Original Article 591
mixture was then heated at reflux for 2h. The solution was
cooled in an ice bath and the resulting crystals were washed
with cold water and filtered under vacuum, giving a yield of 85%
of the titer compound.
fungi were cultivated on Sabouraud dextrose agar (SDA – Difco).
For the filamentous fungi, the suspensions were obtained
according to the reported procedures, and were adjusted to
between 1.0×106 and 5.0×106 spores/mL by microscopic enu-
meration using a hemocytometer [14]. The yeasts were prepared
according to Pfaller et al. [15], adjusting the suspension to give a
final concentration of between 1.0×106 and 5.0×106 cell/mL,
also standardized with 0.5 on the McFarland scale (λ=530nm).
All the chalcones were synthesized by addition of 50% w/v aq.
NaOH solution (1.5mL) to a well stirred solution of appropriate
substituted acetofenone (1.7mmol) and the appropriate substi-
tuted benzaldehyde (1.7mmol) in methanol (50mL). The reac-
tion mixture was stirred overnight at room temperature,
according to the methodology described previously [12]. The
mixture was then neutralized with 1N HCl and the product fil-
tered and extracted with chloroform. The combined organic lay-
ers was dried (Na2SO4), filtered and evaporated. The products
were purified by column chromatography or recrystallization
from ethyl alcohol or ethyl alcohol/water.
Antimicrobial evaluation
The minimum inhibitory concentration (MIC) was determined
for the organisms by the agar dilution method, which was car-
ried out on slants (1mL). Stock solutions of each compound in
dimethylsulfoxide (DMSO) was diluted to give serial 2-fold dilu-
tions which were added to each medium (MHA for bacteria and
SDA for fungi), resulting in 10 different concentrations ranging
from 10 to 100μg/mL. Afterwards, a volume of 1μL of inoculum
suspension, prepared previously, was inoculated with a sterile
loop to each slant, with the exception of the sterile control. The
antibacterial and antifungal agents, vancomycin (Sigma V2002)
and ketoconazole (Sigma K1003), respectively, were included in
the assay as positive control. The final concentration of DMSO in
the assay did not exceed 2%. A drug-free saline solution (0.86%)
was used as a blank control. Each assay was repeated 3 times.
The slants were incubated at 35°C for the bacteria and yeasts
and at 25°C for the hialohyphomycete and dermatophyte strains.
MICs were visually recorded at 48h for yeasts, and at a time
according to the control fungus growth, for the rest of fungi.
Chemistry – General experimental procedures
Melting points were determined with a Microquimica AP-300
apparatus (Florianópolis, Brazil) and were uncorrected. IR spec-
tra were recorded with a Bomem 100 (Québec, Canadian) on KBr
disks. The 13C and 1H-NMR spectra were recorded on a Brucker
200MHz (Karlsruhe, Germany). Elemental analysis was deter-
mined with a Perkin Elmer 2400 (Norwalk, USA). The percent-
ages of C and H were in agreement with the product formula
(within±0.4% of theoretical values). The compounds were dis-
solved in deuterated solvents from commercial sources with
TMS as the internal standard. The purity of the synthesized sub-
stances was monitored by thin-layer chromatography (TLC)
using Sigma (St. Louis, USA) silica pre-coated plastic plates of
200μm in thickness with several solvent systems of different
polarities. Spots were visualized by short-wave UV light and
iodine vapor. Spectral data (IR, 1H- and 13C- NMR) and elemental
analysis were in good agreement with the structures. The log P
values were obtained from the on-line JME molecular editor
program Molinspiration, courtesy of Peter Ertl of Novartis, avail-
bin/properties.
Cytotoxicity activity
The brine shrimp lethality test (BST) (Artemia salina Leach) was
used for cytotoxicity determination of the compounds. Dried
brine shrimp eggs (1g/L)(Maramar, Arraial do Cabo, RJ, Brazil)
were placed in artificial sea water [16]. The pH was adjusted to
9.0 using Na2CO3 and incubated at 22–29°C with strong aera-
tion, under a continuous light regime (2000 Lux). Approximately
12h after hatching the phototropic nauplii were collected for the
cytotoxicity assay. Serial dilutions were made in the wells of
96-well microplates in triplicate in 100μL artificial sea water.
Control wells with DMSO were included in each experiment. A
suspension of nauplii containing 10 organisms was added to
each well and the plate covered and incubated at 22–29°C. The
toxicity was determined after 24h of exposure. The numbers of
survivors were counted. Larvae were considered dead if they did
not exhibit any internal or external movement during several
seconds of observation [17].
Biological assay – Microorganisms
For the antimicrobial evaluation, strains from the American Type
Culture Collection (ATCC), Rockville, MD, USA, and CEREMIC (C),
Centro de Referencia Micológica, Facultad de Ciencias Bioquími-
cas y Farmacéuticas, Rosario, Argentina, and Control Lab (CL),
Rio de Janeiro, Brazil, were used; Bacteria used were Bacillus
cereus ATCC 14579, Bacillus subtilis ATCC 23858, Enterobacter
cloacae ATCC 35030, Escherichia coli ATCC 11775, Proteus mirabi-
lis ATCC 25933, Pseudomonas aeruginosa ATCC 27853, Salmo-
nella typhimurium ATCC 14028, Staphylococcus aureus ATCC
6538P, Staphylococcus saprophyticus ATCC 35552 and Strepto-
coccus agalactie ATCC 13813, and the fungi used were Aspergillus
flavus ATCC 9170, Aspergillus fumigatus ATCC 26934, Aspergillus
niger ATCC 9092, Rhizopus sp CL 35, Microsporum canis C112,
Microsporum gypseum C115, Trichophyton mentagrophytes ATCC
9972, Trichophyton rubrum C137, Cryptococcus neoformans
ATCC 32264, Candida albicans ATCC 10231 and Candida krusei
ATCC 6582.
Statistical analysis
Antimicrobial activity assays were performed in triplicate and
repeated twice and the arithmetic means of the MIC were calcu-
lated and reported. Cytotoxicity assays were carried out in
duplicate, and repeated twice for each compound. Lethal dose
(LC50) values and 95% confidence intervals were determined
from 24h counts using the probit analysis method decribed by
Finney [18]. In cases where data were insufficient for this tech-
nique, the dose-response data were transformed into a straight
line by means of a logic transformation [19].
Media and inocula
The bacteria used were cultivated on Mueller-Hinton agar (MHA
– Difco) at 35°C for 24h. Cell suspension in saline (0.86%) was
adjusted to give a final concentration of 1.5×108 cell/mL, stand-
ardized with 0.5 on the McFarland scale (λ=530nm) [13]. The
Tristão TC et al. Antimicrobial and Cytotoxicity of Chalcones… Arzneimittelforschung 2012; 62: 590–594