In vitro evaluation of the activity of thiosemicarbazone derivatives against mycotoxigenic fungi affecting cereals

Article abstract of DOI:10.1016/j.ijfoodmicro.2015.02.009

With a steadily increasing world population, a more efficient system of food production is of paramount importance. One of the major causes of food spoilage is the presence of fungal pathogens and the production and accumulation of mycotoxins. In the pres

Full text of DOI:10.1016/j.ijfoodmicro.2015.02.009

In vitro evaluation of the activity of thiosemicarbazone derivatives against
mycotoxigenic fungi affecting cereals
Francesca Degola, Caterina Morcia, Franco Bisceglie, Francesca Mussi,
Giorgio Tumino, Roberta Ghizzoni, Giorgio Pelosi, Valeria Terzi, Annamaria
Buschini, Francesco Maria Restivo, Tiziana Lodi
PII:
DOI:
Reference:
S0168-1605(15)00079-3
doi: 10.1016/j.ijfoodmicro.2015.02.009
FOOD 6806
To appear in:
International Journal of Food Microbiology
Received date:
Revised date:
Accepted date:
24 September 2014
26 January 2015
5 February 2015
Please cite this article as: Degola, Francesca, Morcia, Caterina, Bisceglie, Franco, Mussi,
Francesca, Tumino, Giorgio, Ghizzoni, Roberta, Pelosi, Giorgio, Terzi, Valeria, Buschini,
Annamaria, Restivo, Francesco Maria, Lodi, Tiziana, In vitro evaluation of the activity of
thiosemicarbazone derivatives against mycotoxigenic fungi affecting cereals, International
Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.02.009
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In vitro evaluation of the activity of thiosemicarbazone derivatives against mycotoxigenic
fungi affecting cereals.
Francesca Degolaa, Caterina Morciac, Franco Bisceglieb, Francesca Mussia, Giorgio Tuminoc,
Roberta Ghizzonic, Giorgio Pelosib, Valeria Terzic, Annamaria Buschinia, Francesco Maria
Restivoa**, Tiziana Lodia
a) Dipartimento di Bioscienze, Università di Parma, Parma, Italy
b) Dipartimento di Chimica, Università di Parma, Parma, Italy
c) Consiglio per la Ricerca e la sperimentazione in Agricoltura, CRA-GPG, Genomics Research
Centre, Fiorenzuola d’Arda, Italy
** to whom correspondence should be addressed
Francesco Maria Restivo, Dipartimento di Bioscienze, Viale delle Scienze 11A, 43124, Parma ,
Italy
e-mail: francescomaria.restivo@unipr.it
phone +39-0521-905603
fax: +39-0521-905604
key words: thiosemicarbazone, Fusarium, Aspergillus flavus, mycotoxin, cytotoxicity, cereals
Highlights
 A panel of thiosemicarbazones derivatives were synthesized
 The compounds were tested for their effect on fungi (Fusarium and Aspergillus) affecting
cereal crops
 Species-specific effects on fungal growth and mycotoxins biosynthesis were observed
 Two molecules were considered promising for fungal growth and mycotoxin containment
 Potential deleterious effects on seed germination and on human cells were tested

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Abstract
With a steadily increasing world population, a more efficient system of food production is of
paramount importance. One of the major causes of food spoilage is the presence of fungal
pathogens and the production and accumulation of mycotoxins. In the present work we report a
study on the activity of a series of functionalized thiosemicarbazones (namely cuminaldehyde,
trans-cinnamaldehyde, quinoline-2-carboxyaldehyde, 5-fluoroisatin thiosemicarbazone and 5-
fluoroisatin N4-methylthiosemicarbazone), as antifungal and anti-mycotoxin agents, against the two
major genera of cereal mycotoxigenic fungi, i.e. Fusarium and Aspergillus. These
thiosemicarbazones display different patterns of efficacy on fungal growth and on mycotoxin
accumulation depending on the fungal species. Some of the molecules display a greater effect on
mycotoxin synthesis than on fungal growth.
1. Introduction
Plant diseases are probably the greatest obstacle to the increase of global crop production and also
one of the major factors limiting crop quality. Crop protection plays a key role in ensuring food
security and, in turn, is directly related to food preservation, a key step to increasing both food
supply and food safety. In fact about 40% of the food produced worldwide is lost or spoiled (Oerke
and Dehne, 2004) and this not only reduces its availability, but also has an impact on global climate
change by causing excess consumption of fresh water and fossil fuels.
One of the major causes of food spoilage is the presence of micro-organisms. In particular, fungal
pathogens associated with various crop diseases can lead not only to significant production losses,
but can also result in the production and accumulation of secondary metabolites - mycotoxins -
hazardous to human and animal health. In cereals, the main mycotoxin producers belong to the
genera Aspergillus, Penicillium and Fusarium. Fusarium are “field” fungi with worldwide
distribution that can colonize the cereal plant during flowering causing Fusarium Head Blight
(FHB), a destructive disease that leads to severe yield losses and to the accumulation of
mycotoxins in the grains (Goswam and Kistler, 2005). Types A and B trichothecenes, and
zearalenone are the most important toxic products that can occur individually or in combination. In
European environments, the most common species in the FHB complex are F. graminearum, F.
culmorum, F. sporotrichioides and F. langsethiae. As far as Aspergillus is concerned, a few species
belonging to this genus (mainly A. flavus and A. parasiticus) may colonise various crops intended
for human and animal nutrition. As these fungi are well known mycotoxin producers (mainly

