(OAc)2·2H2O with
the amino acid ligands (1:2 mol ratio) in the aqueous solu-
tions (Scheme 1). The acetate anion can act as a weak base
and remove a proton from the free neutral amino acids.
The synthesized Zn(II) amino acid complexes were stable
in the aqueous solutions for 48 h and no precipitation was
observed even after long storage at room temperature, which
indicates the stability of these complexes. In biology, the
essential metal ions such as Zn(II) most frequently bind to
donor ligands according to the hard–soft acid–base theory
(HSAB). The overall guidelines for the coordination of
Zn(II) ion in proteins have been described in the literatures
[38, 39]. According to the Pearson’s principle of HSAB,
Zn(II) is a borderline metal, so it can bind well to both hard
(nitrogen and oxygen) and soft (sulfur) donor atoms. As can
be seen in Scheme 1, the amino acid ligands are coordinated
Scheme 1 General synthesis route and molecular structures of the Zn(II) amino acid complexes
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Journal of the Iranian Chemical Society
to Zn(II) ion as N,O-chelating agents in the synthesized
complexes.
region around 195–280 nm due to ligand-centered n → π* or
π → π* transitions of amino acid ligands, which is slightly
shifted compared to the free amino acid ligands.
IR and UV–Vis spectroscopic studies
Crystal structure description of the zinc(II) amino
acid complexes
The primary information about the zinc ion coordination
was obtained by comparison of the IR frequencies of the
uncoordinated ligands and the zinc amino acid complexes.
The amino acids are as zwitterions in the crystalline state.
The predominant vibrations for the free amino acid ligands
X-ray quality crystals of Zn(Gln)2 and [Zn(Arg)2(OAc)]
OAc·3H2O complexes were obtained by slow diffusion of
ethanol into a saturated aqueous solution of each complex,
while efforts to obtain the suitable single crystals of other
complexes were unsuccessful. A white single crystal of
Zn(Gln)2 with dimensions of 0.13×0.07×0.03 mm was cho-
sen for the single-crystal X-ray diffraction study. An ORTEP
view of Zn(Gln)2 is shown in Fig. 2, and the selected bond
lengths and angles are listed in Table 2. As shown in Fig. 3,
its structure consists of the [Zn(Gln)2] units, which lie in the
crystallographic a–c planes. In addition, the individual units
are linked by long-range Zn–O contacts. Figure 3 shows that
each Zn(II) ion is surrounded by four glutamine moieties,
while each glutamine ligand is connected to two zinc atoms.
The geometry around each Zn(II) ion is roughly octahedral
with four short bonds and two long bonds, a configuration
similar to that found for hexacoordinated copper complexes
[45, 46]. Li and co-workers have also recently reported the
same crystal structure for the [Zn(Gln)2] complex with a
little difference in the unit cell parameters [47].
+
are related to ν(COO¯), ν(NH3 ), and ν(CCN). The amino
acids coordinate to the metal ion in the N,O-chelating mode,
and act as the bidentate ligands. It has been reported that the
ν(COO¯) vibration of the amino acid complexes is affected
by bonding of the non-coordinating C=O group with the
metal of the neighboring complex, or its hydrogen bonding
with the neighboring complex or lattice water molecules
[40].
The FT-IR spectra of the Zn(II) amino acid complexes
show the absorption patterns in the 4000–400 cm−1 region
similar to those observed for the free amino acid ligands.
Selected vibrations of these complexes and their assign-
ments have been reported in the “Experimental” section.
In general, the intense and broad absorption bands, which
appear around 3000–3400 cm−1, are ascribable to the
ν(N–H) vibrations of the coordinated amino acid ligands.
As compared to the free amino acids, these bands are shifted
toward higher frequencies, which confirm the involvement
of the –NH2 group in the coordination to Zn(II) and complex
formation [41]. Because the carboxylic group of amino acid
ligands binds to the Zn(II) ion via covalent bonding, the
ν(C=O) is metal-sensitive, so that its stretching vibration
is shifted toward higher frequencies [42]. Furthermore, the
observed absorption band at 2915 cm−1 for Zn(Met)2 com-
plex is attributed to the symmetric and asymmetric stretch-
ing vibrations of CH2–S and CH3–S bonds. This vibration
band is not shifted compared to the free methionine ligand
(2915 cm−1), indicating that the methionine ligand is not
coordinated to Zn(II) by the sulfur atom. In addition, the
narrow peak at 1242 cm−1 is due to the wagging vibration
of the CH2–S bond for Zn(Met)2 complex [43].
