Synthesis of chitosan derivatives with organoselenium and organosulfur compounds: Characterization, antimicrobial properties and application as biomaterials

Carbohydrate Polymers 219 (2019) 240–250

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Synthesis of chitosan derivatives with organoselenium and organosulfur
compounds: Characterization, antimicrobial properties and application as
biomaterials

T

Matheus S. Gulartea, João M. Anghinonib, Laura Abenanteb, Guilherme T. Vossc,
Renata L. de Oliveirac, Rodrigo A. Vaucherd, Cristiane Luchesec, Ethel. A. Wilhelmc,
⁎
⁎
Eder J. Lenardãob, , André R. Fajardoa,
a
Laboratório de Tecnologia e Desenvolvimento de Compósitos e Materiais Poliméricos (LaCoPol), Universidade Federal de Pelotas (UFPel), Campus Capão do Lễo, 96010900, Pelotas, RS, Brazil
b
Laboratório de Síntese Orgânica Limpa (Lasol), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil
c
Laboratório de Pesquisa em Farmacologia Bioqmica (LaFarBio), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil
d
Laboratório de Pesquisa em Bioqmica e Biologia Molecular de Micro-organismos (LaPeBBioM), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão,
96010-900, Pelotas, RS, Brazil

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article:
Chitosan (PubChem CID: 71853)
Citronellal (PubChem CID: 7794)
Citral (PubChem CID: 638011)
Poly(vinyl alcohol) (PubChem CID: 3083375)
Glutaraldehyde (PubChem CID: 3485)
2,4-dinitrochlorobenzene (PubChem CID: 6)
Tris-hydrochloride (PubChem CID: 93573)
N,N,N′,N′-tetramethylbenzidine (PubChem
CID: 9702)

In this study, Schiff bases of chitosan (CS) were synthesized using citronellal, citral, and their derivatives containing selenium and sulfur. Organoselenium and organosulfur compounds show attractive biological and
pharmaceutical activities, which can be beneficial to CS-based materials. From the characterization analyses, it
was found that the CS-derivatives containing organoselenium and organosulfur compounds exhibited the highest
conversion degrees (23 and 28%). Biological assays were conducted using films prepared by the blending of CSderivatives and poly(vinyl alcohol). The antimicrobial evaluation indicated that the film prepared with the
sulfur-containing CS was the most active against the tested pathogens (Escherichia coli, Staphylococcus aureus, and
Candida albicans) since it reduced considerably their counts (42.5%, 17.4%, and 18.7%). Finally, in vivo assays
revealed that this film attenuates atopic dermatitis-like symptoms in mice by suppressing the increase of myeloperoxidase (MPO) activity and reactive species (RS) levels induced by 2,4-dinitrochlorobenzene (DNCB). In
summary, CS-derivatives containing chalcogens, mainly organosulfur, are potential candidates for biomedical
applications such as for the treatment of chronic skin diseases.

Keywords:
Chitosan
Organoselenium
Organosulfur
Schiff base
Antimicrobial activity
Atopic dermatitis

1. Introduction
In the past few years, the use of natural compounds to development
of functional materials for a wide range of application has attracted
great attention, which can be assigned to the attractive and versatile
properties of such compounds (e.g. biocompatibility, biodegradability,
low-toxicity, easy availability, among others) (Brodin, Vallejos, Opedal,
Area, & Chinga-Carrasco, 2017; Dugmore, Clark, Bustamante,
Houghton, & Matharu, 2017; Mika, Csefalvay, & Nemeth, 2018). In

particular, polysaccharides, which are claimed as potential substitutes
for oil-derived polymers, have been used to elaborate several kind of
materials applicable in different areas (biomedicine, pharmacy, agriculture, hygiene, oil prospecting, among others) (Ali et al., 2018;
Guilherme et al., 2015; Huang et al., 2018; Lessa, Gularte, Garcia, &
Fajardo, 2017; Ma, Liu, Dong, Wang, & Hou, 2015). Chitosan is one
example of polysaccharide largely used for development of novel materials.
Chitosan (CS), a well-known chitin derivative, exhibits interesting

⁎
Corresponding authors at: Programa de Pús-graduaỗóo em Quớmica (PPGQ), Centro de Ciờncias Quớmicas, Farmacêuticas e de Alimentos, Universidade Federal de
Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil.
E-mail addresses: (E.J. Lenardão), (A.R. Fajardo).

/>Received 20 November 2018; Received in revised form 11 February 2019; Accepted 10 May 2019
Available online 11 May 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Scheme 1. The synthesis of CS-derivatives (3a-d) via Schiff base preparation. *Note: Ph = phenyl (C6H5).

2. Materials and methods

properties such as biodegradability, biocompatibility, nontoxicity, antimicrobial activity, among others (Rinaudo, 2006). Despite these intrinsic properties, there has been a growing interest in the chemical
modification of CS in order to improve some features and widen its
applicability (Alves & Mano, 2008; Martins et al., 2015). CS is a linear
polysaccharide composed of repeated β-(1–4) linked units of either 2amino-2-deoxy-β-D-glucopyranose (glucosamine) or 2-acetamido-2deoxy-β-D-glucopyranose (glucosacetamide), depending on the degree
of N-acetylated units (Rinaudo, 2006). It exhibits two types of reactive
groups bound to the main backbone: free amine groups in the deacetylated units, and hydroxyl groups in the C3 and C6 carbon atoms in
acetylated or deacetylated units (Alves & Mano, 2008). The presence of
these groups leads to the possibility of various chemical modifications,
such as the formation of Schiff bases by the reaction with aldehydes or
ketones (Jin, Wang, & Bai, 2009; Yue et al., 2017), the acylation of the
hydroxy groups by reaction with acyl (Santos et al., 2015), the biocatalytic oxidation of the hydroxyl groups to aldehydes and carboxylic
acids (da Silva, Krolicka, van den Broek, Frissen, & Boeriu, 2018) and
the sulfonation of the amino and hydroxyl groups, by the reaction with
ClSO3, (Khan & Siddiqui, 2015) among others (Tharanathan & Kittur,
2003). In particular, the free amine groups allow preparing Schiff bases
by reacting them with aldehydes or ketones (linear or aromatics) (Jin
et al., 2009; Yue et al., 2017). Herein, we investigated the synthesis of
Schiff bases by reacting CS with citronellal, citral, and their derivatives
containing selenium and sulfur in order to obtain CS-derivatives with
enhanced biological properties. Previous studies demonstrated that
organoselenium and organosulfur compounds have several attractive
biological and pharmaceutical activities (e.g. antibacterial, antifungal,
and antioxidant), which can be associated with the ability of selenium
and sulfur to stabilize free radicals (Bhattacherjee et al., 2017; Vogt
et al., 2018). Moreover, the graft of this kind of compound on CS

backbone was not reported in the literature so far.
The efficacy of the CS-derivations in biomedical applications was
tested using films prepared by blending of such derivatives with poly
(vinyl alcohol) (PVA), a synthetic polymer with hydrophilic and biocompatible properties (Choo, Ching, Chuah, Julai, & Liou, 2016;
Teodorescu, Bercea, & Morariu, 2018). In addition, PVA shows an excellent film-forming ability, which confers to the final material desirable mechanical properties. As demonstrated in the literature, materials
formulated by crosslinked CS usually exhibit poor mechanical properties (e.g. lack of flexibility, low mechanical strength, etc.), which restricts their application (Kiuchi, Kai, & Inoue, 2008; Vieira, da Silva, dos
Santos, & Beppu, 2011). Taking into account the development of materials for wound dressing purposes, a promising strategy to overcome
this limitation is to blend CS and PVA to obtain the combined properties
of both polymers. Here, in vitro and in vivo experiments were performed
in order to investigate the antimicrobial activities and potential uses of
these CS-derivatives/PVA films for the treatment of atopic dermatitislike skin lesions.