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aflatoxins; AFs), they consitute a serious threat to humans and animals. In particular, Aspergillus
flavus, can be found in corn fields throughout the world and is responsible of severe economic
losses for the farmers and the stakeholders of the post-harvest phase (Abbas et al., 2009; Williams,
2008; Wu and Khlangwiset, 2010). In fact, the AF risk for human health comes not only from direct
consumption of crop materials, but may persist in processed products (milk, cheese, etc) derived
from animals fed with contaminated feed. Several and complementary strategies have been adopted
to control FHB and AF, starting from the breeding of resistant cereal varieties, to the adoption of
more suitable agronomic practices including the application of fungicides (for reviews see Abbas et
al., 2009; Terzi et al., 2014). Currently, fungicides are required to increase food availability, to
reduce food waste and to increase food safety (Cooper and Dobson, 2007). However the use of
fungicides can lead, in the medium and long term, to the evolution of populations of pathogens
resistant to the antimicrobial agents used against them (Anderson, 2005; Ma and Michailides,
2005). Hence, the identification of new molecular targets becomes of paramount importance for
novel strategies in crop protection.
A secondary but no less important issue that should be considered when testing new fungicides for
their activity on growth inhibition concerns a possible contradictory and unwanted effect on
mycotoxin biosynthesis; i.e. inhibition of mycelium growth resulting in an increase of toxin
production (Schmidt-Heydt et al., 2013). In some cases, such as Aspergillus flavus colonization of
maize crops, the prevalent economic damage emerges from the sanitary risk deriving from toxin
contamination of the food matrix rather than to a negative effect on plant yield (van Egmond and
Jonker, 2004).
Thiosemicarbazones, an important class of nitrogen and sulfur containing compounds, have
received great attention because of their chemical and biological activities, such as antibacterial,
antivirus, antiamoebic, and antitumor activities in human pharmacology (Pelosi, 2010). Numerous
thiosemicarbazone derivatives are also active for the control of some human pathogens, however
there are only a limited number of studies concerning their applications [see patents: CN 102219769
(2011) CN 103694155 (2014)]. Recently, data on the effect of some thiosemicarbazone metal
complexes on a subset of plant pathogens were published (Tyagi et al., 2014); however the study
was mainly aimed at characterizing the coordination compounds and investigating the influence of
the metal on the features of the compounds, and a very limited part of the work was dedicated to
mycological studies.
The aim of the present work was therefore to evaluate the potential of a panel of
thiosemicarbazones, differing in their functionality, for crop protection and food spoilage control,

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with a particular focus on the biological activity of these compounds as antifungal and anti-
mycotoxin agents against the two major genera of cereal mycotoxigenic fungi, i.e. Fusarium and
Aspergillus.
2. Material and methods
2.1. Thiosemicarbazones synthesis
Figure 1 depicts the synthesized thiosemicarbazones for which the acronyms reported in
parentheses are used: cuminaldehyde thiosemicarbazone (Htcum), trans-cinnamaldehyde
thiosemicarbazone (Htcin), quinoline-2-carboxyaldehyde thiosemicarbazone (Htisq), 5-fluoroisatin
N4-methylthiosemicarbazone (HtmeFis) and 5-fluoroisatin thiosemicarbazone (HtFis). All reagents
used in the syntheses were purchased from Aldrich. C, H, N analyses were obtained with a Carlo-
Erba 1108 instrument. IR spectra were recorded using KBr pellets on a Nicolet 5PC FTIR
spectrophotometer, or using the compounds directly on the ATR accessory in the 4000-400 cm-1
range. The relative intensity of reported FT IR signals are defined as s = strong, br = broad, m =
medium, and w = weak. 1H NMR spectra were recorded on a Bruker Avance spectrometer at 300
MHz with TMS as the internal reference. The splitting of proton resonances in the reported 1H
NMR spectra are defined as s = singlet, br s = broad singlet, d = doublet, t = triplet, and m =
multiplet. Melting points were determined with a Gallenkamp instrument (Weiss–Gallenkamp). UV
measurements were performed on a Varian UV-vis Cary 50 spectrometer with quartz cuvettes. The
ligands were prepared following a modified procedure based on the following refs (Bisceglie et al.,
2014; Chiyanzu et al., 2003; Karali et al., 2007; Yi et al., 2011). Htcum and Htcin. were
synthesized by respectively adding 0.39 mL (2.3 mmol) of cuminaldehyde (p-isopropyl
benzaldehyde) or 0.29 mL (2.3 mmol) of trans-cinnamaldehyde (3-phenyl-2-propenal) to 20 mL of
a hot stirred ethanolic solution of thiosemicarbazide (0.21 g, 2.3 mmol). The mixture was then
refluxed for 7-9 h and left cooling down to room temperature. Htisq was obtained by dissolving
thiosemicarbazide (337.6 mg, 3.7 mmol) in 80 mL of methanol at room temperature under stirring
and adding an equimolar amount of quinoline-2-carboxaldehyde (582.1 mg, 3.7 mmol). After
adding a few drops of glacial acetic acid to catalyze the reaction, the solution became clear and its
color turned to orange. This reaction mixture was kept under stirring and placed in an ice bath and
left standing for 20 hrs. The white precipitate was filtered on a Buchner and washed using ethanol.
HtmeFis was synthesized by dissolving, in about 70 mL of methanol,350.6 mg (3.3 mmol) of 4-
methyl-3-thiosemicarbazide together with an equimolar amount of 5-fluoro-isatin (389.6 mg).

