Chitosan nanogels condensed to ferulic acid for the essential oil of Lippia origanoides Kunth encapsulation

Carbohydrate Polymers 188 (2018) 268–275

Contents lists available at ScienceDirect

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

Chitosan nanogels condensed to ferulic acid for the essential oil of Lippia
origanoides Kunth encapsulation

T

Regiamara Ribeiro Almeidaa, Elisa Tatiana Silva Damascenoa,
Stephanne Yonara Barbosa de Carvalhoa, Gustavo Senra Gonỗalves de Carvalhob,

Leiriana Aparecida Pinto Gontijoa, Luiz Gustavo de Lima Guimarãesa,
a
b

Federal University of São João del-Rei, Natural Science Department, CEP 36301160, São João del-Rei, MG, Brazil
Federal University of Juiz de Fora, Chemistry Department, Institute of Exact Sciences, CEP 36036-900, Juiz de Fora, MG, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords:
Biopolymer
Nanoencapsulation
Carvacrol
Bioactive compounds

In order to enable the applicability of the essential oil of Lippia origanoides Kunth, increasing its stability and
dispersion in aqueous base, were prepared nanogels of chitosan modified with ferulic acid to be used in their
encapsulation. The results obtained by FTIR and 13C SSNMR revealed the formation of CS-FA link in the different
synthesized nanogels, while the studied by DLS showed particles with varied sizes and positive charge. A satisfactory encapsulation capacity of the essential oil was obtained for the nanogels. However, the nanogel
synthesized with the highest proportion of ferulic acid in relation to chitosan 0.760 g of ferulic acid (CF1)
showed the highest encapsulation efficiency of 20%. The results indicate the CF1 nanogel potential to encapsulate important components of the essential oil of L. origanoides, being able to guarantee the constituents,
preservation, moreover facilitate the dispersion and release, expanding its use.

1. Introduction

when exposed to environmental factors and in antifungal performance
(Beyki et al., 2014; Zhaveh et al., 2015).
The nanogels are constituted by a three-dimensionally reticulated
polymer grid composed by particulate units of hydrogel in nanometric
size and large number of hydrophilic groups. These materials present
long useful life, good biocompatibility, good dispersibility in water,
exhibit high load capacity, controlled release of active compounds and
well-defined structure (Li, Maciel, Rodrigues, Shi, & Tomás, 2015;
Pujana et al., 2013; Tiwari & Tiwari, 2013; Zhang, Zhai, Wang, & Zhai,
2016).
Ferulic acid shows favorable characteristics for the chitosan nanogels synthesis with biological applicability. The 4-hydroxy-3-methoxycinnamic acid phenolic compound presents in its structure a carboxylic group which shows the ability to react with the amino groups of
chitosan, improving their properties (Woranuch & Yoksan, 2013). Although the structural modification of chitosan with ferulic acid has
already been described in the literature, there are no reports about the
use of these materials for the essential oils encapsulation.
The Lippia origanoides Kunth plant, easily founded in Brazilian vegetation, produces an essential oil with great medicinal potential.
Innumerable biological activities have been reported in literature for
the essential oil of this species, such as antimicrobial, anti-

Chitosan presents many favorable characteristics for medicinal,

pharmaceutical and feeding applications, including biocompatibility,
biodegradability, abundance, easy obtainment and non-toxicity (Hu &
Luo, 2016; Younes & Rinaudo, 2015). In addition to these properties,
the presence of functional groups along its polymer chain extends the
possibilities of chemical modifications, making possible the obtainment
of materials with improved physico-chemical properties (Jennings &
Bumgardner, 2017; Pujana, Pérez-Álvarez, Cesteros, & Katime, 2013;
Ramimoghadam, Bagheri, & Hamid, 2014).
Changes in the chitosan structure allows the development of several
hybrid materials in the most different conformations, such as fibres,
powders, films, gels and capsules, being primordial for its using advancement (Agnihotri, Mallikarjuna, & Aminabhavi, 2004; Hu & Luo,
2016; Jennings & Bumgardner, 2017; Younes & Rinaudo, 2015). Recent
studies revealed the nanogels obtain through chemical modification in
the structure of chitosan using cinnamic acid derivatives, leading new
materials formation with the ability to encapsulate essential oils.
Highlighting the promising characteristics that these materials presented, such as encapsulation efficiency, slow release of active compounds and a considerable improvement in the essential oil stability

