Comparative study on the antioxidant and anti-Toxoplasma activities of vanillin and its resorcinarene derivative

Article abstract of DOI:10.3390/molecules19055898

A resorcinarene derivative of vanillin, resvan, was synthesized and characterized by spectroscopic techniques. We measured the cytotoxicity (in vivo and in vitro), antioxidant and anti-Toxoplasma activities of vanillin and the resorcinarene compound. Here we show that vanillin has a dose-dependent behavior with IC₅₀ of 645 μg/mL through an in vitro cytotoxicity assay. However, we could not observe any cytotoxic response at higher concentrations of resvan (IC₅₀ > 2,000 μg/mL). The in vivo acute toxicity assays of vanillin and resvan exhibited a significant safety margin indicated by a lack of systemic and behavioral toxicity up to 300 mg/kg during the first 30 min, 24 h or 14 days after administration. The obtained derivative showed greater antioxidative activity (84.9%) when comparing to vanillin (19.4%) at 1,000 μg/mL. In addition, vanillin presents anti-Toxoplasma activity, while resvan does not show that feature. Our findings suggest that this particular derivative has an efficient antioxidant activity and a negligible cytotoxic effect, making it a potential target for further biological investigations.

Full text of DOI:10.3390/molecules19055898

Molecules 2014, 19, 5898-5912; doi:10.3390/molecules19055898
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Comparative Study on the Antioxidant and Anti-Toxoplasma
Activities of Vanillin and Its Resorcinarene Derivative
Claudio B. S. Oliveira 1, Ywlliane S. R. Meurer 1,2, Marianne G. Oliveira 1,
Wendy M. T. Q. Medeiros 3, Francisco O. N. Silva 3, Ana C. F. Brito 3, Daniel de L. Pontes 3 and
Valter F. Andrade-Neto 1,*
1
Laboratory of Malaria and Toxoplasmosis Biology/LABMAT, Department of Microbiology and
Parasitology, Bioscience Center, Federal University of Rio Grande do Norte, Av. Salgado Filho,
s/n, Lagoa Nova, Natal/RN, CEP 59078-970, Brazil
2
Memory Studies Laboratory, Physiology Department, Bioscience Center, Federal University of Rio
Grande do Norte, Av. Salgado Filho, s/n, Lagoa Nova, Natal/RN, CEP 59078-970, Brazil
3
Laboratory of Coordination Chemistry and Polymers, Institute of Chemistry, Federal University of
Rio Grande do Norte, Av. Salgado Filho, s/n, Lagoa Nova, Natal/RN, CEP 59078-970, Brazil
* Author to whom correspondence should be addressed; E-Mail: aneto@cb.ufrn.br;
Tel.: +55-84-3215-3437; Fax: +55-84-3211-9210.
Received: 7 February 2014; in revised form: 26 April 2014 / Accepted: 28 April 2014 /
Published: 7 May 2014
Abstract: A resorcinarene derivative of vanillin, resvan, was synthesized and characterized
by spectroscopic techniques. We measured the cytotoxicity (in vivo and in vitro),
antioxidant and anti-Toxoplasma activities of vanillin and the resorcinarene compound.
Here we show that vanillin has a dose-dependent behavior with IC50 of 645 µg/mL through
an in vitro cytotoxicity assay. However, we could not observe any cytotoxic response at
higher concentrations of resvan (IC50 > 2,000 µg/mL). The in vivo acute toxicity assays of
vanillin and resvan exhibited a significant safety margin indicated by a lack of systemic
and behavioral toxicity up to 300 mg/kg during the first 30 min, 24 h or 14 days after
administration. The obtained derivative showed greater antioxidative activity (84.9%)
when comparing to vanillin (19.4%) at 1,000 μg/mL. In addition, vanillin presents
anti-Toxoplasma activity, while resvan does not show that feature. Our findings suggest
that this particular derivative has an efficient antioxidant activity and a negligible cytotoxic
effect, making it a potential target for further biological investigations.
Keywords: vanillin; resorcinarene; antioxidant; anti-Toxoplasma