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Similarly, HtFis was obtained by reacting 110.6 mg (1.1 mmol) of thiosemicarbazide with an
equimolar amount of 5-fluoro-isatin (200.3 mg) in about 60 mL of methanol. To the resulting
orange solutions, few drops of glacial acetic acid were added and the resulting mixture was heated
to reflux temperature under magnetic stirring for 24 h. A yellow powder was isolated by filtration
using a Buchner funnel and washed 3 times with methanol.
2.1.1. Cuminaldehyde thiosemicarbazone (Htcum)
Yield: 86%. Mp: 144 °C. FT-IR (KBr, cm-1)3411, m, 3280, m, ν (NH); 3013, m, ν(CHaromatic);2957,
mw, ν(CHaliphatic), 1586, s, ν (C=N); 820, s, ν (CS). Anal. Calc. for C11H15N3S: C 59.64; H 6.78; N
18.98; S 14.48. Found: C 59.54; H 6.62; N 18.67; S 14.19 %. 1H NMR data (δ, ppm; DMSO d6):
1.23 (d, 6 H, CH3); 2.89 (ept, 1H, CH isopropyl); 7.27, 7.30 (2s, 2H, aromatic); 7.70 (m, 2H,
aromatic); 7.93 and 8.03 (2s, 2H, NH2); 8.17 (s, 1H, CH=N);11.34 (br, 1H, NHCS). UV−visible
(solvent MeOH): 312 nm (n → π*), 223 nm (n → σ*); (solvent DMSO): 311 nm (n → π*), 240 nm
(n → σ*).
2.1.2. trans-Cinnamaldehyde thiosemicarbazone (Htcin)
Yield: 91%. Mp: 139 °C. FT-IR (KBr, cm-1)3416, s, 3260, m, 3154, m, ν (NH); 3042, mw,
ν(CHaromatic); 1630, ms, ν (C=N); 1621, ms, ν (C=C); 820, w, ν (CS). Anal. Calc. for C10H13N3OS: C
53.78; H 5.87; N 18.81; S 14.36. Found: C 53.65; H 5.35; N 19.12; S 14.53 %. 1H NMR data (δ,
ppm; DMSO d6): 6.68 (m, 1 H, CH=C); 7.06 (d, 1 H, CH=C); 7.30-7.60 (m, 6 H, aromatic o and m,
and NH2); 7.90 (d, 1H, aromatic p); 8.17 (m, 1 H, CH=N); 11.38 (br, 1H, NHCS). UV−visible
(solvent MeOH): 297 nm (n → π*), 246 nm (n → σ*); (solvent DMSO): 324 nm (π → π*), 241 nm
(n → σ*).
2.1.3. Quinoline-2-carboxyaldehyde thiosemicarbazone (Htisq)
Yield: 77%. Mp: 180 °C. FT-IR (cm-1): 3395, 3262 and 3152  N-H, 3066  C-H arom., 2986  C-
H aliph., 1609  C=N, 1533 and 1503  C=C, 1110 and 839  C=S. Anal. Calc. for C11H10N4S: C
57.37, H 4.38, N 24.33. Found: C 57.38, H 4.22, N 24.55 %. 1H NMR data (δ, ppm; DMSO d6)
11.79 ppm (1H, s, C=N-NH), 8.45 ppm (2H, d, J = 8.7 Hz, NH2 ), 8.35 ppm (2H, m, CH arom.),
8.23 ppm (1H, s, CH=N), 8.00 ppm (2H, t, J = 8.7 Hz, CH arom.), 7.78 ppm (1H, t, J = 8.4 Hz, CH
arom.), 7.62 ppm (1H, t, J = 8.1 Hz, CH arom.).
2.1.4. 5-fluoroisatin N4-methylthiosemicarbazone (HtmeFis)

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Yield: = 34%. Mp: 150 °C. FT-IR (cm-1): 3196. ν (N-H), 1693 ν (C=O), 1670 ν (C=O), 1552 ν
(C=N), 842 and 743 ν (C=S). Anal. Calc. for C10H9FN4OS: C 47.6, H 3.6, N 22.2. Found: C 47.48,
H 3.57, N 21.85 %.1H NMR data (δ, ppm; DMSO d6) 12.50 ppm (1H, s, C=N-NH), 9.35 ppm (1H,
s, CH3-NH ), 7.48-6.95 ppm (3H, m, CH arom.), 3.10 ppm (3H, m, CH3-NH).
2.1.5. 5-fluoroisatin thiosemicarbazone (HtFis)
Yield: = 58%. Mp: 135 °C. FT-IR (cm-1): 3435-3261 ν (N-H), 1685 ν (C=O), 1482 ν (C=N), 863
and 754 ν (C=S). Anal. Calc. for C9H7FN4OS: C 45.37, H 2.96, N 23.52. Found: C 45.36, H 2.96, N
23.58 %.1H NMR data (δ, ppm; DMSO d6) 12.36 ppm (1H, s, C=N-NH), 11.21 ppm (1H, s, CH
arom.), 9.13-8.77 ppm (2H, 2s, NH2), 7.52-6.90 ppm (3H, m, CH arom.).
2.2. Fungi
Strains of Fusarium graminearum and Fusarium sporotrichioides (provided by the Università
Cattolica del Sacro Cuore, Piacenza, Italy) isolated from bread wheat plants in Northern Italy in
2002 and 2010 respectively were used. Strains of Aspergillus flavus were isolated from corn kernels
collected in the fields of the Po Valley and have been described previously (Degola et al., 2011).
The toxigenic A. flavus strain Fri2+ and the atoxigenic strain To were mainly used in the
experiments reported in this work.
2.3. Evaluation of the effects of thiosemicarbazones on in vitro mycelial growth
2.3.1. Fusarium
The mycelial growth of F. graminearum and F. sporotrichioides was evaluated on solid PDA
medium (Liofilchem, Teramo, Italy), amended with 0.5% Tween 20 (Sigma, St Louis, MO, USA)
and 100 μM of Htcum, Htcin, Htisq, HtmeFis, HtFis thiosemicarbazones. Moreover, Htcum and
Htcin molecules were tested in the range from 0 to 300 μM. The experiments were conducted twice
in triplicate in 60-mm Petri dishes, inoculated with 8-mm PDA plugs from actively growing
cultures. Mycelial growth was evaluated as the mean diameter measured each day from fungal
inoculation up to 6 days post-inoculation. The effect of different molecules and of different
concentrations was expressed as percentage inhibition, calculated according to the formula:
I=[(C-T)/C] x 100
where I is the percentage of inhibition, C is the control plate colony diameter in mm, T is the treated
plate colony diameter in mm.