The base plane of the octahedron is formed by nitrogen
and oxygen atoms, N(l), O(1), N(2), and O(3), from two
independent glutamine ligands. The chelating glutamine
ligands are arranged in a trans-configuration. The bond
lengths are in the range of Zn–O and Zn–N distances usu-
ally observed for the other zinc amino acid complexes [48,
49]. Furthermore, the axial sites are occupied by two oxygen
atoms of carboxyl groups, O(4) and O(6), from two other
molecules of complex. The bond lengths of Zn–O(4) and
Absorption in the UV–Vis region is related to the elec-
tronic transitions within the molecule. The electronic absorp-
tion spectra of the synthesized complexes were taken in the
aqueous or DMF solutions and data have been presented in
the “Experimental” section. Since the Zn(II) complexes have
a d10 configuration, there is no ligand field transition (d →
d). In general, the UV–Vis spectra of the Zn(II) complexes
show the intraligand transitions, including σ → σ*, n → σ*,
n → π*, and π → π*. The σ → σ* transitions are observed
only in the vacuum UV (λ˂190), because they are very high
energy [44]. In this work, the synthesized Zn(II) amino acid
complexes show a high energy absorption band in the UV
Fig. 2 ORTEP view of Zn(Gln)2 with displacement ellipsoids drawn
at the 40% probability level
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Journal of the Iranian Chemical Society
Fig. 3 View of the polymeric
nature of Zn(Gln)2 complex
Zn–O(6) are 2.521 and 2.304 Å, respectively, which are
comparable to those reported previously for similar com-
plexes, including Zn(l-glutamate) [50], Zn(l-serine)2 [51],
and Zn(dl-histidine)2 [52].
In vitro antifungal activity and minimum inhibition
concentration (MIC) of the complexes
A wide range of properties of amino acids and zinc ion
promoted us to examine some biological activities of the
zinc amino acid complexes. Fortunately, all of these Zn(II)
amino acid complexes except Zn(Cys)2, are sufficient solu-
ble in water to investigate their in vitro antifungal activities.
Therefore, the inhibitory effect of the synthesized complexes
on the growth of Aspergillus flavus fungus and aflatoxin B1
production was studied. In this respect, the aqueous solu-
tions of the compounds at different concentrations includ-
ing 5, 10, 15, 20, 25, 30, and 50 µg mL−1 were added to
PDA medium consisting of Aspergillus flavus fungus. Each
experiment was simultaneously repeated in four times to
ensure the accurate results. The produced aflatoxin B1 was
determined using ELISA, after the incubation of the culture
media at 25–30 °C for 6 days (Table 3). Furthermore, two
blank assays were used for cultures. One of them was con-
sidered as a positive control to calculate the normal growth
of fungus, without addition of the inhibitors to contaminated
PDA medium with spores of Aspergillus flavus. The most
amount of produced aflatoxin (3233 µg L−1) was found for
this blank, in the absence of the Zn(II) amino acid complexes
As can be seen in Fig. 2, Zn(II) ion displays a slightly
distorted octahedral, because the transoid angles are all
within the 169.80 (11)°–176.98 (9)° range, and the cisoid
angles are in the range of 81.02 (9)°–98.76 (9)°. The chelate
bite angles of O(3)–Zn–N(2) and O(1)–Zn–N(1) are 81.03
(95)° and 82.18 (93)°, respectively, which force the other
“in-plane” angles to differ from 90° and 180°. In addition,
the mean planes of two glutamine ligands form the dihedral
angle of 9.76 (11) to each other, which confirms that they
are not co-planar.
The hydrogen-bonding interactions in Zn(Gln)2 complex
are much less extensive than the free glutamine ligand. Many
of hydrogen-bonding interactions, which link the individual
molecules into sheets in the free glutamine, are eliminated
by long- and short-range coordination of the metal atom in
Zn(Gln)2 complex. Only one hydrogen atom of –NH2 group
shows the evidence of hydrogen bonding.
The [Zn(Arg)2(OAc)]OAc·3H2O complex was also char-
acterized crystallographically, matching the unit cell param-
eters reported in the literature [53] (see ESI).
Table 3 Amount of produced
Concentration of Zn(II) com-
5
10
15
20
25
30
50
aflatoxin B1 (µg L−1) in the
culture media in the presence
of 10 mL of Zn(II) compounds
solutions at different
pounds solutions (µg mL−1
)