2.1. Materials
All materials utilized in this work are described in Supporting information.
2.2. Synthesis of CS-derivatives
CS-derivatives were synthesized via the preparation of Schiff bases
by the reaction of raw CS (1) with different aldehydes (2a-d). The CS
(100 mg) was solubilized in acetic acid aqueous solution (40 mL, 1.0 v/
v-% of acetic acid) into a 100 mL round-bottomed flask and heated up
to 55 °C. Consequently, an excess of the aldehyde (2 mmol, 2a-d),
previously solubilized in ethanol (2 mL), was added to the reaction
mixture, which was kept at 55 °C under magnetic stirring for 3 h (see
Scheme 1). The amount of aldehydes was calculated based on the
amount of the amino groups presented in the CS structure (0.02 mmol
in 100 mg). The resulting CS-derivatives (3a-d) were recovered after
the evaporation of the solvent and exhaustively washed with ethanol to
remove the non-reacted starting materials. Finally, the products were
vacuum-dried at 70 °C. Trying to know the yield, the excess of aldehyde
was quantified and in every cases almost half has been recovered. This
suggests that the other half reacted with the CS. On average of

200 ± 40 mg of the products 3a-3d were obtained.
2.3. Characterization of the CS-derivatives (3)
The products 3a-d were characterized using Fourier transform infrared (FTIR) spectroscopy, Nuclear Magnetic Resonance (NMR), and
energy-dispersive X-ray (EDX) analysis. FTIR analyses were performed
in a Shimadzu IR-Affinity-1 (Japan) equipment operating in the spectral
region of 4000–400 cm−1. Before the spectra acquisition, the products
were ground with spectroscopic grade KBr and pressed into disks.
Hydrogen (1H) NMR spectra were recorded using a Bruker Avance DPX
400 spectrometer at 400 MHz. All spectra were acquired using a mixture of deuterated solvents (D2O/acetic acid-d4 10–20 wt-%) and tetramethylsilane (TMS) was used as internal standard. EDX spectra were
recorded using a Jeol JSM-6610LV Scanning Electron Microscopy
(SEM) microscope (USA) equipped with an EDX analyzer. Before SEM
visualization, the samples were gold-coated by sputtering.
2.4. Synthesis of films based on CS-derivatives
To investigate the potential of the CS-derivatives 3 in practical uses,
films based on the products 3a-d blended with PVA were synthesized by
a solvent casting method. In general lines, each CS-derivative 3
(100 mg) was solubilized in 20 mL of acetic acid solution (1.0 v/v-%)
and, then, blended with a PVA solution (150 mg in 30 mL of distilled
water). The resulting system was homogenized at room temperature for
1 h. Next, 25 μL of glutaraldehyde (crosslinker agent) was added
dropwise to the polymeric solution, which was gently poured into a
Petri dish (polystyrene, round-shape). After the solvent casting (vacuum-oven, 40 °C for 24 h) the as-prepared film was peeled off from the
241


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10231)

were diluted to adjust a microbial count of 2 × 108 CFU/mL. For the
experiments, film samples were aseptically sectioned (3 × 3 cm) and
distributed in sterile Petri dishes. These samples were inoculated with
1 mL of tested bacterial solution. Immediately after inoculation, ca.
15–20 mL of the sterile standard counting agar (PCA) medium was
added to the Petri dishes, which were incubated for 24 h at 37 °C under
aerobic conditions. Petri dishes without the film samples (inoculum
only) were used as the control. After the incubation period, microbial
colonies were counted using a colony counter equipment and the mean
log10 CFU/mL was calculated. The bacterial reduction to evaluate the
effectiveness of the tested materials was calculated as the difference
between the log10 CFU of the inoculum and the log10 CFU recovered
from the film-containing samples.

Petri dish, immediately washed with distilled water and then ovendried (40 °C for 24 h). The film samples synthesized using the different
CS-derivatives were labeled as CS3a-PVA, CS3b-PVA, CS3c-PVA, and
CS3d-PVA, respectively. For comparative purposes, a film sample was
prepared using the raw CS (1) and PVA (labeled here as CS1-PVA).
2.5. Characterization of the films
FTIR analyses were performed as aforementioned. SEM images were
recorded using a Jeol JSM-6610LV microscope using an acceleration
voltage of 8–10 kV. Prior to the SEM visualization, the surface of the
samples was gold-coated by sputtering.
2.6. Swelling and stability experiments
Swelling experiments were performed in order to investigate the
liquid uptake capacity of the as-prepared films. These experiments were
performed using dry samples previously weighed (w0), which were
placed into vials filled with 50 mL of simulated wound fluid (SWF)
(2 w/v-% of bovine albumin, 0.02 mol/L calcium chloride, 0.4 mol/L
sodium chloride, pH 7.4). This system was kept at 37 °C under mild

stirring (100 rpm) and at pre-determined time intervals, the samples
were withdrawn, blotted carefully with tissue paper to remove the
surface-adhered liquid droplets and, then, reweighed (wt). The swelling
degree at different immersion times was calculated using Eq. (1) (Iqbal
et al., 2015). Each swelling experiment was performed in triplicate.

w − w0 ⎤
Swelling (%) = ⎡ t
x 100
⎢
⎦
⎣ w0 ⎥

2.8. In vivo assays
All in vivo assays performed in this work are described in Supporting
information.
2.9. Statistical analysis
All the collected data in this study were expressed as means ±
standard deviation regarding a minimum of three replicates (n ≥ 3).
The statistical analysis was performed by using OriginPro® 8.5 software.
For in vivo assays, data were expressed as mean ± standard error
medium (S.E.M.). Statistical analysis was performed using one-way
ANOVA followed by the Newman-Keuls test when appropriated
(GraphPad Prism software). Values of p < 0.05 were considered statistically significant.

(1)

Using a similar gravimetric approach, the stability of the CS-derivatives films in SWF was investigated. For this, pre-weighed samples
were placed in sealed tubes filled with 10 mL of SWF and kept at 37 °C.
At desired time intervals (1, 2, and 3 weeks), the samples were withdrawn, washed with ultrapure water to remove undesired compounds

and dried to a constant weight (oven, 50 °C). The stability of each film
sample was calculated using the Eq. (2).

w
Stability (%) = ⎡ 1 ⎤ x 100
⎢
⎣ w0 ⎥
⎦

3. Results and discussion
3.1. Characterization of CS-derivatives (3)
In this contribution, FTIR and 1H NMR spectroscopic techniques
were used to characterize the CS-derivatives (3a-d) as well as to
quantify the percentage of substitution in each case. The FTIR spectra of
raw CS (1) exhibited the characteristic bands associated with this
polysaccharide, as noticed in the literature (Fig. 1a) (Lawrie et al.,
2007). In this spectrum, it was observed a broad band centered at
3417 cm−1 assigned to OeH and NeH stretching vibrations and bands
at 2920 cm-1, 1664 cm-1, and 1600 cm−1 due to C–H stretching, amide I
and NeH bending from amine and amide II bonds (Lawrie et al., 2007).
Additional bands at 1461 cm-1, 1380 cm-1, 1155 cm-1 and 1031 cm-1 are
associated with the −CH2– bending, −CH3 symmetrical deformation,
antisymmetric stretching of C–O-C and C–N bonds and skeletal