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1. Introduction
Toxoplasma gondii is an intracellular protozoan of the phylum Apicomplexa. It is one of the most
widespread parasites in Nature, which can infect a wide range of host species, including one-third of
the World’s human population [1]. As an opportunistic human pathogen, T. gondii may cause severe
disease in immunocompromised individuals, such as HIV positive patients, and congenitally infected
newborns [2]. In these patients, upon reactivation of a latent infection, this parasite can cause
retinochoroiditis or CNS lesions [3,4].
Infection can occur congenitally or it can be acquired through any of the forms of the parasite, such
as oocysts ingested from contaminated food with cat feces and across cysts in undercooked meat. In
addition, it can also be transmitted through acute maternal infection during pregnancy [5,6].
Nowadays, toxoplasmosis is being treated with a combination of sulfadiazine and pyrimethamine,
both key enzyme inhibitors in the biosynthesis of pyrimidines in T. gondii [7]. However, these
treatments may cause adverse reactions, including suppression of the bone marrow, teratogenic effects
in the first trimester of pregnancy and sulfadiazine hypersensitivity [5,8,9]. Thus, there is an increasing
need for new drugs and treatment regimens against toxoplasmosis. Manipulations of natural products
mainly derived from plants and insects might be a good choice in order to diminish the damage caused
by conventional treatment or even improve treatment outcome.
Cultural knowledge about medicinal plants handling represent a vital role in the discovery of novel
natural products with chemotherapeutic properties [10–15]. The use of medicinal herbs in developed
countries kept its popularity due to historical and cultural reasons even with the access to modern
medicine for most of the population [16]. On the other hand, in the developing countries, 65%–80% of
the population depends exclusively on the medicinal plants for basic healthcare. It corresponds to 80%
of the population in Africa, 71% in Chile, 40% in Colombia and 80% in Brazil [17,18].
Thereby, vanillin (4-hydroxy-3-methoxybenzaldehyde, Figure 1), a compound isolated from the
bean and pod of tropical vanilla orchid is widely used in the food and beverage industry and is
responsible for the characteristic vanilla flavor. This substance is also relevant for the synthesis of
different agrochemicals, antifoaming and pharmaceutical products [19].
Figure 1. Vanillin structure.
Besides its industrial and food application this compound has been the subject of several scientific
investigations in the last years, such as the identification of antioxidant properties [20], antimicrobial

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activity [21–23] as well as antimutagenic [24,25] and anticarcinogenic actions [26]. Conversely,
vanillin may also induce oxidative stress in yeast cells [27].
Part of these biological properties can be attributed to the fact that vanillin is a phenolic compound.
The antioxidant activity of phenols is attributed to their ability to scavenge free radicals. Other
important phenol compounds include the dihydroxybenzenes, and its derivatives [28].
Indeed, vanillin presents different functional groups, including ether and aldehyde moieties, besides
the phenolic group. The presence of a methoxy group adjacent to the phenol hydroxyl gives origin to a
group of compounds isolated from plants called vanilloids, which includes substances such as vanillin,
eugenol and capsaicin [29].
The presence of the aldehyde functional group turns possible the synthesis of vanillin derivatives by
condensation reactions. It is known from the works of Niederl and Vogel [30] that the reaction of
aldehyde with resorcinol (1,3-dihydroxybenzene) produces macrocyclic structures called
resorcinarenes (Figure 2). These structures generally adopt a bowl or vase-like conformation
characterized by the presence of a wide upper rim (resorcinol hydroxyl groups) and a narrow lower rim
(R groups from the starting aldehydes). This class of compounds is particularly interesting because the
structural organization and the presence of moieties with different polarities: polar region due the eight
hydroxyl groups in the upper rim and nonpolar region originated by the benzene rings.
The condensation of vanillin and resorcinol (Scheme 1) enables the preparation of the compound
C-4-hydroxy-3-methoxyphenylcalix[4]resorcinarene, here identified as resvan (Figure 2), with a large
number of phenolic groups.
Figure 2. Resorcinarene general structure.
Scheme 1. Resvan synthesis scheme.
Our interest in studying substances isolated from plants and its derivatives, and knowledge of
antiprotozoal activity of the vanilloids compounds [31] led us to examine the antioxidant properties of
the resorcinarene synthesized from vanillin and to evaluate the toxicity in vivo and in vitro and the
antioxidant and the anti-Toxoplasma activity of this compound compared to vanillin.

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2. Results and Discussion
2.1. Infrared Analysis
The assignments of the signals observed in the infrared (IR) vibrational spectra of resvan were
made in a qualitative form by comparison with similar data reported in the literature [32] for related
compounds and with the signals observed in the vibrational spectra of the starting materials vanillin
and resorcinol (Table 1).
Table 1. Infrared spectra and assignments of the main bands (cm−1) of the compounds
resorcinol, vanillin and resvan.
Assignment
νO-H (phenol)
Resorcinol
3447
Vanillin
3447
Resvan
3411
νC-H (aromatic)
νC-H (aldehyde)
νC=O (aldehyde)
νC-C (benzene)
νCH3 (methoxy)
δCH (methine bridge)
νC-CHO (aldehyde)
νC-O (ether)
3012
3013
3008
2855
1666
1616
1150
1596
1616 and 1518
1463
1463
1430
1265
1200
1172
1155
1123
1028
732
1204
δCH3 (methoxy)
νC-OH
1154
1123
1030
δCCH (in-plane)
νO-CH3
δC-C-CHO (out-of-plane)
δC-C=O (in-plane)
588
The IR spectrum of the compound has a broad absorption at 3411 cm−1 related to phenolic hydroxyl
stretching (νOH) of the upper and lower rim. Absorptions at 1616 and 1518 cm−1 are assigned to C-C
stretching of the aromatic rings present in the structure. As resvan formation involves the condensation
of the vanillin aldehyde group with resorcinol, the infrared peaks related to the aldehyde functional
group are no longer expected in the IR spectrum. This result was confirmed since the aldehyde bands
at 1666 (νC=O), 1267(νC-CHO), 734 (δC-CO out-of-plane) and 590 cm−1 (δC-CO in-plane) present in
the vanillin IR spectrum were not observed in the resorcinarene spectrum [33]. On the other hand, the
IR retains the peaks related to the methoxy group also observed in vanillin, however with a slight
variation in wavenumber. The peak corresponding to the methine carbon of the macrocyclic structure
was observed at 1430 cm−1.
2.2. Electronic Spectra Analysis
The electronic spectrum of the resvan in water solution presented two bands at 198 (ε = 1.7 ×
104 M−1 cm−1) and 288 nm (ε = 3.5 × 103 M−1 cm−1), besides two shoulders around 233 nm and 316 nm
(Figure 3). Spectra of benzene derivatives usually have at least three bands assigned to π-π* electronic
transitions in the UV-Vis region. The resorcinol spectrum shows these bands at 194, 218 and 272 nm.