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2.3.2. Aspergillus
Conidia suspensions were obtained from 10-day YES-agar [2% (w⁄v) yeast extract (Difco, Detroit,
MI), 5% (w⁄v) sucrose (Sigma, St Louis, MO), 2% (w⁄v) agar (Difco)] cultures incubated at 28 °C;
conidia concentration (quantified by OD600) and viability (>90%) were determined according to
Degola et al. (2011). Determination of A. flavus radial growth was performed in YES-agar amended
with 100 µM of B, C, I, M, O thiosemicarbazones: three single spots (5 µL of a 107 conidia/ml
suspension each) of aflatoxigenic strain Fri2 were equidistant inoculated in 90-mm Petri dishes,
plates were incubated for 4 days at 25 °C and the mycelial growth was evaluated daily by
measuring the reverse of colonies along two orthogonal diameters. Radial growth was expressed as
cm/d ± SD. Additionaly, conidial germination rate and post germination hyphal outgrowth were
assessed analyzing changes in optical density of spore suspensions over time: in a 96 well microtiter
plate (Sarstedt, Newton, NC, USA) 104 spores were inoculated in a final volume of 200 µL of YES
liquid medium amended with Htcum, Htcin, Htisq, HtmeFis, HtFis thiosemicarbazones and
incubated at 28 °C. The optical density at 620 nm (OD620) was recorded for each well every 120
min with a microplate reader (MULTISKAN EX, Thermo Electron Corporation, Vantaa, Finland)
without shaking. Samples were inoculated in triplicate. Preliminary experiments indicated that OD
values, recorded between 38 and 46 h after inoculum, could reliably be used to compare growth rate
of the different samples. Results obtained according to radial growth and optical density
determinations were comparable, as far as the inhibitory effect of the thiosemicarbazones are
concerned. Biomass variation was also determined in some experiments involving inoculation in
CCM medium (see 2.4.2): fresh or dry mycelium weight were obtained, for each sample,
withdrawing the mycelium from eight wells of the microplate and pooling them. The pooled
mycelia were then blotted dry on clean paper and weighed (fresh weight determination) and then
incubated at 80 °C for 48 h before dry weight determination. Samples were inoculated in triplicate.
2.4. Evaluation of the effect of molecules on mycotoxin production
2.4.1. Fusarium
The entire mycelium was collected from control and treated plates inoculated with Fusarium
graminearum and Fusarium sporotrichioides six days after inoculation. The deoxynivalenol
concentration produced by F. graminearum mycelium was determined using Ridascreen Fast DON
ELISA kit (R-Biopharm AG, Darmstadt, Germany) according to the manufacturer’s instructions.
The T2-HT2 concentration produced by F. sporotrichioides mycelium was determined using
VERATOX ELISA kit (Diessechem srl, Milan, Italy), according to the manufacturer’s instructions.

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2.4.2. Aspergillus
A highthroughput procedure performed in multiwell plate was used to assess aflatoxin accumulation
in a coconut milk-derived medium (CCM) (Degola et al., 2012, 2011). Briefly: 400 mL of
commercial coconut cream was diluted to the final volume of 1.2 L with bidistilled water, sterilized
by autoclaving (10 min, 120 °C), cooled at 4 °C overnight and clarified by centrifugation (15 min at
3200 g). The residual floating material and the pellet were discarded, and the intermediate phase
was then recovered and used as culture medium in the aflatoxin inhibition assays. The effect on
aflatoxin biosynthesis was assessed by the microplate fluoresescence-based procedure described in
Degola et al. (2012). Standard flat-bottom 96-well microplates (Sarstedt, Newton, NC, USA) were
used. Suspensions of conidia were diluted to the appropriate concentrations and brought to the final
concentration of 5x102 conidia⁄well; cultures were set in a final volume of 200 µL/well of CCM
medium added with Htcum, Htcin, Htisq, HtmeFis, HtFis thiosemicarbazones. The plates were
incubated in the dark under stationary conditions for up to 6 days at 25 °C. Aflatoxin accumulation
was monitored by fluorescence emission determination: readings were performed directly from the
bottom of wells of the culture plate with a microplate reader (TECAN SpectraFluor Plus,
Männedorf, Switzerland) using the following parameters: λex = 360 nm; λem = 465 nm; manual
gain = 83; lag time = 0 µs; number of flashes = 3; integration time = 200 µs).
2.5. Effects of thiosemicarbazones on seed germinability
2.5.1. Barley
Four hulled barley cultivars (Cometa, Martino, Nure and Tremois) and three hulless cultivars
(Astartis, Fior7341 and Zacinto) were used for germination tests. Stocks of 100 seeds/cultivar were
treated for 2 h with solutions of 0.05% Irol Plus in sterile distilled water containing 0 M, 300 M
and 600 M of Htcum and Htcin molecules. After the treatments, the seeds were placed on a
wetted filter paper (2 mL sterile distilled water) in a Petri dish at room temperature and the
percentage of germination was evaluated after 1 week. Two replicates of the experiment were done.
A chi-squared analysis was performed to compare germination percentages of treated and non-
treated seeds.
2.5.2. Corn
Effect of thiosemicarbazones on corn germination was assessed according to the International Seed
Testing Association (ISTA) rules (http://seedtest.org/en/home.html). Maize kernels soaked for 5
min in a solution containing Htcum or Htcin thiosemicarbazone to the final concentration of 100