(2)

where w0 (mg) is the sample initial weight and w1 (mg) is the weight
after soaking. Again, this experiment was performed in triplicate.
2.7. In vitro antimicrobial activity
The antimicrobial activity of the as-synthesized films was evaluated

using the Pour plate method with minor modifications (Iqbal et al.,
2015). Briefly, overnight cultures of Escherichia coli (ATCC 25922),

Fig. 1. FTIR spectra of raw Cs and Cs-derivatives. (A) Full and (B) zoom-in views of the spectra.
242


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

3.2. Characterization of the CS-derivatives based films

vibration of C–O stretching (Lawrie et al., 2007; Lessa, Nunes, &
Fajardo, 2018). Overall, the spectra of CS-derivatives (3a-d) exhibited
the bands proceeding from CS with some discrepancies. As noticed, the
bands assigned to OeH and NeH stretching vibrations (3600-3100 cm1
) are sharpened while bands assigned to NeH bending disappeared
indicating that the amine groups of CS (1) have reacted with the aldehydes (2a-d). Moreover, the appearance of shoulder-type bands at
1628 cm-1 and bands at 1562 cm-1 confirms the imine (C]N) bond
formation, suggesting that the aldehydes were covalently bound to the
CS backbone (Fig. 1b). These data corroborate other similar studies
dealing with the derivatization of CS via Schiff base preparation (Jin
et al., 2009; Marin, Simionescu, & Barboiu, 2012; Tamer et al., 2016).
Furthermore, the spectra of the CS-derivatives (3a-d) also showed noticeable changes in bands associated with the C–H and = C–H vibration
modes (3000-2800 cm-1 stretching, 1480-1350 cm-1 bending and 1000650 cm-1 out of plane bending) and the appearing of bands at 1652 cm-1
due to C]C stretching of alkenes. In particular, the spectra of CS-derivatives containing the –SePh (3c) and –SPh (3d) groups presented
additional bands at 1542 cm-1 associated with the C]C stretching of
aromatic rings and bands at 536 cm-1 and 648 cm-1 assigned to Se-C and
SeC bonds (Devillanova, Sathyanarayana, & Verani, 1978; Vien,

Cotthup, Fatoley, & Crasselli, 1991).
The 1H NMR spectrum of raw CS 1 (Fig. S1) presents the typical
resonance signals assigned to this polysaccharide and their chemical
shifts are in agreement with previous studies (Heux, Brugnerotto,
Desbrieres, Versali, & Rinaudo, 2000). The 1H NMR spectra of the CSderivatives (3a-d) (Figs. S2–S5) exhibited the resonance signals expected for the CS backbone with some noticeable modifications. In
general lines, the CS-derivatives spectra exhibited new resonance signals in the chemical shift ranges of 1.0–2.0 ppm (−CH3 groups),
1.5–3.5 (aliphatic CH and CH2 groups), and 5.0–5.5 ppm (vinyl hydrogen atom). For the CS-derivatives containing the –SePh (3c) and
–SPh (3d) groups, the NMR spectra exhibited new resonance signals in
the region of 7.0–8.0 ppm due to the hydrogen atoms of the phenyl ring.
Moreover, all spectra showed new resonance signals attributed to the
imine hydrogen in the region of 9.0–10.0 ppm confirming the CS-derivatization. This inference is corroborated by the FTIR data and other
similar studies from literature (Jin et al., 2009; Marin et al., 2012). The
degree of conversion (DC) of –NH2 groups of CS in imine (–N = CH–)
units has been estimated using the equation DC = (AN=CH)/(AH1 x
0.85) x 100, where AN=CH and AH1 are the integrated areas of these
respective resonance signals and 0.85 reflects the degree of deacetylation of the CS used in this study (Marin et al., 2012). The DC values
calculated for 3a, 3b, 3c, and 3d were 11%, 18%, 23% and 28%, respectively. Table 1 summarizes a general description of the experimental conditions and the calculated DC values for each CS-derivative.
As noticed, the highest conversion values were verified to the aldehydes containing the –SePh (2c) and –SPh (2d) groups suggesting a
higher reactivity of such compounds as compared with citronellal (2a)
and citral (2b). This result is agrees with previous data reported by
Marin et al. (2012), which demonstrated that citral shows low reactivity with CS when the reaction is performed in aqueous medium. On
the other hand, considering the further use of CS in the development of
biomaterials, it is expected that the modification of the CS structure be
restrict to a degree where its original physicochemical and biological
properties remain preserved (Mourya & Inamdar, 2008).
Finally, SEM/EDS analysis was used to investigate the elemental
composition of the CS-derivatives containing the –SePh and –SPh
groups. The EDS spectra recorded to 3c and 3d compounds (Fig. S6)
revealed the presence of signals associated with the elements that
compose the CS chains (C, O, and N) and signals confirming the presence of the elements Se or S in derivatives 3c and 3d. Taking together,

these findings confirm the successful synthesis of the CS-derivatives,
including examples of organoselenium and organosulfur compounds.

The chemical crosslinking of the as-synthesized films with glutaraldehyde was characterized using FTIR spectroscopy. The spectra recorded to each film sample are depicted in Fig. 2. Glutaraldehyde is a
dialdehyde commonly used as a crosslinking agent due to its ability to
react with different functional groups. Considering the polymers used
through in this study (CS and PVA), glutaraldehyde may react primarily
with the amino groups of CS and, subsequently, with the hydroxyl
groups of PVA. As highlighted in the literature, glutaraldehyde converts
the amino groups of CS in imine groups and forms acetal rings with PVA
(Hoffmann, Seitz, Mencke, Kokott, & Ziegler, 2009; Lessa et al., 2018).
The CS1-PVA spectrum shows the characteristic bands of raw CS and
PVA with some discrepancies. For instance, the broadband assigned to
OeH stretching vibration is shifted to higher wavenumber region suggesting the decreasing of intramolecular H-bonds due to the crosslinking process. Moreover, the obvious increase of intensity regarding
the bands associated with the C–H stretching (2900–2800 cm−1) and
C–H bending (1460-1380 cm−1) is assigned to the presence of aliphatic
CH and CH2 groups proceeding from glutaraldehyde and PVA. The
shoulder band at 1710 cm−1 is due to residual acetate groups remaining on PVA, the main component of the films, while the band at
1652 cm−1 is associated with the imine bonds (C]N) formed between
the CS and glutaraldehyde, confirming the crosslinking process
(Hoffmann et al., 2009; Lessa et al., 2018). The increase of intensity
observed for the band at 1458 cm−1 can be associated with the increase
of C–N bonds as a result of the incorporation of glutaraldehyde structure in the film matrix. Finally, the bands at 1200-900 cm−1 region are
broadened due to the increase of C–O and CeOeC bonds due to the
acetal ring formed from the reaction with glutaraldehyde. In light of
these results, it can be inferred that the CS1-PVA matrix is a full interpenetrating network that results from the crosslinking of both
polymers with glutaraldehyde. A similar analysis can be done from the
recorded FTIR spectra of the formulated films using the CS-derivatives.
As compared with the Schiff bases (3a-d) (see Fig. 1), these spectra
exhibited the bands that confirm the blending of Cs-derivatives with