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Such bands are shifted around 10 nm to longer wavelength in the vanillin spectrum due to the presence
of different chemical groups bonded to the benzene ring. For this substance these bands were observed
at 204, 229 and 279 nm. Additionally the presence of a fourth band at 309 nm assigned to n-π*
transitions involving the carbonyl group of the aldehyde substituent can be verified.
The resvan electronic spectrum had a similar trend showing bands in the ultraviolet region at 198,
233 and 288 nm, all assigned to π-π* transitions, and a shoulder at approximately 316 nm that extends
to the visible region assigned to n-π* transitions.
Figure 3. Electronic spectrum in water of resvan (5.5 × 10−5 mol L−1) and vanillin
(5.8 × 10−5 mol L−1).
2.3. Citotoxicity and Acute Toxicity
The cytotoxicity assay with murine macrophage cells was performed to compare the effect of
vanillin and its derivative molecule, resvan (Figure 4). In the cytotoxicity assay analysis, the T-test
revealed significant differences between resvan and vanillin treatment (t = 2.692, p = 0.0192). In the
multiple T-test, differences between vanillin and resvan treatment were detected for 500, 1,000 and
2,000 µg/mL concentrations. In this test vanillin presented an IC50 of 645 µg/mL. In other studies,
vanillin shows a dose-dependent activity, where a high concentration induces a reduction of 50% in
cell viability, including tumoral cells [26]. However, no cytotoxic effects were observed when cells
were treated with different concentrations of resvan (IC50 > 2,000 µg/mL), a vanillin derivative. The
low cytotoxicity found for vanillin in vitro corroborates the results obtained by other authors for
vanillin and the o-vanillin isomer [26,34]. The cytotoxicity results obtained suggest compounds
vanillin and resvan can be used safely in other tests in order to investigate their biological activity.

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Figure 4. Influence of vanillin and resorcinarene in RAW 264.7 macrophage cell viability
5903
after treatment for 24 h. Results are expressed in percentage of control. Three independent
experiments are performed in triplicate. (# p < 0.001 compared to 500, 1,000 and
2,000 µg/mLvanillin treatment).
μg/mL
The results suggest better biological activity and altered toxicity properties after molecular
alterations in the vanillin structure. Thus, modifications seem to be promising for other biological and
chemical studies. The acute toxicity in vivo studies of vanillin and resvan were performed based on
OECD guidelines, with modifications [35], where both exhibited a significant safety margin as
indicated by a lack of systemic and behavioral toxicity up to 500 mg/kg, that is, no related adverse
effects for animals treated with 500 mg/kg during first 30 min, 24 h and even up to 14 days after
administration of chemicals. Thus, the result indicates that resvan had no significant impact regarding
the in vivo toxicity when compared to vanillin. Also, no adverse effects were observed in the control
group receiving DMSO 8% (data not shown).
The divergent toxicity (in vivo and in vitro models) presented by vanillin, in relation to resvan,
suggests a different metabolic route for this compound. Resvan was nontoxic in both in vivo and
in vitro evaluation. This result provides further evidence of involvement of the liver in
biotransformation the vanillin into a nontoxic metabolite.
2.4. Antioxidant Activity
The DPPH method was used to evaluate the antioxidant activity of vanillin and its resorcinarene
derivative. It is an appropriate method when radical scavenging abilities of compounds are investigated
at room temperature. The measure is widely recognized for this purpose and presents high correlation
with other methods such as ferric reducing power and the total phenolics content [36,37]. The results
suggest that resvan presented an excellent free radical scavenging activity, 84.9%, when compared to
vanillin, 19.4%. The resvan activity was similar to that observed for the ascorbic acid, 93.6%, used as a
standard product for the assay (Figure 5).