Zn(OAc)2·2H2O
Zn(Gln)2
Zn(His)2
Zn(Gly)2
[Zn(Arg)2(OAc)]OAc·3H2O
Zn(Met)2
2340
756
2118
305
447
831
1120
1336
1966
123
168
571
776
857
1785
N. D.
N. D.
84
138
280
1720
N. D.
N. D.
N. D.
22
1686
N. D.
N. D.
N. D.
N. D.
14
1630
N. D.
N. D.
N. D.
N. D.
N. D.
concentrations after incubation
at 25–30 °C for 6 days
930
1280
1338
1645
154
N. D. no detectable
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Journal of the Iranian Chemical Society
as the inhibitors. In the other blank, only PDA medium was
used as the negative control. The latest confirmed that the
medium was not polluted before the addition of fungus, and
it was found to be completely sterilized. As can be seen
in Table 3, these water soluble complexes (1–5) showed
the remarkable inhibitory effect on aflatoxin B1 production
by Aspergillus flavus fungus. The results revealed that the
measured aflatoxin B1 content was significantly changed at
the different concentrations of these complexes. In general,
the growth of Aspergillus flavus decreased with increasing
concentration of the Zn(II) complexes. As a consequence,
the aflatoxin production was decreased. The minimal inhib-
itory concentration (MIC) of Zn(II) compounds against
Aspergillus flavus is also shown in Table 4. Therefore, in
the presence of the high concentration of these compounds,
the amount of aflatoxin B1 produced in vitro by the fungus
was the minimum, so that was no detectable using ELISA.
At any certain concentration, Zn(Gln)2 complex exhibited
the most inhibitory effect in comparison with the other com-
plexes. These complexes are arranged in order according
to the antifungal activity as Zn(Gln)2, Zn(His)2, Zn(Gly)2,
[Zn(Arg)2(OAc)]OAc·3H2O, and Zn(Met)2. For example,
Fig. 4 shows the images of plates at the different concentra-
tion of Zn(Gln)2 as the inhibitor, after 6 days of incubation
at 25–30 °C. As can be seen, the growth of the Aspergillus
flavus has significantly decreased with the increasing con-
centration of Zn(Gln)2 complex.
To compare, the inhibitory effect of Zn(OAc)2∙2H2O on
the growth of Aspergillus flavus fungus was also studied
under the same experimental conditions (entry 1, Tables 3,
4). The results indicate that the synthesized Zn(II) amino
acid complexes are more antifungal active than the zinc
ion for the Aspergillus flavus. In general, the polarity of the
metal ion is noticeably reduced after coordination of the
ligand, due to the overlap of its valence orbitals with the
ligand orbitals. This effect decreases the positive charge of
metal ion and π-electron delocalization in the ligand chelat-
ing ring, which would enhance the lipophilic character of the
metal ion center. The increased lipophilicity character leads
to more diffusion of the complexes into the cell membrane
and blocks the metal coordination sites of the enzymes in
the fungi structures [54, 55]. Therefore, it is considerable
that the complexation of Zn(II) ion with amino acids as the
ligands significantly improves its antifungal activity.
Table 4 Minimal inhibitory concentration (MIC) of the Zn(II) com-
pounds against Aspergillus flavus
Conclusions
In this paper, synthesis and characterization of Zn(II) amino
acid complexes including Zn(Gln)2 (1), [Zn(Arg)2(OAc)]
OAc∙3H2O (2), Zn(His)2 (3), Zn(Gly)2 (4), Zn(Met)2 (5),
and Zn(Cys)2 (6) are reported. In addition, the Zn(Gln)2 and
[Zn(Arg)2(OAc)]OAc∙3H2O complexes have been structur-
ally characterized. The structure of Zn(Gln)2 reveals the
distorted octahedral environment around the zinc center.
The synthesized Zn(II) amino acid complexes exhibit the
Compounds
MIC (µg mL−1
)
Zn(OAc)2·2H2O
Zn(Gln)2
Zn(His)2
Zn(Gly)2
[Zn(Arg)2(OAc)]OAc·3H2O
Zn(Met)2
150
20
20
25
30
50
Fig. 4 Images of plates after 6
days of incubation at 25–30 °C
in the presence of different
concentration of Zn(Gln)2, 0
(positive control blank), 5, 10,
15, 20, 25, 30, and 50 µg mL−1
which are, respectively, shown
as a–h images
,
1 3

Journal of the Iranian Chemical Society
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effect of the synthesized Zn(II) complexes on aflatoxin B1
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growth of Aspergillus flavus and the amount of toxin pro-
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centration of these Zn(II) complexes. Among the synthe-
sized Zn(II) amino acid complexes, Zn(Gln)2 and Zn(Met)2
showed the most and least of the antifungal activity at any
certain concentration, respectively. The minimal inhibitory
concentration (MIC) of Zn(II) complexes against Aspergillus
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Supporting information Electronic supplementary material available:
CIF files giving the crystal data for Zn(Gln)2 and [Zn(Arg)2(OAc)]
OAc·3H2O. CCDC 1556782 and 1558139 contain the supplementary
crystallographic data for Zn(Gln)2 and [Zn(Arg)2(OAc)]OAc·3H2O,
respectively. These data can be obtained free of charge via http://www.
ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
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