PVA and the crosslinking of such polymers with glutaraldehyde. It is
worth to note that the bands assigned to the imine bonds formed from
the reaction between the remaining amino groups from the CS-derivatives and the crosslinker are overlapped by the imine bands proceeding
from the starting compounds (3a-d).
SEM images of the synthesized films were utilized to investigate
their surface morphology (Fig. S7, Supporting information). Overall, all
films exhibited homogeneous surfaces with poor roughness and without
the presence of agglomerates and pores. At high magnification, there is
not indicative of phase separation in these blended materials suggesting
good compatibility between the PVA and the CS/CS-derivatives. This
good compatibility is likely due to the crosslinking process, which can
often generate materials with stable morphologies (Rumer &
McCulloch, 2015). It should be mentioned that the presence of some
marks on the surfaces of CS3c-PVA and CS3d-PVA films is due to slots in
the Petri dishes (polystyrene) utilized as a mold. From a macroscopic
viewpoint, all the film samples are visually similar, all of them with
some flexibility and mechanically robust for handling. This feature can
be assigned to PVA, which improves the mechanical performance of
polysaccharides-based materials that sometimes fail to meet specific
requirements due to their poor mechanical properties (Teodorescu
et al., 2018).
3.3. Antimicrobial activity
The interest in materials expressing antimicrobial activity has increased in the last years, especially in materials designed for biomedical
applications. For instance, antimicrobial activity is particularly interesting for biomaterials applied in the treatment of chronic skin diseases
or skin wounds, because they preventing contamination and infection
243


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Table 1
Experimental molar amounts used in this study and the calculated DC values for each CS-derivative.
Molar amount of amino
groups (mmol)a

Molar amount of
aldehyde
(mmol)

Amino groups/aldehyde
molar ratio

Degree of conversion
(%)b

Citronellal (2a)

0.02

0.2

1:10

11

3b

Citral

(2b)

0.02

0.2

1:10

18

3c

Phenylselanyl citronellal
(2c)

0.02

0.2

1:10

23

3d

Phenylthio citral (2d)

0.02

0.2

1:10

28

CS-derivatives

Tested aldehyde

3a

a
b

Aldehyde structure

Considering 100 mg of CS (deacetylation degree of 85%).
Calculated from 1H NMR data.

Rasheed, Iqbal, Hu et al., 2017; Iqbal et al., 2015). Overall, the use of
natural compounds such as plant-based extracts (or their derivatives)
seems to be a promising strategy (Iqbal, Kyazze, Locke, Tron, &
Keshavarz, 2015; Iqbal, Kyazze, Locke, Tron, & Keshayarz, 2015).
Herein, the antimicrobial activities of the prepared films against E.
coli (Gram-negative bacteria), S. aureus (Gram-positive bacteria), and C.
albicans (fungal pathogen) were examined. The quantity of microbe
after the incubation was evaluated by CFU counts, and the results are
displayed in Fig. 3.
As noticed, the film synthesized using raw CS (CS1-PVA) exhibited a
neglected effect on the reduction of both bacteria counts (E. coli and S.

aureus) and C. albicans counts when compared with the control groups.
CS shows a well-known antimicrobial activity, which is linked to its
cationic groups that interact electrostatically with negatively charged
microbial cell membranes inhibiting the growth of microorganisms and
leading to the leakage of intracellular electrolytes (Goy, de Britto, &
Assis, 2009; Sahariah & Masson, 2017). Generally, the blending of CS
with PVA renders films with antimicrobial activity, since this property
is not observed for films prepared only with PVA (Bonilla, Fortunati,
Atares, Chiralt, & Kenny, 2014). Here, CS was blended with PVA and,
further, this blend was crosslinked (i.e. reducing the free amino groups
of CS), which probably reduced the positive charge density within the
film matrix and impaired the antimicrobial activity. In a similar way,
the film sample synthesized using compound 3b (CS3b-PVA) did not
present a noticeable reduction in the microbial counts as compared
with the control. Although some studies have demonstrated the antimicrobial activity of citronellal (2b) against some strains of bacteria
and fungi, such activity is associated with the use of significant amounts
of this monoterpenoid (Lopez-Romero, Gonzalez-Rios, Borges, &
Simoes, 2015). According to the NMR data, the percentage of 2b

Fig. 2. FTIR spectra of the CS and CS-derivatives based films.

of the treated sites and, also, control the spread of pathogens
(Daeschlein, 2013; Dai, Tanaka, Huang, & Hamblin, 2011). Currently,
different approaches have been utilized to endow biomaterials with
antimicrobial properties (e.g. chemical modification, incorporation of
synthetic antimicrobials, metal nanoparticles, and so on) (Bilal,
Rasheed, Iqbal, Hu et al., 2017; Bilal, Rasheed, Iqbal, Lib et al., 2017; K.
S. Huang et al., 2016). One of the challenges faced by the researchers is
to overcome inherent cytotoxicity issues due to the modification of
these biomaterials. In this sense, some elegant strategies have been

reported in the literature to overcome this eventual drawback (Bilal,

244


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Fig. 3. (a) E. coli, (b) S. aureus and (c) C. albicans counts (log10 CFU/mL) after incubation with different CS-based films. Data are reported as mean ± standard error
of the mean (S.E.M.) (One-way analysis of variance/Newman-Keuls test). **p < 0.05 denotes significant levels when compared with control and CS1-PVA groups.

applied in wound healing is the capability of such material to absorb
and retain the exudate secreted from the wound (Boateng, Matthews,
Stevens, & Eccleston, 2008). Materials with low absorption ability are
inefficient to remove the wound exudate, leading to its accumulation at
the wound surface, which results in microbial attacks and further
complications. Additionally, hydrophilic materials are able to keep the
local moisture, which is perfect for keeping skin and tissue hydrated
(Hoque, Prakash, Paramanandham, Shome, & Haldar, 2017). In this
sense, swelling experiments were performed in order to investigate the
liquid uptake capacity of the selected film samples (CS3a-PVA, CS3cPVA, and CS3d-PVA) when exposed to SWF. The swelling curves built
for each film sample are shown in Fig. 4a.
As observed in Fig. 4, all film samples exhibited fast swelling rate
just after their immersion in SWF, which can be assigned to the hydrophilic nature of PVA and CS-derivatives. In particular, the liquid
uptake in this kind of blend generally increased thanks to the presence
of PVA, which contains a remarkable amount of hydroxyl groups distributed along their molecules (Kamoun, Chen, Eldin, & Kenawy, 2015).
Here, the swelling degree of all samples increased rapidly with increasing swelling time and close to 60 min after the beginning of the
experiment, a discrepant behavior between the film samples was observed. For CS3a-PVA, the swelling rate slows down and the equilibrium was achieved close to 120 min after immersion in SWF. The
maximum swelling degree calculated for this sample under these experimental conditions was 700% (i.e. this sample is able to absorb a

liquid amount 7-folds higher than its dry weight). In contrast, CS3c-PVA
and CS3d-PVA achieved the equilibrium earlier (˜80 min after

grafted in the CS backbone was restricted to 18%, which can explain the
absence of antimicrobial activity in this film. Considering the CS3a-PVA
and CS3c-PVA films, these films presented some reduction on the microbe counts; however the difference compared with the control group
and CS1-PVA film was not statistically significant. In parallel, Cs3d-PVA
reduced the E. coli, S. aureus and C. albicans counts by 42.5%, 17.4%,
and 18.7% (p < 0.05). This result can be associated with the highest
grafting percentage of 2d in the CS backbone as compared with the
other CS-derivatives. As demonstrated in the literature, organoselenium
and organosulfur compounds usually exhibit antimicrobial properties
(Schneider et al., 2011; Victoria et al., 2009, 2012). In light of this, it
has been noted that the presence of Se or S atoms in the terpenoids (e.g.
citral and citronellal) generally enhances the antimicrobial activities of
such compounds as compared with the natural (i.e. unsubstituted) ones
(Victoria et al., 2012). As observed from this experiment, it can be
hypothesized that the films synthesized from the CS-derivatives containing organoselenium and organosulfur compounds (CS3c-PVA and
CS3d-PVA) can be efficient devices to control these tested bacteria and
fungi. Furthermore, the grafting of such compounds in the CS backbone
allows prolonging the antimicrobial activity of the synthesized films,
thereby preventing microbial re-colonization and proliferation. Considering these results, the films CS3a-PVA, CS3c-PVA, and CS3d-PVA,
were selected for further experiments.