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Figure 5. Antioxidant activity of ascorbic acid, vanillin and resvan assessed by DPPH
5904
radical scavenging activities method (# p < 0.001 compared to 125 µg/mL Ascorbic acid
treatment; ° p < 0.01 compared to 500 µg/mL ascorbic acid treatment; * p < 0.05 compared
to 1,000 µg/mL ascorbic acid treatment).
μg/mL
The reducing property indicates that the antioxidant compounds are electron donors and can reduce
the oxidized intermediates of the lipid peroxidation process, so that they can act as primary and
secondary antioxidants [36].
The reduction of DPPH as indicated is followed by monitoring the decrease in its absorbance at a
characteristic wavelength during the reaction. In its radical form, DPPH absorbs at 517 nm, but upon
reduction by an antioxidant (AH) or a radical species (Re), the absorption disappears [37]. From the
results obtained in the present study, it is evident that the interaction of a potential antioxidant with
DPPH* depends on its structural conformation. Certain compounds react very rapidly with the DPPH
reducing the number of DPPH molecules which corresponding to the number of available hydroxyl groups.
However, for the majority of the compounds tested the mechanism seems to be more complex [38,39].
In this paper, the results corroborate to those obtained in other studies [37,40,41]. Here, have
attempted to explain the results obtained using the DPPH method for the compound as well as ascorbic
acid. Even if antioxidant activity is extremely beneficial for scavenging free radicals, it’s even more
important in cell signaling and in defense against pathogens, an can be very harmful as they react with
several important molecules, altering the structure and function of proteins and initiating the process of
oxidation of fatty acids, which are the cell membrane lipids [42].
This higher antioxidant activity found in resvan is extremely relevant especially when compared to
vanillin, already recognized as an antioxidant substance [20,43] and resvan has more activity. This
effect is possibly due to the higher number of phenolic hydroxyls found in resvan (Figure 2) compared
to vanillin (Figure 1). These phenolic hydroxyls probably act in scavenging of the free radicals.
Another advantage of this result is that the resvan, beyond its improved antioxidant activity, also
showed lower toxicity for cultured macrophages. Hence, the structural change generated an efficient
less toxic and more active compound. These encouraging data were expected considering that the

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chemical structure of resvan presents several hydroxyl groups, which could act in the clearance of free
radicals in the DPPH assay.
2.5. Determination of in Vivo Antiprotozoal Activity of the Compounds
Mice were infected with T. gondii ME49 strain and treated for six consecutive days. The 30-day
observation period showed that mortality only occurred in the infected and untreated control groups
(19th and 23th days) and in mice treated orally with resvan (10th day). The groups of animals treated
with vanillin and sulfadiazine displayed a higher survival trend compared to the untreated control
group (Figure 6), evidencing the antitoxoplasmic activity of these regimens. Vanillin also caused a
reduction in loss body weight in mice, indicating also its contribution to reduce the disease morbidity
(Figure 7).
Figure 6. Survival curves of Swiss female mice after infection with the ME49 strain of
T. gondii and treated with sulfadiazine (200 mg/Kg), resvan (500 mg/Kg) or vanillin
(500 mg/Kg).
Figure 7. Change in body weight in Swiss mice infected with 25 cysts of T. gondii ME49
strain and treated with sulfadiazine (200 mg/Kg), resvan (500 mg/Kg) or vanillin (500 mg/Kg).
The survival time for the vanillin and sulfadiazine groups was significantly longer (p < 0.05)
compared to the infected control group. Treatment of the mice with vanillin significantly increased

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their survival rate on the 30th day after challenge by approximately 50% compared with the infected
control group (p < 0.05). These data are similar to those observed with Artemisia annua tea infusion [44],
which produced a similar effect to that achieved with the reference drug, sulfadiazine, in reducing
morbidity (Figure 8) and mortality (Figure 7), but in this work brains cysts were increased in groups
treated with vanillin‒4,533 cysts, compared to sulfadiazine treatment group‒4 cysts (Figure 8). For the
number of cysts in mice brain, one way ANOVA analysis revealed significant differences between
treatment groups [F(3,12) = 5.084, p = 0.0168). Post hoc analysis revealed an increased number of
cysts in mice brains for infected control and vanillin treatments when compared with sulfadiazine
treatment. However, no differences between sulfadizine and resvan treatment were detected by the
Bonferroni’s pos hoc test . The ME49 strain is a better alternative in in vivo antitoxoplasmic activity
tests, since it is less virulent, facilitating the chronic phase of infection [45], thereby allowing more
time to assess different treatment regimes.
The importance of vanillin in inhibiting the mortality in mice with toxoplasmosis is remarkable.
However, it is important to remark that, in the surviving animals, there was an increase in the number
of brain cysts, probably due formation of a parasite-stressed environment, important to cyst
formation [46–49]. The lower therapeutic efficiency for resvan may be due its larger antioxidant activity,
which can inhibit the formation of free radicals essential for the destruction of the parasite [50]. There
are no studies that report the action of vanillin or resvan in control of toxoplasmosis, which is
unprecedented. There are papers that report several biological activities for vanillin and its
derivatives [20–23,26,27,43], however this anti-protozoan activity was only just recently reported
among vanilloid compounds [29]. Further studies to assess the antiparasitic effects in vitro are required.
Figure 8. Number of brain cysts in Swiss mice infected with 25 cysts of T. gondii
ME49 strain and treated with sulfadiazine (200 mg/Kg), resvan (500 mg/Kg) or vanillin
(500 mg/Kg) (* p < 0.05 compared to sulfadiazine treatment).