Abbreviation: EE, encapsulation efficiency; L. origanoides, Lippia origanoides Kunth; CS-FA, chitosan linked to ferulic acid
⁎
Corresponding author at: Natural Science Department, Campus Dom Bosco, Federal University of São João del Rei, São João del Rei, Minas Gerais, Brazil.
E-mail address: (L.G. de Lima Guimarães).
/>Received 16 November 2017; Received in revised form 18 December 2017; Accepted 30 January 2018
Available online 04 February 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 188 (2018) 268–275

R.R. Almeida et al.

The low molecular weight chitosan (75–85% deacetylation degree
and molecular weight of 50,000–190,000 Da), the trans ferulic acid
(99%) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
(≥97.0% and density of 0.877 g mL−1 at 20 °C) were supplied by Sigma
Aldrich. All other reagents used in this study were of analytical grade.

The trans-ferulic acid was coupled to low molar mass chitosan by a
reaction mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) according to the methods proposed by Chen, Lee, & Park (2003)
and Woranuch and Yoksan (2013), followed by modifications. Initially,
1.20 g of chitosan was dissolved in 100 mL of a 1% (v/v) aqueous solution of acetic acid under magnetic stirring at 1000 rpm for 24 h.
Subsequently, a solution of EDC and ferulic acid in 10 mL of ethanol
was prepared. Then, this solution was slowly added to the chitosan
solution under stirring at 1000 rpm, the resulting mixture was kept
under stirring for 24 h in the same rate used for the addition. Then, it
was diluted with 85 mL methanol and its pH adjusted to 8.5–9.0 using
sodium hydroxide solution (1 mol L−1), and kept under refrigeration at
4 °C for 24 h. The nanogel was centrifuged at 3000 rpm for 5 min, and
submitted to a series of washes with distilled water until neutral pH. In
the sequel, the nanogel was submitted to three washes with ethanol, the
blend being centrifuged at 5100 rpm for 15 min. Finally, the supernatant was removed, the nanogel being placed for drying under vacuum
at natural temperature in a desiccator containing silica gel.
In order to observe the effects of the relative ratio of ferulic acid to
chitosan, the same methodology was used in the obtainment of all the
nanogels by altering only the ferulic acid and EDC amount, maintaining
the 0.4 mmol proportion between these compounds in all solutions
(Table 1).

2.2. Essential oil extraction

2.5. Encapsulation of L. origanoides essential oils in CS–FA nanogel

The leaves of L. origanoides were collected in the month of
November 2016 in the morning in the municipality of Itumirim/MG
(21° 12′58 “S, 44° 51′21” W, 837 m). The extraction using L. origanoides
leaves, was performed by the hydrodistillation technique in modified
Clevenger apparatus, according to the 5th edition of the Brazilian
Pharmacopoeia (Brazil, 2010) recommended method. The obtained
hydrolate after extraction was taken to a bench centrifuge, separating
the organic and aqueous phases, the essential oil being collected and
kept under refrigeration (4 °C).

In order for the CS-FA nanogels to incorporate the essential oil,
100 mg of each nanogel were dissolved in 10 mL of aqueous acetic acid
solution (pH = 3.5–4.0), under stirring at 500 rpm. The essential oil
(100 mg) was dispersed in ethanol (1:1 w/w). After, still under 500 rpm
stirring, the essential oil solution was dripped into the nanogel solution.
The resulting mixture was taken into an ultrasonic bath at a frequency
of 40 kHz for 10 min. After that, the nanogel containing the essential oil
had the pH adjusted to 8.5–9.0 using sodium hydroxide (1 mol L−1).
The precipitated material was submitted to sequel washes with distilled
water until neutral pH, and centrifuged at 5100 rpm for 15 min. The
supernatant was removed and the CS-FA nanogels containing encapsulated essential oil (CF1EO, CF2EO, CF3EO and CF4EO) nanogels
containing essential oil were placed under vacuum at natural temperature in a desiccator containing silica gel. They were stored at 4 °C.