3.4. Swelling and stability experiments
One of the most important features of an efficient dressing material
245


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Fig. 4. (a) Swelling curves and (b) stability of CS3a-PVA, CS3c-PVA, and CS3d-PVA in SWF (pH 7.4) at 37 °C.

Fig. 5. Effect of CS-derivatives based films on DNCB-induced atopic dermatitis-like symptoms in mice. (a) Images of skin and ear lesions from the groups of mice
taken on the last day of the experiment (day 30). (b) Dermatitis scores evaluated on day 30. Data represent the mean ± S.E.M. (one-way ANOVA followed by the
Newman-Keuls' test). ∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group.

246


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Fig. 6. Effect of CS-derivatives based films on (a) DNCB-induced scratching incidence and (b) ear swelling. Scratching time and ear swelling were evaluated on day
30. Data represent the mean ± S.E.M. (one-way ANOVA followed by the Newman Keuls' test). ∗ p < 0.05 compared with the control group, # p < 0.05 compared
with the DNCB group.

immersion) and the maximum swelling degree of these films were
580% and 610%, respectively. After the equilibrium, both swelling
curves exhibited a plateau indicating that the liquid absorption leaves
off. The highest liquid uptake capacity demonstrated by CS3a-PVA can
be explained due to its lower substitution degree as compared with
other tested CS3c-PVA and CS3d-PVA. As aforementioned, compound
3a presented a DS value of 11%, while 3c and 3d have DS values of
23% and 28%. Therefore, the film formulated with 3a has more hydrophilic groups (i.e. amino groups) available in its matrix to interact
with water molecules. Sobahi et al. (2014) have reported that the
swelling ability of CS-derivatives decrease as a function of their degree

of substitution. Despite these finds, it should be mentioned here that
these CS-derivatives/PVA based films exhibited remarkable swelling
properties as compared with other similar devices claimed as wound
dressing materials (Alves et al., 2016; Singh & Dhiman, 2015; Zheng
et al., 2014).
Another desirable feature that a wound dressing material should
possess is an adequate stability. Since swellable materials may degrade,
eventually disintegrate and dissolve, it is important to evaluate its
stability when exposed to the wound exudates for a prolonged period.
The stability of the selected films in SWF for different periods of time
was examined using gravimetric analyses. The results depicted in
Fig. 4b, reveal that the CS3a-PVA film has the lowest stability against
degradation as compared with the other film samples. Three weeks
after its immersion in SWF, the CS3a-PVA sample had just 21% of its
initial weight. On the other hand, at the end of the experiment (i.e. after
3 weeks), the samples CS3c-PVA and CS3d-PVA still preservers 49%
and 56% of their initial weights. Generally, water absorbing materials
with high swelling ability degrade faster, which may explain the results
observed in this experiment, since CS3a-PVA swell more than the other
two film samples in similar conditions (Meyvis, De Smedt, Demeester, &
Hennink, 2000). As an additional comment, the imine bonds that keep
the CS moiety crosslinked could be hydrolyzed to the initial amine and
carbonyl groups in the aqueous medium. This process is relatively fast
under acidic conditions; however, under neutral or alkaline pH, imine
hydrolysis is relatively slow (Monteiro & Airoldi, 1999), which explains
the results presented in Fig. 4b. On the other hand, PVA should be a
secondary role in this process, since its synthetic polymer shows noticeable resistance against aqueous solubility (Baker, Walsh, Schwartz,
& Boyan, 2012). In summary, it is possible to suggest that the matrix
formed by CS3c-PVA and CS3d-PVA are less susceptible to this hydrolysis process than CS3a-PVA.

3.5. In vivo assays
3.5.1. Clinical skin severity scores
Atopic dermatitis induces edema, erythema, itching, skin pigmentation, thickening, eczematous lesions, and excoriation of the skin
(Lipozencic & Wolf, 2007). Here, we evaluated the effects of CS-derivatives based films treatment on the severity of skin lesions by appearance and clinical skin severity scores in mice exposed to 2,4-dinitrochlorobenzene (DNCB) (Fig. 5a and b) [F4,30 = 31.15, p < 0.0001].
DNCB-induced skin lesion on the murine model is wildly used to study
the pathological mechanism of atopic dermatitis or to evaluate the
therapeutic candidates for this disease (Jiang & Sun, 2018; Voss et al.,
2018). One-way ANOVA followed by Newman-Keuls' post-hoc test
showed that DNCB significantly increased skin severity scores when
compared with the control group (p < 0.0001) (Fig. 5b). CS3c-PVA and
CS3d-PVA treatments partially reduced the severity of skin lesions induced by DNCB (p < 0.001) (Fig. 5b). In addition, no significant difference in severity of skin lesions was observed after treatment with
CS3a-PVA when compared with DNCB group (p > 0.05) (Fig. 5a and
b).
3.5.2. Scratching behavior
In atopic dermatitis, cutaneous abnormalities such as dryness may
initiate an itching sensation that leads to mechanical injury form
scratching, and a recurrent itching-scratching cycle can worsen the
disease by increasing the release of pro-inflammatory cytokines and
chemokine production (Kabashima, 2013). In this sense, with the objective of evaluating the severity of skin lesions caused by DNCB and to
provide scientific evidence for CS-derivatives based films anti-pruritus
effects, we evaluated the scratching behavior.
Effect of CS-derivatives based films on the scratching behavior is
showed in Fig. 6a [F4,30 = 37.18, p < 0.0001]. One-way ANOVA
followed by Newman-Keuls' post-hoc test revealed that DNCB-exposed
animals increased the scratching time, when compared with the control
group (p < 0.0001). CS3a-PVA (p < 0.01) and CS3d-PVA (p <
0.001) treatments were partially effective in reducing scratching time,
when compared with DNCB group. Additionally, CS3c-PVA treatment
did not alter the scratching time when compared with DNCB group
(p > 0.05) (Fig. 6a). Pruritus is a representative feature of atopic

dermatitis and triggers a vicious cycle of barrier dysfunction and skin
inflammation, leading to a decrease in the quality of life. Here, we
verified that CS3a-PVA and CS3d-PVA exert anti-pruritus effects. Since
there are limited drugs with low side effects and effective inhibition of
247


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

F4,30 = 19.16, p < 0.0001 (back)]. One-way ANOVA followed by
Newman-Keuls' post-hoc test revealed that DNCB significantly increased the MPO activity in the mouse ear (p < 0.0001; Fig. 7a) and
back (p < 0.0001; Fig. 7b) when compared with the control group.
The results presented in Fig. 7a and b demonstrated that treatment with
CS3a-PVA, CS3c-PVA or CS3d-PVA (p < 0.0001) reduced the activity
of MPO (ears and back) to levels similar to those in the control group,
respectively.
The importance of neutrophils in the pathogenesis of a number of
autoimmune diseases and the lack of safe and efficient strategies to
specifically target them, makes MPO a potential therapeutic target.
Since neutrophils are important contributors to autoimmune disease
pathogenesis it is not surprising that MPO is generally regarded as
pathogenic during autoimmune disease progression. Herein, in both
tissues, all treatments decreased MPO activity induced by repeated
DNCB challenges, indicating a reduction in inflammation and corroborating with the ear swelling results. It is important highlight that
treatments were applied in the back of animals, suggesting a systemic
action. In line with these results, we can suggest that the MPO is an
important therapeutic target on atopic dermatitis. Here, we verify that
the modulation of the MPO activity contributes to the protective effects

of CS-derivatives based films on atopic dermatitis.