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3. Experimental
3.1. Chemicals
Vanillin and resorcinol were purchased from Vetec Química Fina Ltda – Sigma-Aldrich
Corporation (Rio de Janeiro, Brazil). MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide)] and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). All reagents were used without further purification. Other trivial chemicals used were all
of analytical grade. Reaction progress was monitored by TLC using Fluka silica gel HF254.
3.2. Synthesis of C-4-Hydroxy-3-Methoxyphenylcalix [4] Resorcinarene (Resvan)
The compound was prepared in one-step reaction pathway (Scheme 1) using a similar procedure to
that reported by Rose [51]. Vanillin (0.766 g, 5 mmol) and resorcinol (0.552 g, 5 mmol) were
dissolved in ethanol (30 mL) and to the resulting solution concentrated hydrochloric acid (0.5 mL) was
added dropwise with continuous stirring. The solution was kept 15 h under reflux and the beige
precipitate obtained was filtered, washed with ethanol and dried. Yield: 75%.
3.3. Apparatus
Electronic spectra in the ultraviolet and visible (UV-Vis) regions were acquired with a Shimadzu
model UV 1800 spectrophotometer using water as solvent. Infrared spectra were obtained in KBr
pellets with a Thermo Nicolet Nexus 470 FT-IR spectrophotometer. When necessary (MTT Method)
the optical density (OD) at 570 nm was determined in a Uv-Vis spectrophotometer (Thermoplate).
3.4. Cytotoxicity Assay
For the cytotoxicity assay, an adapted methodology from Tempone [52] was used. RAW 264.7 cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO Inc., NY, USA)
supplemented with 40 mg/L of gentamicin and 10% fetal bovine serum (FBS; GIBCO Inc., NY, USA).
The cells were incubated in an atmosphere of 5% CO2 at 37 °C and were sub-cultured every 5 days.
RAW 264.7 murine macrophage cells were seeded at 1 × 104 cells per well in 96-well microplates
and incubated at 37 °C for 24 h in the presence of the compounds, dissolved previously in DMSO at a
concentration lower than 1% and twofold serial diluted with DMEM medium to the concentration
between 31.2 µg/mL and 2,000 µg/mL. The microplates were incubated for 24 h at 37 °C in a 5% CO2
humidified incubator. Control cells were incubated in the presence of dimethylsulfoxide (DMSO)
without drugs (1%).
The viability of the macrophages was determined with the Diphenyltetrazolium (MTT) assay.
Initially, MTT (0.5 mg/mL) was dissolved in Phosphate Buffered Saline (PBS) and sterilized through
0.22 µm membranes, and then 100 µL/well was added to a 96-well plate and left at 37 °C for 3 h.
Formazan extraction was performed with DMSO (200 µL/well) at 25 °C, and the (OD) at 570 nm was
determined in a Uv-Vis spectrophotometer.

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3.5. Animals
Swiss-Webster albino (6–8 weeks of age) were used for the toxicity and anti-Toxoplasma tests and
received water and food ad libitum. In vivo tests were performed using Guidelines for Ethical Conduct
in The Care and Use of Animals from the Federal University of Rio Grande do Norte (CEUA, Protocol
number 43/2010).
3.6. Acute Toxicity
Acute toxicity studies were performed for the compounds following, with modifications, OECD
guidelines [35]. Female Swiss mice of 8–12 weeks weighing around 23–25 g were used. Chemicals
were orally administered to the animals in a dose of 500 mg/kg. The animals were observed
continuously for 2 h for any symptoms of toxicity (piloerection, weight loss and postural
abnormalities) and /or death. They were under observation for further 2 weeks.
3.7. Scavenging Activity on 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radicals
Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by vanillin and resvan was
measured according to the method described by Ye [53], with adaptations, in which decrease in
absorbance of reaction mixture containing DPPH and compounds were measured after fixed
time interval.
Accordingly, 100 µL of vanillin, resvan and ascorbic acid in different concentrations (31.25 µg/mL
up to 1,000 µg/mL) were plated with 200 µL of DPPH solution (0.7 mM) in each well. The
microplates were incubated for 30 min in room temperature and then the absorbance was measured at
517 nm in a Uv-Vis spectrophotometer.
Percent scavenger was calculated as follows:
% S = [1 − (A1/A0)] × (100%)
where A1 corresponds to the absorbance of the samples and A0 is the absorbance of the control.
3.8. Determination of in Vivo Antiprotozoal Activity of the Compounds
In vivo tests were performed with female Swiss mice (n = 4), as previously described [44]. Initially
mice were infected orally with 25 cysts. After 24 h, animals received orally their respectively treatment
with a daily dose of vanillin (diluted in distilled water), resvan (500 mg/kg, diluted in DMSO 8%) or
sulfadiazine (200 mg/kg) for 6 days after infection. The animals were observed daily for mortality and
morbidity. All surviving animals were euthanized at thirty days of infection for the quantification of
the parasite burden in brain tissues.
3.9. Statistical Analysis
Data were analysed by Multiple T-test or one way analysis of variance (ANOVA) followed
Bonferroni’s pos hoc test when appropriate. The Mantel-Cox log-rank test was performed for survival
time analysis using GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, CA, USA).