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µM were placed and rolled in wet paper towels (that increases the surface contact area between the
seeds and the moisture supply),which were then placed in an upright position (MacKay, 1972).
Rolls were incubated up to 4-5 days at 25 °C. Seeds were considered to have germinated after the
emergence and development from the embryo of dicots and root. Root number per seed and mean
root lenght were also recorded.
2.6. DPPH radical scavenging activity assay
Measurement of scavenging activity of thiosemicarbazones against the 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) was performed in accordance with Choi et al. (Choi et al., 2002)
measuring the bleaching of a purple colored methanol solution of the stable DPPH radical. Briefly,
to a 1 mL aliquot of freshly prepared 85 µM DPPH was added a 4 mL solution containing the
selected compound. The resulting mixture was stirred and then incubated for 30 min at room
temperature The thiosemicarbazone concentrations were 10 µM, 100 µM and 1000 µM.The
decrease in absorbance measured at 518 nm depicts the scavenging activity of the compound
against DPPH. Ascorbic acid (30 mM) was used as positive control to determine the maximal
decrease in DPPH absorbance (Choi et al., 2002). Values were expressed in percentage of inhibition
of DPPH absorbance in relation to the control values without the thiosemicarbazone (ascorbic acid
maximal inhibition was considered 100% of inhibition).
2.7. Effect of thiosemicarbazones on Aspergillus flavus growth and mycotoxin production on maize
kernels
Autoclaved maize kernels (100 seeds, 30 g) were inoculated by soaking in a suspension (0.1%
Tween in distilled water) containing 103 conidia of A. flavus, transferred wet to a sterile Petri plate
(diam. = 15 cm) and incubated at 28 °C in the dark for 5-7 days. For the treatment with the
thiosemicarbazones, the conidial suspension was amended with Htcum or Htcin molecules to the
final concentration of 100 M. Controls received an equivalent concentration of the
thiosemicarbazone solvent (DMSO; 1% final concentration). The experiments were performed
twice in triplicate. Data are reported as mean ± SD of the number of infected seeds as detected by
visual inspection. For aflatoxin determination, infected maize kernels were added to 150 mL of a
80% MetOH solution and ground for 3 min in a blender. The maize methanol mixture was then
passed through a paper filter, diluted 1:10 in bidistilled water and filtered with a microfiber filter
(1.5 μm, VICAM, Watertown, MA, USA). Aflatoxin concentration was determined by HPLC as
described by Degola et al. (2009) with minor modifications. The filtrate was diluted 1:100 in the
mobile phase before HPLC injection (Spotti et al., 1999). The chromatographic analyses were

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performed using a JascoModel PU-1580 pump, equipped with a Vertex column (150×4.6 mm,
Eurospher 100-5C18, Kramer, Berlin, Germany), a Jasco Model AS-1555 autosampler (loop=0.1
ml), and a Jasco Model FP-1520 fluorescence detector (λex=365 nm and λem=440 nm).
2.8. Toxicity of thiosemicarbazones on human cells
The growth inhibitory effect towards a human foetal lung fibroblast cell line was evaluated by the
MTS assay (CellTiter96RAQueousOne Solution Cell Proliferation Assay, Promega Corporation,
Madison, WI, USA).
Briefly, 5×103cells/well were seeded in 96-well plates in 100 μL of DMEM medium supplemented
with 1% glutamine, 0.5% penicillin/streptomycin and 5% foetal bovine serum and then incubated at
37 °C in a humidified (95 %) CO2 (5 %) incubator. After seeding (24 h), cells were treated, in
quadruplicate, with increasing concentrations of the compounds (Htcum and Htcin) for 24 h. The
cytotoxicity assay was performed by adding 20 μL of the CellTiter96RAQueousOne Solution Cell
Proliferation Assay directly to the culture wells, incubating for 4 h and then recording the
absorbance at 450 nm with a 96-well plate reader (MULTISKAN EX, Thermo Electron
Corporation, Vantaa, Finland).
3. Results and Discussion
3.1. Chemistry
Antifungal activity associated to thiosemicarbazones has already been described in the literature
(Beraldo and Gambino, 2004). However, in all previous reports, only MIC value against few fungal
genera were determined (Kasuga et al., 2006; Rodríguez-Argüelles et al., 2009, 2007, 2005) and no
data were shown on the effect on mycotoxin production. In this work, a panel of
thiosemicarbazones differing in their derivatization were synthesized and tested for their antifungal
and antimycotoxinogenic activity against strains of Fusarium graminearum, Fusarium
sporotrichioides and Aspergillus flavus. In particular, we focused our attention on five molecules
(Htcum, Htcin, Htisq, HtmeFis and HtFis) derived from natural aldehydes/ketones differing in
shape and in the pattern of the potential donor-acceptor atoms, in order to gain information about
possible structure-activity relationships. Their chemical structure and data concerning the synthesis