itching, well-controlled clinical studies are warranted to demonstrate
the beneficial effects of CS-derivatives based films on atopic dermatitis.
Our results of skin lesions and scratching behavior (Figs. 5 and 6)
suggested that the animals exposed to DNCB have developed clinical
signs of atopic dermatitis that begin with scratching behavior followed
by the onset of eczematous skin lesions. Importantly, CS3d-PVA treatment was effective in reducing both parameters evaluated, suggesting
that this CS-derivative based film has a potential therapeutic in the
treatment and management of atopic dermatitis.
3.5.3. Ear swelling
The edema formation is a characteristic feature of atopic dermatitis.
In this sense, the anti-edematogenic and anti-inflammatory potentials of
CS-derivatives based films were investigated. Fig. 6b illustrates the effects of CS-derivatives based films on the ear swelling in mice
[F4,30 = 52.02, p < 0.0001]. One-way ANOVA followed by NewmanKeuls' post-hoc test demonstrated that DNCB substantially increased ear
swelling as compared with the control group (p < 0.0001) (Fig. 6b).
Treatments with CS3a-PVA, CS3c-PVA and CS3d-PVA (p < 0.0001)
partially reduced the ear swelling induced by DNCB (Fig. 6b). Our results indicate that CS-derivatives based films treatments led to a reduction of ear swelling induced by DNCB challenge, reflecting an inhibition of the edema and cell infiltration in ears. Thus, the current
findings suggest that CS3a-PVA, CS3c-PVA and CS3d-PVA exert antiinflammatory and anti-edematogenic actions. Indeed, other studies
have highlighted the close linkage of inflammation in the pathophysiology of atopic dermatitis (Devos et al., 2018; Heratizadeh, 2016;
Voss et al., 2018). In line with our results, it is imperative to understand
the mechanisms that are associated with the anti-inflammatory and
anti-edematogenic actions of CS-derivatives based films. For this purpose, the myeloperoxidase (MPO) activity and reactive species (RS)
levels were evaluated.

3.5.5. RS levels
RS is considered as one of the important biomarkers of oxidative
damage and act as a secondary messenger that can induce the generation of pro-inflammatory and Th2 cytokines during inflammatory
signaling (Dormandy, 1978; Kannan & KJain, 2000). Lastly, to explore
the linkage of redox imbalance in the pathophysiology of atopic dermatitis, we analyzed the dorsal skin RS levels in mice.

Results depicted in Fig. 8 show the effects of CS-derivatives based
films on dorsal skin RS levels in mice [F4,30 = 10.01, p < 0.0001].
One-way ANOVA followed by Newman-Keuls' post-hoc test revealed
that DNCB-exposed animals increased the RS levels when compared
with the control group (p < 0.001). CS3d-PVA treatment protected
against the increase of RS levels induced by DNCB exposure (p <
0.001). CS3a-PVA and CS3c-PVA had no effect in decreasing the RS
levels compared with the DNCB group (p > 0.05).
It is well established that skin cells produce RS. Atopic dermatitis
can disrupt the redox balance, resulting in the overproduction of RS,
and high levels of lipid peroxidation products. This process exacerbates
the disease state and shifts the response toward a Th2 skewed immune
response (Briganti & Picardo, 2003). Here, CS3d-PVA treatment alleviates the increase on RS levels, indicating that its antioxidant activity

3.5.4. MPO assay
MPO is a myeloid-lineage restricted enzyme with strong antibacterial properties. This enzyme is largely expressed by neutrophils,
during myeloid cell diff ;erentiation, which is located within azurophilic granules (Oren & Taylor, 1995). Elevated MPO levels and activity are observed in several diseases and the mechanisms whereby
MPO is thought to contribute to disease pathogenesis include tuning of
adaptive immune responses and/or the induction of vascular permeability (Strzepa, Pritchard, & Dittel, 2017).
Fig. 7 illustrates the effects of CS-derivatives based films on the ear
and back MPO activities [F4,30 = 14.58, p < 0.0001 (ear);

Fig. 7. Effect of CS-derivatives based films on (a) ear and (b) back MPO activities in mice. Data represent the mean ± S.E.M. (one-way ANOVA followed by the
Newman-Keuls' test). ∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group.
248


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Alves, N. O., da Silva, G. T., Weber, D. M., Luchese, C., Wilhelm, E. A., & Fajardo, A. R.
(2016). Chitosan/poly(vinyl alcohol)/bovine bone powder biocomposites: A potential biomaterial for the treatment of atopic dermatitis-like skin lesions. Carbohydrate
Polymers, 148, 115–124.
Baker, M. I., Walsh, S. P., Schwartz, Z., & Boyan, B. D. (2012). A review of polyvinyl
alcohol and its uses in cartilage and orthopedic applications. Journal of Biomedical
Materials Research Part B-Applied Biomaterials, 100B, 1451–1457.
Bhattacherjee, D., Basu, C., Bhardwaj, Q., Mal, S., Sahu, S., Sur, R., et al. (2017). Design,
synthesis and anti-cancer activities of benzyl analogues of garlic-derived diallyl disulfide (DADS) and the corresponding diselenides. ChemistrySelect, 2, 7399–7406.
Bilal, M., Rasheed, T., Iqbal, H. M. N., Hu, H. B., Wang, W., & Zhang, X. H. (2017).
Macromolecular agents with antimicrobial potentialities: A drive to combat antimicrobial resistance. International Journal of Biological Macromolecules, 103, 554–574.
Bilal, M., Rasheed, T., Iqbal, H. M. N., Lib, C. L., Hu, H. B., & Zhang, X. H. (2017).
Development of silver nanoparticles loaded chitosan-alginate constructs with biomedical potentialities. International Journal of Biological Macromolecules, 105,
393–400.
Boateng, J. S., Matthews, K. H., Stevens, H. N. E., & Eccleston, G. M. (2008). Wound
healing dressings and drug delivery systems: A review. Journal of Pharmaceutical
Sciences, 97, 2892–2923.
Bonilla, J., Fortunati, E., Atares, L., Chiralt, A., & Kenny, J. M. (2014). Physical, structural
and antimicrobial properties of poly vinyl alcohol-chitosan biodegradable films. Food
Hydrocolloids, 35, 463–470.
Briganti, S., & Picardo, M. (2003). Antioxidant activity, lipid peroxidation and skin diseases. What’s new. Journal of the European Academy of Dermatology and Venereology,
17, 663–669.
Brodin, M., Vallejos, M., Opedal, M. T., Area, M. C., & Chinga-Carrasco, G. (2017).
Lignocellulosics as sustainable resources for production of bioplastics - A review.
Journal of Cleaner Production, 162, 646–664.
Choo, K., Ching, Y. C., Chuah, C. H., Julai, S., & Liou, N. S. (2016). Preparation and
characterization of polyvinyl alcohol-chitosan composite films reinforced with cellulose nanofiber. Materials, 9, 1–6.
da Silva, F. D., Pinz, M., de Oliveira, R. L., Rodrigues, K. C., Ianiski, F. R., Bassaco, M. M.,
et al. (2017). Organosulfur compound protects against memory decline induced by
scopolamine through modulation of oxidative stress and Na+/K+ ATPase activity in