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4. Conclusions
Thus, we can conclude that the chemical modification proposal was efficient in increasing the
antioxidant and reduce cytotoxicity, but just vanillin presented anti-Toxoplasma activity. These results
show this derivative attractive as an efficient antioxidant with negligible cytotoxic effects and
potentially interesting for further biological trials. We also highlight the importance of the vanillin
acting to combat parasite infections.
Acknowledgments
This work was supported by grants (476637/2012-0 and 301837/2012-0) from National Counsel of
Technological and Scientific Development (CNPq).
Author Contributions
C.B.S.O.; D.L.P. and V.F.A.N. participated in the conception and design of the study. C.B.S.O;
Y.S.R.M. and M.G.O. carried out the bulk of the biological assays. W.M.T.Q.M.; O.A.O.; F.O.N.S.;
A.C.F.B. and D.L.P. carried out the structural changes in compounds and chemical analysis. D.L.P.
and VFAN are the guarantors of the paper.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Tenter, A.M.; Heckeroth, A.R.; Weiss, L.M. Toxoplasma gondii: From animals to humans. Int. J.
Parasitol. 2000, 30, 1217–1258.
2. Montoya, J.G.; Liesenfeld, O. Toxoplasmosis. Lancet 2004, 363, 1965–1976.
3. Ferreira, M.S.; Borges, A.S. Some aspects of protozoan infections in immunocompromised
patients—a review. Mem. Inst. Oswaldo. Cruz. 2002, 97, 443–457.
4. Lang, C.; Grob, U.; Luder, C.G.K. Subversion of innate and adaptative immune responses by
Toxoplasma. gondii. Parasitol. Res. 2007, 100, 191–203.
5. Degerli, K.; Kilimcioğlu, A.A.; Kurt, Ö.; Tamay, A.T.; Özbilgin, A. Efficacy of azithromycin in a
murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey. Acta Trop.
2003, 88, 45–50.
6. Carruthers, V.B.; Suzuki, Y. Effects of Toxoplasma gondii infection on the brain. Schizophr. Bull.
2007, 33, 745–751.
7. Kaye, A. Toxoplasmosis: Diagnosis, treatment, and prevention in congenitally exposed infants.
J. Pediatr. Health. Care. 2011, 25, 355–364.
8. Schmidt, D.R.; Hogh, B.; Andersen, O.; Hansen, S.T.; Dalhoff, K.; Petersen, E. Treatment of
infants with congenital toxoplasmosis: Tolerability plasma concentrations of sulfadiazine and
pyrimethamine. Eur. J. Pediatr. 2006, 165, 19–25.