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are reported in Fig.1 and in the Materials and Methods section. Compounds Htcum and Htcin are
thiosemicarbazones containing aromatic fragments deriving from cuminaldehyde and
cinnamaldehyde (essential oil components, known for their natural mycostatic properties), while
Htisq, HtmeFis and HtFis are thiosemicarbazones of quinoline and fluoroisatine, compounds
known for their antiproliferative properties (Adsule et al., 2006; Hall et al., 2011). Since
thiosemicarbazones are known to be metal ion chelators and in many cases, their activity has been
associated with their ability to sequester metal ions essential to life, such as iron, (Pelosi, 2010) we
have chosen molecules Htisq and HtmeFis because they possess in the aromatic moiety a further
donor atom, which increases the denticity to three donor atoms (N, N, S or O, N, S) favouring the
chelating properties of these molecules. In addition, in order to understand if the amino group of the
thiosemicarbazide plays a determinant role in the activity, we have also synthesised compound
HtFis, which differs from HtmeFis for the presence of a methyl on the terminal nitrogen.
3.2. Antifungal and antimycotoxin activity
A preliminary assay was performed to assess the relative effect of the different thiosemicarbazones
on growth and on mycotoxin accumulation of the different fungi. Using 100 M as a starting
concentration for testing, some of the thiosemicarbazones here analysed were very effective in
inhibiting mycelium growth (Table 1). In particular, treatments with molecules Htcum and Htcin
were more effective than those with HtmeFis, Htisq or HtFis (see also Fig. 2d which shows a
visual comparison of the differential effect of Htcum and Htisq thiosemicarbazones on A. flavus
mycelium development). These preliminary observations showed that the cuminaldehyde and trans-
cinnamaldehyde moieties are essential in the activity of these compounds, while the chelating
properties, which characterize molecules HtmeFis, Htisq and HtFis, do not play a key role in the
activity mechanisms of these thiosemicarbazones. This fact allows us to suggest that the inhibitory
activity is not due to a sequestering action on essential metal ions present in the medium. An
increased sensitivity of A. flavus to both Htcum and Htcin thiosemicarbazones as compared to the
two Fusarium species here analysed, was observed. For example, thiosemicarbazone Htcin exerted
a much stronger inhibitory activity on A. flavus mycelium growth (58%) than on F. sporotrichioides
or F. graminearum (around 25% for both species). Similarly, A. flavus growth was strongly affected
(76%) by Htcum, whereas both Fusaria experienced a limited inhibition of mycelium biomass
increase (25-39%). Next, we determined the effect of the various molecules on mycotoxin
production. Here again, a different pattern of efficacy of the thiosemicarbazones on toxin
production by the two genera of fungi was observed. Compounds Htcum and Htcin were found be

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very effective in decreasing T2-HT2 and aflatoxin B accumulation by F. sporotrichioides and A.
flavus respectively, whereas deoxynivalenol (DON) production by F. graminearum was nearly
unaffected (Table1). On the other hand, Htisq, HtmeFis and HtFis molecules did not affect toxin
accumulation in either Fusarium species (inhibition < 6%). Surprisingly this was not the case for A.
flavus: aflatoxin accumulation in the culture medium decreased by 44%, 60% and 35% with Htisq,
HtmeFis and HtFis thiosemicarbazones respectively. A favorable outcome of our survey is the
absence of mycotoxin inducing activity by the majority of the tested thiosemicarbazones, either in
the Fusarium or Aspergillus species here analysed.
Our analysis focused subsequently on the effect of the two thiosemicarbazones that provided the
greatest efficacy in preventing fungal growth and/or mycotoxin accumulation by the three species
analysed. Serial dilution of Htcum and Htcin molecules were performed to assess their range of
activity. Increasing Htcum or Htcin concentration, up to 300 M, in the culture medium affected
the growth of both Fusarium species. However, the inhibition (40% - 65% for Htcum and 50% -
70% for Htcin for F. sporotrichioides and F. graminearum respectively) at the higher
concentrations used indicated a different sensitivity of the Fusarium species to the two
abovementioned thiosemicarbazones (Fig. 2a, 2b). Concentrations of thiosemicarbazones over 100
M were not considered for A. flavus treatments, since an inhibitory effect of the solvent (DMSO,
present in the stock solution) on mycelium growth was observed in this conditions. In particular A.
flavus, and to a lesser extent F. graminearum, displayed a greater sensitivity to Htcum than to
Htcin over the entire range of concentrations tested (Fig. 2c), whereas no differential effect of the
relevant molecules was observed for F. sporotrichioides. When considering the effect of Htcum
and Htcin molecules on mycotoxin production by the fungal strains, here again, A. flavus was the
most responsive: treatment of conidial suspensions with as low as 25 M concentrations of either
thiosemicarbazone strongly inhibited aflatoxin accumulation in the medium (Fig. 2c). This was not
the case for F. sporotrichioides (74% inhibition of T2-HT2 with both molecules) (Fig. 2a) and for
F. graminearum (73% and 68 % inhibition of DON with Htcum and Htcin respectively) (Fig. 2b).
To sum up, these findings indicate that the two Fusarium species are less susceptible than A. flavus
to Htcum and Htcin thiosemicarbazones, F. sporotrichioides and F. graminearum being the most
resistant, to mycotoxin biosynthesis inhibition and growth inhibition respectively. In Fig. 1S, the
data concerning aflatoxin inhibition in A. flavus cultures grown in CCM medium are reported on a
mycelium dry weight basis as an additional confirmation of the observed effect of Htcum and
Htcin.