mice. Metabolic Brain Disease, 32, 1819–1828.
da Silva, S. B., Krolicka, M., van den Broek, L. A. M., Frissen, A. E., & Boeriu, C. G. (2018).
Water-soluble chitosan derivatives and pH-responsive hydrogels by selective C-6
oxidation mediated by TEMPO-laccase redox system. Carbohydrate Polymers, 186,
299–309.
Daeschlein, G. (2013). Antimicrobial and antiseptic strategies in wound management.
International Wound Journal, 10, 9–14.
Dai, T. H., Tanaka, M., Huang, Y. Y., & Hamblin, M. R. (2011). Chitosan preparations for
wounds and burns: Antimicrobial and wound-healing effects. Expert Review of AntiInfective Therapy, 9, 857–879.
Devillanova, F. A., Sathyanarayana, D. N., & Verani, G. (1978). Infrared spectroscopic
investigation on N-methyl-1,3-thiazolidine-2-thione and N-methyl-1,3-thiazolidine-2selone. Journal of Heterocyclic Chemistry, 15, 945–947.
Devos, M., Mogilenko, D. A., Fleury, S., Gilbert, B., Becquart, C., Quemener, S., et al.
(2018). Keratinocyte expression of A20/TNFAIP3 controls skin inflammation associated with atopic dermatitis and psoriasis. Journal of Investigative Dermatology, 139,
135–145.
Dormandy, T. L. (1978). Free-radical oxidation and antioxidants. Lancet, 1(8065),
647–650.
Dugmore, T. I. J., Clark, J. H., Bustamante, J., Houghton, J. A., & Matharu, A. S. (2017).
Valorisation of biowastes for the production of green materials using chemical
methods. Topics in Current Chemistry, 375, 1–8.
Goy, R. C., de Britto, D., & Assis, O. B. G. (2009). A review of the antimicrobial activity of
chitosan. Polimeros-Ciencia E Tecnologia, 19, 241–247.
Guilherme, M. R., Aouada, F. A., Fajardo, A. R., Martins, A. F., Paulino, A. T., Davi, M. F.
T., et al. (2015). Superabsorbent hydrogels based on polysaccharides for application
in agriculture as soil conditioner and nutrient carrier: A review. European Polymer
Journal, 72, 365–385.
Heratizadeh, A. (2016). Atopic dermatitis: New evidence on the role of allergic inflammation. Current Opinion in Allergy and Clinical Immunology, 16, 458–464.
Heux, L., Brugnerotto, J., Desbrieres, J., Versali, M. F., & Rinaudo, M. (2000). Solid state
NMR for determination of degree of acetylation of chitin and chitosan.
Biomacromolecules, 1, 746–751.
Hoffmann, B., Seitz, D., Mencke, A., Kokott, A., & Ziegler, G. (2009). Glutaraldehyde and

oxidised dextran as crosslinker reagents for chitosan-based scaffolds for cartilage
tissue engineering. Journal of Materials Science-Materials in Medicine, 20, 1495–1503.
Hoque, J., Prakash, R. G., Paramanandham, K., Shome, B. R., & Haldar, J. (2017).
Biocompatible injectable hydrogel with potent wound healing and antibacterial
properties. Molecular Pharmaceutics, 14, 1218–1230.
Huang, J. J., Deng, Y. M., Ren, J. A., Chen, G. P., Wang, G. F., Wang, F., et al. (2018).
Novel in situ forming hydrogel based on xanthan and chitosan re-gelifying in liquids
for local drug delivery. Carbohydrate Polymers, 186, 54–63.
Huang, K. S., Yang, C. H., Huang, S. L., Chen, C. Y., Lu, Y. Y., & Lin, Y. S. (2016). Recent
advances in antimicrobial polymers: A mini-review. International Journal of Molecular
Sciences, 17, 1578–1589.
Ianiski, F. R., Alves, C. B., Bassaco, M. M., Silveira, C. C., & Luchese, C. (2014). Protective
effect of ((4-tert-butylcyclohexylidene) methyl) (4-methoxystyryl) sulfide, a novel
unsymmetrical divinyl sulfide, on an oxidative stress model induced by sodium nitroprusside in mouse brain: Involvement of glutathione peroxidase activity. Journal of
Pharmacy and Pharmacology, 66, 1747–1754.
Iqbal, H. M. N., Kyazze, G., Locke, I. C., Tron, T., & Keshavarz, T. (2015). In situ

Fig. 8. Effect of CS-derivatives based films on RS levels of back of mice. Data
represent the mean ± S.E.M. (one-way ANOVA followed by the NewmanKeuls' test). ∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group.

probably also contributes to a protective effect on atopic dermatitis.
Indeed, the antioxidant activity of organosulfur compounds has an
important contribution in their pharmacological actions (da Silva et al.,
2017; Ianiski, Alves, Bassaco, Silveira, & Luchese, 2014).
4. Conclusion
In conclusion, we have described the synthesis and characterization
of new chalcogen-containing CS-derivatives. Films were prepared by
blending CS-derivatives with PVA, they characterized in detail and
subjected to antimicrobial and pharmacological assays. Our finds reveal
that the film based on CS-modified with the organosulfur compound

(CS3d-PVA) attenuates atopic dermatitis-like symptoms in mice by
suppressing the increase of MPO activity and RS levels induced by
DNCB. In addition, its antimicrobial activity seems to contribute to its
pharmacological effect in atopic dermatitis model. The present new
finding emphasizes the potential of CS-modified with an organosulfur
compound as a lead material for the development of new agents for the
treatment of atopic dermatitis, a chronic skin disease.
Acknowledgments
The authors are grateful for the financial support and scholarships
from the Brazilian agencies CNPq (Grant. number 305974/2016-5).
CNPq is also acknowledged for the fellowship to A.R.F., E.J.L., E.A.W.
and C.L. This study was nanced in part by the Coordenaỗóo de
Aperfeiỗoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi: />References
Ali, A., Xie, F. W., Yu, L., Liu, H. S., Meng, L. H., Khalid, S., et al. (2018). Preparation and
characterization of starch-based composite films reinfoced by polysaccharide-based
crystals. Composites Part B-Engineering, 133, 122–128.
Alves, N. M., & Mano, J. F. (2008). Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications. International Journal of
Biological Macromolecules, 43, 401–414.

249


Carbohydrate Polymers 219 (2019) 240–250

M.S. Gularte, et al.

Mourya, V. K., & Inamdar, N. N. (2008). Chitosan-modifications and applications:

Opportunities galore. Reactive & Functional Polymers, 68, 1013–1051.
Oren, A., & Taylor, J. M. G. (1995). The subcellular-localization of defensins and myeloperoxidase in human neutrophils - Immunocytochemical evidence for azurophil
granule heterogeneity. Journal of Laboratory and Clinical Medicine, 125, 340–347.
Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer
Science, 31, 603–632.
Rumer, J. W., & McCulloch, I. (2015). Organic photovoltaics: Crosslinking for optimal
morphology and stability. Materials Today, 18, 425–435.
Sahariah, P., & Masson, M. (2017). Antimicrobial chitosan and chitosan derivatives: A
review of the structure-activity relationship. Biomacromolecules, 18, 3846–3868.
Santos, J. C. C., Moreno, P. M. D., Mansur, A. A. P., Leiro, V., Mansur, H. S., & Pego, A. P.
(2015). Functionalized chitosan derivatives as nonviral vectors: Physicochemical
properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes. Soft
Matter, 11, 8113–8125.
Schneider, T., Baldauf, A., Ba, L. A., Jamier, V., Khairan, K., Sarakbi, M. B., et al. (2011).
Selective antimicrobial activity associated with sulfur nanoparticles. Journal of
Biomedical Nanotechnology, 7(3), 395–405.
Singh, B., & Dhiman, A. (2015). Designing bio-mimetic moxifloxacin loaded hydrogel
wound dressing to improve antioxidant and pharmacology properties. Rsc Advances,
5, 44666–44678.
Sobahi, T. R. A., Abdelaal, M. Y., & Makki, M. S. I. (2014). Chemical modification of
Chitosan for metal ion removal. Arabian Journal of Chemistry, 7(5), 741–746.
Strzepa, A., Pritchard, K. A., & Dittel, B. N. (2017). Myeloperoxidase: A new player in
autoimmunity. Cellular Immunology, 317, 1–8.
Tamer, T. M., Hassan, M. A., Omer, A. M., Baset, W. M. A., Hassan, M. E., El-Shafeey, M.
E. A., et al. (2016). Synthesis, characterization and antimicrobial evaluation of two
aromatic chitosan Schiff base derivatives. Process Biochemistry, 51, 1721–1730.
Teodorescu, M., Bercea, M., & Morariu, S. (2018). Biomaterials of poly(vinyl alcohol) and
natural polymers. Polymer Reviews, 58, 1–40.
Tharanathan, R. N., & Kittur, F. S. (2003). Chitin - The undisputed biomolecule of great
potential. Critical Reviews in Food Science and Nutrition, 43, 61–87.