Molecules 2014, 19
5910
9. Haverkos, H.W. Assessment of therapy for toxoplasma encephalitis: The TE study group. Am. J. Med.
1987, 82, 907–914.
10. Rahman, M.A.; Mossa, J.S.; Al-Said, M.S.; Al-Yahya, M.A. Medicinal plant diversity in the flora
of Saudi Arabia 1: A report on seven plant families. Fitoterapia 2004, 75, 149–161.
11. Emami, S.A.; Asgary, S.; Naderi, G.A.; Ardekani, M.R.S.; Aslani, S.; Airin, A.; Kasher, T.;
Sahebkar, A. Investigation of antioxidant and anti-glycation properties of essential oils from fruits
and branchlets of Juniperus oblonga. Rev. Bras. Farmacogn. 2012, 22, 985–993.
12. Hussain, A.I.; Anwar, F.; Rasheed, S.; Nigam, P.S.; Janneh, O.; Sarker, S.D. Composition,
antioxidant and chemotherapeutic properties of the essential oils from two Origanum species
growing in Pakistan. Rev. Bras. Farmacogn. 2011, 21, 943–952.
13. Moresco, H.H.; Queiroz, G.S.; Pizzolatti, M.G.; Brighente, I.M.C. Chemical constituents and
evaluation of the toxic and antioxidant activities of Averrhoa carambola leaves. Rev. Bras.
Farmacogn. 2012, 22, 319–324.
14. Kavitha, N.; Noordin, R.; Kit-Lam, C.; Sasidharan, S. Real Time Anti-Toxoplasma gondii
Activity of an Active Fraction of Eurycoma. longifolia Root Studied by in Situ Scanning and
Transmission Electron Microscopy. Molecules 2012, 17, 9207–9219.
15. Zhai, B.; Clark, J.; Ling, T.; Connelly, M.; Medina-Bolivar, F.; Rivas, F. Antimalarial Evaluation
of the Chemical Constituents of Hairy Root Culture of Bixa. orellana L. Molecules 2014, 19,
756–766.
16. The World Health Organization (WHO). The World Health Report 1999: Making a difference;
WHO: Geneva, Switzerland, 1999; pp. 31–63.
17. Oliveira, F.Q.; Andrade-Neto, V.; Krettli, A.U.; Brandão, M.G.L. New evidences of antimalarial
activity of Bidens pilosa roots extract correlated with polyacetylene and flavonoids.
J. Ethnopharmacol. 2004, 93, 39–42.
18. Silva, N.C.C.; Fernandes Júnior, A. Biological properties of medicinal plants: A review of their
antimicrobial activity. J. Venom. Anim. Toxins. Incl. Trop. Dis. 2010, 16, 402–413.
19. Walton, N.J.; Mayer, M.J.; Narbad, A. Vanillin. Phytochemistry 2003, 63, 505–515.
20. Tai, A.; Sawano, T.; Yazama, F.; Ito, H. Evaluation of antioxidant activity of vanillin by using
multiple antioxidant assays. BBA-Gen. Subjects. 2011, 1810, 170–177.
21. Fitzgerald, D.J.; Stratford, M.; Narbad, A. Analysis of the inhibition of food spoilage yeasts by
vanillin. Int. J. Food. Microbiol. 2003, 86, 113–122.
22. Vaghasiya, Y.K.; Nair, R.; Soni, M.; Baluja, S.; Chanda, S. Synthesis, structural determination
and antibacterial activity in compounds derived from vanillin and 4-aminoantipyrine. J. Serb.
Chem. Soc. 2004, 69, 991–998.
23. Rakchoy, S.; Suppakul, P.; Jinkarn, T. Antimicrobial effects of vanillin coated solution for coating
paperboard intended for packaging bakery products. As. J. Food. Ag-Ind. 2009, 2, 138–147.
24. Imanishi, H.; Sasaki, Y.; Matsumoto, K.; Watanabe, M.; Ohta, T.; Shirasu, Y.; Tutikawa, K.
Suppression of 6-TG-resistant mutations in V79 cells and recessive spot formations in mice by
vanillin. Mutat. Res. Lett. 1990, 243, 151–158.
25. Ohta, T.; Watanabe, M.; Watanabe, K.; Shirasu, Y.; Kada, T. Inhibitory effects of flavourings on
mutagenesis induced by chemicals in bacteria. Food Chem. Toxicol. 1986, 24, 51–54.