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The results described above prompted us to verify whether they could be validated in an “in field”
simulation. To this purpose, maize kernels previously treated with 100 M of Htcum or Htcin
molecules were inoculated with a conidial suspension of A. flavus and incubated at 28 °C for 7-10
days. As reported in Fig. 3, both thiosemicarbazones significantly lowered seed contamination by A.
flavus: less than 30% of the seeds were infected when compared with the untreated control. No
significant increase in the level of fungal infection was observed extending the time (10 days) of the
incubation (data not shown). Aflatoxin accumulation (Fig. 3) was also drasticaly reduced, as
compared to the untreated control, when the seeds were treated either with Htcum (2%) or Htcin
(36%). Analogous experiments were not performed on Fusarium species as inflorescence (and not
kernels, as in maize) are the main infected organ in barley.
3.3. Antioxidant activity
It is well established that, in several fungal species, an oxidative imbalance may trigger mycotoxin
biosynthesis (Reverberi et al., 2012, 2010). In contrast, the oxidative stress that may be experienced
by fungi during their interaction with the host plant, lowers the production of toxin (Barberis et al.,
2010; Chulze, 2010; Farnochi et al., 2005; Magan et al., 2010; Nesci et al., 2008, 2003; Torres et
al., 2003). We were then prompted to analyse if the observed inhibitory effect of the synthesized
thiosemicarbazones on toxin accumulation may, at least in part, be attributed to a possible
antioxidant activity. In order to elucidate the ability of the compounds to act toward a reactive
radical species, we carried out the DPPH radical scavenging assay. The Htcum, Htcin, Htisq,
HtmeFis and HtFis compounds were tested at 10, 100, and 1000 µM concentrations. As reported in
Fig. 4 a remarkable radical scavenging activity of the compounds was reached at 1000 µM where
all the compounds inhibited DPPH by more than 90%. At the lowest concentrations tested Htcin,
Htisq, HtmeFis and HtFis inhibited the radical already by more than 80%, whereas Htcum showed
20% of scavenging activity as compared to the control. It is apparent that no correlation between the
antioxidant efficacy of the different molecules and their inhibitory activity on mycotoxin
biosynthesis may be observed: all the molecules here tested exhibited antioxidant activity but
differentially affected mycotoxin accumulation in the different fungal species. Since in vitro and in
vivo assays are sensitive to different factors, it is not surprising that the antioxidant capacity
determined by chemical analyses does not correlate well with cell-based assays (López-Alarcón and
Denicola, 2013). In agreement with the modern concept of antioxidant, as “a compound able to
modulate the redox state of the cell”, we may speculate that in vivo more subtle mechanisms are
involved in the oxidative stress defense network and at cascade in the regulation of the biosynthetic
pathways of the various toxins.

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3.4. Phytotoxicty and toxicity to human cells
The early identification of the toxicological properties of new chemical substances is a fundamental
step in protecting human and environmental health. A rapid identification of toxicity and
toxicological risk related to the possible release of new chemicals in the environment is a strategic
tool to improve the chemical synthetic design. In addition, if the molecules are intended to be used
to prevent fungal growth on seeds during storage, it is also important to ascertain the absence of
negative effects on seed viability. In our case, no negative effects on seed germination, root and
epicotyl elongation, as determined according to ISTA procedure, were observed when corn seeds
were treated with 100 M of Htcum or Htcin thiosemicarbazones (data not shown). Similarly, no
inhibition of germination could be detected when the relevant molecules were tested at fungicidal
concentrations on both hulless and hulled barley seeds. The different cultivars showed variability in
the response to the highest concentrations (600 M) of Htcum and Htcin, with a significant, albeit
negligible inhibition of germination mainly of hulless seeds (Fig. 5). The cytotoxic activity of the
selected thiosemicarbazones on human cells (HFL-1 cell line) was assessed by treating normal
embryonic lung fibroblasts at different concentrations (0.5; 1.0; 5.0; 10.0; 50.0; 100.0 µM) of
Htcum or Htcin. The two molecules presented a different toxicological behaviour (Fig. 6). Htcum
induced a strong cytotoxicity with a dose-response relationship, its IC50 was 49 µM. On the
contrary, in the range of the tested doses, Htcin showed only a slight antiproliferative activity
starting from 50 µM with an IC50> 100 µM
4. Conclusions
In this paper we have provided evidence that thiosemicarbazones depending on the
functionalization of the molecules and on the fungal species considered display different patterns of
activity against fungal growth and mycotoxin accumulation. With these molecules no negative
effect was recorded against seed germination and low toxicity was observed against human cells (at
least for one of the two molecules considered). These findings open up the way to the design of
more specific molecules to be used according to the target of interest avoiding unwanted effects on
biological processes and/or organisms that occupy the same environmental niche. Moreover we
consider particularly interesting those molecules that display a prominent effect on mycotoxin
synthesis rather than on fungal growth. These molecules could also be profitably used as probes to
gain information on the regulatory mechanism affecting toxin biosynthesis, and, as such, providing
insight into developing improved and more specific inhibitors or new strategies to fight mycotoxin
contamination of food and feed commodities. As concerns possible structure/activity relationships,
we can affirm that an increased capacity to sequester ions does not correspond to a more effective