Victoria, F. N., Radatz, C. S., Sachini, M., Jacob, R. G., Alves, D., Savegnago, L., et al.
(2012). Further analysis of the antimicrobial activity of alpha-phenylseleno citronellal and alpha-phenylseleno citronellol. Food Control, 23, 95–99.
Victoria, F. N., Radatz, C. S., Sachini, M., Jacob, R. G., Perin, G., da Silva, W. P., et al.
(2009). KF/Al2O3 and PEG-400 as a recyclable medium for the selective alpha-selenation of aldehydes and ketones. Preparation of potential antimicrobial agents.
Tetrahedron Letters, 50, 6761–6763.
Vieira, M. G. A., da Silva, M. A., dos Santos, L. O., & Beppu, M. M. (2011). Natural-based
plasticizers and biopolymer films: A review. European Polymer Journal, 47, 254–263.
Vien, D., Cotthup, N., Fatoley, W., & Crasselli, J. (1991). The handbook of infrared and
Raman characteristic frequencies of organic Molecules. Orlando, Florida: Academic
Press.
Vogt, A. G., Voss, G. T., de Oliveira, R. L., Paltian, J. J., Duarte, L. F. B., Alves, D., et al.
(2018). Organoselenium group is critical for antioxidant activity of 7-chloro-4-phenylselenyl-quinoline. Chemico-Biological Interactions, 282, 7–12.
Voss, G. T., Oliveira, R. L., de Souza, J. F., Duarte, L. F. B., Fajardo, A. R., Alves, D., et al.
(2018). Therapeutic and technological potential of 7-chloro-4-phenylselanyl quinoline for the treatment of atopic dermatitis-like skin lesions in mice. Materials Science &
Engineering C-Materials for Biological Applications, 84, 90–98.
Yue, L., Li, J. R., Chen, W. W., Liu, X. L., Jiang, Q. X., & Xia, W. S. (2017). Geraniol grafted
chitosan oligosaccharide as a potential antibacterial agent. Carbohydrate Polymers,
176, 356–364.
Zheng, A., Xue, Y., Wei, D., Li, S., Xiao, H., & Guan, Y. (2014). Synthesis and characterization of antimicrobial polyvinyl pyrrolidone hydrogel as wound dressing. Soft
Materials, 12, 297–305.

development of self-defensive antibacterial biomaterials: Phenol-g-keratin-EC based
bio-composites with characteristics for biomedical applications. Green Chemistry, 17,
3858–3869.
Iqbal, H. M. N., Kyazze, G., Locke, I. C., Tron, T., & Keshayarz, T. (2015). Development of
bio-composites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours. Carbohydrate Polymers, 131,
197–207.
Jiang, P., & Sun, H. (2018). Sulfuretin alleviates atopic dermatitis like-symptons in mice
via suppressing Th2 cell activity. Immunologic Research, 66, 611–619.
Jin, X. X., Wang, J. T., & Bai, J. (2009). Synthesis and antimicrobial activity of the Schiff

base from chitosan and citral. Carbohydrate Research, 344, 825–829.
Kabashima, K. (2013). New concept of the pathogenesis of atopic dermatitis: Interplay
among the barrier, allergy, and pruritus as a trinity. Journal of Dermatological Science,
70, 3–11.
Kamoun, E. A., Chen, X., Eldin, M. S. M., & Kenawy, E. R. S. (2015). Crosslinked poly
(vinyl alcohol) hydrogels for wound dressing applications: A review of remarkably
blended polymers. Arabian Journal of Chemistry, 8, 1–14.
Kannan, K., & KJain, S. (2000). Oxidative stress and apoptosis. Pathophysiology, 7,
153–163.
Khan, K., & Siddiqui, Z. N. (2015). An efficient synthesis of tri- and tetrasubstituted
imidazoles from benzils using functionalized chitosan as biodegradable solid acid
catalyst. Industrial & Engineering Chemistry Research, 54, 6611–6618.
Kiuchi, H., Kai, W. H., & Inoue, Y. (2008). Preparation and characterization of poly
(ethylene glycol) crosslinked chitosan films. Journal of Applied Polymer Science, 107,
3823–3830.
Lawrie, G., Keen, I., Drew, B., Chandler-Temple, A., Rintoul, L., Fredericks, P., et al.
(2007). Interactions between alginate and chitosan biopolymers characterized using
FTIR and XPS. Biomacromolecules, 8, 2533–2541.
Lessa, E. F., Gularte, M. S., Garcia, E. S., & Fajardo, A. R. (2017). Orange waste: A valuable
carbohydrate source for the development of beads with enhanced adsorption properties for cationic dyes. Carbohydrate Polymers, 157, 660–668.
Lessa, E. F., Nunes, M. L., & Fajardo, A. R. (2018). Chitosan/waste coffee-grounds composite: An efficient and eco-friendly adsorbent for removal of pharmaceutical contaminants from water. Carbohydrate Polymers, 189, 257–266.
Lipozencic, J., & Wolf, R. (2007). Atopic dermatitis: An update and review of the literature. Dermatologic Clinics, 25, 605–611.
Lopez-Romero, J. C., Gonzalez-Rios, H., Borges, A., & Simoes, M. (2015). Antibacterial
effects and mode of action of selected essential oils components against escherichia
coli and staphylococcus aureus. Evidence-Based Complementary and Alternative
Medicine, 2015, 1–9.
Ma, Q., Liu, Y. F., Dong, Z., Wang, J. L., & Hou, X. (2015). Hydrophobic and nanoporous
chitosan-silica composite aerogels for oil absorption. Journal of Applied Polymer
Science, 132, 1–7.
Marin, L., Simionescu, B., & Barboiu, M. (2012). Imino-chitosan biodynamers. Chemical

Communications, 48, 8778–8780.
Martins, A. F., Facchi, S. P., Monteiro, J. P., Nocchi, S. R., Silva, C. T. P., Nakamura, C. V.,
et al. (2015). Preparation and cytotoxicity of N,N,N-trimethyl chitosan/alginate
beads containing gold nanoparticles. International Journal of Biological
Macromolecules, 72, 466–471.
Meyvis, T. K. L., De Smedt, S. C., Demeester, J., & Hennink, W. E. (2000). Influence of the
degradation mechanism of hydrogels on their elastic and swelling properties during
degradation. Macromolecules, 33, 4717–4725.
Mika, L. T., Csefalvay, E., & Nemeth, A. (2018). Catalytic conversion of carbohydrates to
initial platform chemicals: Chemistry and sustainability. Chemical Reviews, 118,
505–613.
Monteiro, O. A. C., & Airoldi, C. (1999). Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system. International Journal of Biological
Macromolecules, 26, 119–128.

250