Molecules 2014, 19
5911
26. Ho, K.; Yazan, L.S.; Ismail, N.; Ismail, M. Apoptosis and cell cycle arrest of human colorectal
cancer cell line HT-29 induced by vanillin. Cancer Epidemiol. 2009, 33, 155–160.
27. Nguyen, T.T..; Iwaki, A.; Ohya, Y.; Izawa, S. Vanillin causes the activation of Yap1 and
mitochondrial fragmentation in Saccharomyces cerevisiae. J. Biosci. Bioeng. 2014, 117, 33–38.
28. Surjadinata, B.B.; Cisneros-Zevallos, L. Biosynthesis of phenolic antioxidants in carrot tissue
increases with wounding intensity. Food. Chem. 2012, 134, 615–624.
29. Beaudry, F.; Ross, A.; Lema, P.P.; Vachon, P. Pharmacokinetics of vanillin and its effects on
mechanical hypersensitivity in a rat model of neuropathic pain. Phytother. Res. 2010, 24, 525–530.
30. Timmerman, P.; Verboom, W.; Reinhoudt, D.N. Resorcinarenes. Tetrahedron 1996, 52, 2663–2704.
31. Machado, M.; Dinis, A.M.; Salgueiro, L.; Custódio, J.B.A.; Cavaleiro, C.; Sousa, M.C.
Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: Effects on growth,
viability, adherence and ultrastructure. Exp. Parasitol. 2011, 127, 732–739.
32. Ito, H.; Nakayama, T.; Sherwood, M.; Miller, D.; Ueda, M. Characterization and Lithographic
Application of Calix[4]resorcinarene Derivatives. Chem. Mater. 2008, 20, 341–356.
33. Gunasekaran, S.; Ponnusamy, S. Vibrational spectra and normal coordinate analysis on an organic
non-linear optical crystal-3-methoxy-4-hydroxy benzaldehyde. Indian. J. Pure. Ap. Phy. 2005, 43,
838–843.
34. Alonso Moreno, Y.; Trapero Quintana, Y.M. Toxicidad aguda oral de la o-vainillina. Rev. Cubana.
Farm. 2008, 42, 0–0.
35. (OECD) Organization for Economic Cooperation and Development. Guidelines 423, Acute Oral
Toxicity-Acute Toxic Class Method. In OECD Guidelines for Testing of Chemicals; OECD:
Paris, France, 2001; pp. 1–14.
36. Clarke, G.; Ting, K.N.; Wiart, C.; Fry, J. High correlation of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging, ferric reducing activity potential and total phenolics content indicates
redundancy in use of all three assays to screen for antioxidant activity of extracts of plants from
the malaysian rainforest. Antioxidants 2013, 2, 1–10.
37. Mishra, K.; Ojha, H.; Chaudhury, N.K. Estimation of antiradical properties of antioxidants using
DPPH assay: A critical review and results. Food Chem. 2012, 130, 1036–1043.
38. Yen, G.C.; Chen, H.Y. Antioxidant activity of various tea extracts in relation to their antimutagenicity.
J. Agric. Food Chem. 1995, 43, 27–32.
39. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate
antioxidant activity. LWT-Food. Sci. Technol. 1995, 28, 25–30.
40. Liu, D.; Sheng, J.; Li, Z.; Qi, H.; Sun, Y.; Duan, Y.; Zhang, W. Antioxidant activity of
polysaccharide fractions extracted from Athyrium multidentatum (Doll.) Ching. Int. J. Biol.
Macromol. 2013, 56, 1–5.
41. Wang, Z.; Luo, D. Antioxidant activities of different fractions of polysaccharide purified from
Gynostemma pentaphyllum Makino. Carbohyd. Polym. 2007, 68, 54–58.
42. Barla, A.; Ozturk, M.; Kultur, S.; Oksuz, S. Screening of antioxidant activity of three Euphorbia
species from Turkey. Fitoterapia 2007, 78, 423–425.
43. Zhao, T.; Zhang, Q.; Qi, H.; Zhang, H.; Niu, X.; Xu, Z.; Li, Z. Degradation of porphyran from
Porphyra haitanensis and the antioxidant activities of the degraded porphyrans with different
molecular weight. Int. J. Biol. Macromol. 2006, 38, 45–50.

Molecules 2014, 19
5912
44. Azzi, A.; Davies, K.J.; Kelly, F. Free radical biology - terminology and critical thinking.
FEBS Lett. 2004, 558, 3–6.
45. Makni, M.; Chtourou, Y.; Fetoui, H.; Garoui, E.M.; Boudawara, T.; Zeghal, N. Evaluation
of the antioxidant, anti-inflammatory and hepatoprotective properties of vanillin in carbon
tetrachloride-treated rats. Eur. J. Pharmacol. 2011, 668, 133–139.
46. Oliveira, T.C.; Silva, D.A.O.; Rostkowska, C.; Béla, S.R.; Ferro, E.A.V.; Magalhães, P.M.;
Mineo, J.R. Toxoplasma gondii: Effects of Artemisia annua L. on susceptibility to infection in
experimental models in vitro and in vivo. Exp. Parasitol. 2009, 122, 233–241.
47. Silva, T.A.C.; Chioccola, V.L.P. Toxoplasma gondii acute infection: Estimation of humoral
response and blood parasitism in mice AS/n inbred. Sci. Med. 2010, 20, 88–92.
48. Dubey, J.P.; Lindsay, D.S.; Speer, C.A. Structures of Toxoplasma gondii tachyzoites, bradizoytes,
and sporozoytes and biology and development of tissue cysts. Clin. Microbiol. Rev. 1998, 11,
267–299.
49. Sullivan, W.J.; Jeffers, V. Mechanisms of Toxoplasma Gondii persistence and latency.
FEMS Microbiol. Rev. 2012, 36, 717–733.
50. McCarthy, S.M.; Davis, C.D. Prooxidant diet provides protection during murine infection with
Toxoplasma gondii. J. Parasitol. 2003, 89, 886–894.
51. Rose, K.N.; Hardie, M.J.; Atwood, J.L.; Raston, C.L. Oxygen-center laden C2h symmetry
resorcin[4]arenes. J. Supramol. Chem. 2001, 1, 35–38.
52. Tempone, A.G.; Treiger Borborema, S.E.; Andrade, H.F., Jr.; Amorim Gualda, N.C.; Yogi, A.;
Salerno Carvalho, C.; Bachiega, D.; Lupo, F.N.; Bonotto, S.V.; Fischer, D.C.H. Antiprotozoal
activity of Brazilian plant extracts from isoquinoline alkaloid-producing families. Phytomedicine
2005, 12, 382–390.
53. Ye, H.; Wang, K.; Zhou, C.; Liu, J.; Zeng, X. Purification, antitumor and antioxidant activities
in vitro of polysaccharides from the brown seaweed Sargassum pallidum. Food Chem. 2008, 111,
428–432.
Sample Availability: Samples of the compounds are available from the authors.
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