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inhibition of proliferation. We can therefore exclude that the activity of these compounds is based
on, and limited to, essential metal ion sequestration in the growth medium. An additional benefit of
the low cost and high throughput procedures described in this paper emerges from the possibility to
test combinatorial treatments of different molecules in order to release new antifungals that may
withstand a well known and problematic issue: the development of resistance in the population of
treated fungi. In this way we may contribute to the goal of potentiating and/or extending the time of
efficacy of the antifungal product.
In conclusion, with this work we have provided an experimental system potentially useful to study
the molecular mechanisms which underlie the antifungal properties of derivatives of natural
molecules. We have found that there is a remarkable variability both among different fungal genera
and within single species. We have also demonstrated the possibility to evaluate the impact of
molecular fragments and the possibility to modify them and evaluate the effect of the substitution.
The marked inhibitory activity towards the mycotoxin biosynthesis shown by these compounds,
which have exhibited antioxidant properties, can be useful for the identification and the
characterization of regulatory genes involved in mycotoxin biosynthesis, paving the way to the
development of targeted strategies for crop protection.
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Acknowledgements
CRA-GPG has been supported by“SO.FI.A.” (SOstenibilità della FIliera Agroalimentare) Cluster
Agrifood project. We are also indebted with the Stazione Sperimentale per l’Industria delle
Conserve Alimentari (SSICA) of Parma, and in particular to dr Elettra Berni, for using their
facilities and for help in HPLC analysis and to Antonietta Cirasolo and Roberto Silva for their
technical help. FB is grateful to dr Beatrice Bonati for her assistance in performing UV
experiments.
Contribution
F.D. performed all the experiments in the corn-Aspergillus flavus system while C.M., R.G., G.T.
and V.T. equally contributed to the experimental work done in the barley-Fusarium system. G.P.
and F.B. synthesized the thiosemicarbazones and determined their antioxidant activity. F.M. and
A.B. performed the human toxicity tests. T.L. and G.P. conceived the idea. T.L., G.P., V.T., A.B.
and F.M.R. wrote the manuscript and all the authors contributed to editing the manuscript.

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Legend to figures
Figure 1 Structures of the synthesized thiosemicarbazones. (Htcum) cuminaldehyde
thiosemicarbazone; (Htcin) trans cinnamaldehyde thiosemicarbazone; (Htisq) quinoline-2-
carboxyaldehyde thiosemicarbazone; (HtmeFis) 5-fluoroisatin N4-methylthiosemicarbazone;
(HtFis) 5-fluoroisatin thiosemicarbazone.
Figure 2 In vitro mycelium growth inhibition and mycotoxin production of Fusarium graminearum
(A), Fusarium sporotrichioides (B) and Aspergillus flavus (C) treated with the indicated
concentrations (M) of Htcum and Htcin thiosemicarbazones. Mycotoxin released into the culture
medium or accumulated by thiosemicarbazone Htcum (Htcum-M) or Htcin (Htcin-M) treated
micelium, is expressed as ppm for A. flavus and as ppb for both Fusaria species. Growth inhibition
induced by thiosemicarbazone Htcum (Htcum-GI) or Htcin (Htcin-GI) is expressed as percent of
control. A microscopic visualisation (D) of the effect of different concentrations (0, 25, 50 and 100
M) of two thiosemicarbazones (Htcum and Htisq) on A. flavus mycelium development is also
reported.
Figure 3 In vivo mycelium growth inhibition and aflatoxin production of Aspergillus flavus treated
with 100 M of Htcum and Htcin thiosemicarbazones. Growth inhibition is expressed as % of
infected corn seeds (means are presented; n = 100; three replicates). Aflatoxin detected on kernels is
reported as ppm (means are presented; three replicates).
Figure 4 DPPH scavenging activity of thiosemicarbazones. The values are expressed in percentage
of inhibition in relation to ascorbic acid scavenging activity (100%). Data for three
thiosemicarbazones concentrations (10, 100 and 1000 µM) are presented as mean ± S.E.M. (n = 3).
Figure.5 Barley seed germination in presence of 300 μM and 600 μM of Htcum and Htcin
molecules. Significative differences from the control are indicated with asterisk. p<0.05
Figure 6 Antiproliferative activity on Hfl-1 human cell line: growth percentage after 24 hours of
treatment with increasing concentrations of Htcum and Htcin thiosemicarbazones.

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inhibition of mycelium growth (%)
inhibition of mycotoxina production (%)
F. sporot
F. gramin
A. flavus
F. sporot
F. gramin
A. flavus
26.3
24.9
11.7
9.8
38.8
26.2
10.9
7.5
76.3
58.1
2.2
-
86.9
91.4
-
-
3.4
14.3
11.4
-
5.3
5.7
99.0
96.5
44.4
67.9
34.7
Htcum
Htcin
Htisq
HtmeFis
HtFis
6.7
5.8
-
Table 1 Effects of 100 M concentrations of Htcum, Htcin, Htisq, HtmeFis and HtFis molecules
on mycelial growth (expressed as mean % inhibition in comparison with non-treated controls) and
on mycotoxin production (expressed as mean % inhibition in comparison with non-treated controls)
of Fusarium sporotrichioides (F. sporot), Fusarium graminearum (F. gramin) and Aspergillus
flavus (A. flavus).
a
mycotoxins: T2-HT2, DON and aflatoxin B for Fusarium sporotrichioides, Fusarium
graminearum and Aspergillus flavus respectively.

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Highlights
 A panel of thiosemicarbazones derivatives were synthesized
 The compounds were tested for their effect on crops (cereals) affecting fungi (Fusaria and
Aspergillus)
 Species-specific effects on fungal growth and mycotoxins biosynthesis were observed
 Two molecules were considered promising for fungal growth and mycotoxin containment
 Potential deleterious effects on seed germination and on human cells were tested