inflammatory, antioxidant, antiprotozoal, repellent, insecticide and
antimalarial, evidencing the great potential for commercial exploration
(Oliveira, Leitão, & Leitão, 2014; Ribeiro, Andrade, Salimena, & Maia,
2014; Soares et al., 2017). Although the essential oil of this plant exhibits promising properties, some characteristics presented by the
constituents present in the essential oils, such as instability, volatility

and mainly low solubility in water, may limit its applications (Asbahani
et al., 2015; Raut & Karuppayil, 2014).
In this work, we intend modify the chitosan structure with ferulic
acid in order to improve its physicochemical characteristics, resulting in
a material with greater lipophilic molecules affinity, making possible
the essential oil of Lippia origanoides Kunth encapsulation that presents
important biological activities.
2. Materials and methods
2.1. Materials

2.3. Essential oil qualitative and quantitative analysis
The qualitative analysis of the essential oil of L. origanoides was
performed by using an Agilent 7890 B chromatograph coupled to an
Agilent 7000C triple quadrupole mass spectrometer. The equipment
was operated under the following conditions: capillary column of fused
silica with apolar phase; 220 °C injector temperature; charged helium
gas (1 mL min−1); initial column pressure at 100.2 kPa; column temperature was programmed from 60 °C to 240 °C at a rate of 3 °C min−1,
split ratio of 1:50 and injected essential oil volume of 1.0 μL [1% (m/v)
in hexane]. For the mass spectrometer (EM) the following conditions
were used: 70 eV impact energy. The constituents were identified by
comparing their mass spectra with those of NIST library databases, and
by comparing their calculated arithmetic indices with those present in
the Nist webbook and in literature (Adams, 2007).
The constituents quantification was performed using a gas chromatograph Shimadzu, model GC-2010, equipped with a flame ionization detector (FID) and RTX-5MS capillary column (30 mm 0.25 mm x
0.25 μm film thickness). Nitrogen (1.18 mL min−1) was used as carrier
gas; 1:50 split ratio; 115 KPa column pressure and the injected volume
of 1 μ L diluted in hexane (1: 100 v/v). The column temperature was
programmed from 60 °C to 240 °C at a rate of 3 °C min−1, after going, to
a heating rate of 10 °C min−1–300 °C, remaining at that temperature for
10 min. The injector and detector temperatures were set at 220 °C and

300 °C respectively, with the 115 kPa column pressure.

2.6. Characterization of nanogels
2.6.1. Fourier Transform Infrared Spectroscopy (FT-IR) analysis
Absorption spectra in the infrared region were obtained using a
Shimadzu (IRAffinity) spectrometer, being the samples pressed to obtain the pellets (KBr). The spectral range was 400–4000 cm−1, with a
resolution of 4 cm−1. Analyzes of the chitosan and ferulic acid compounds were performed also of the CS-FA nanogels.
2.6.2. 13C Solid State Nuclear Magnetic Resonance (SSNMR)
Solid state 13C NMR experiments were performed on a Bruker
Avance III HD 300 (7.04 T) spectrometer, operated at a Larmor frequency of 75.00 MHz. The analyzes were performed on a MAS probe,
on ZrO2 rotors (and caps of Kel-F) of 4 mm. The spectra were obtained
at a rotation frequency of 10000 Hz, with relaxation time of 3.5 s and a
Table 1
EDC and ferulic acid amounts used in the nanogels preparation.

2.4. Preparation of chitosan linked to ferulic acid (CS–FA) nanogels
In this study, 4 nanogels were synthesized using different amounts
of ferulic acid and maintaining the chitosan concentration (Table 2).
269

Sample

Ferulic acid (g)

EDC (mL)

Acid:EDC (mmol)

CF1
CF2

CF3
CF4

0.760
0.380
0.190
0.095

1.75
0.88
0.44
0.22

3.9:
2.0:
1.0:
0.5:

9.9
5.0
2.5
1.3


Carbohydrate Polymers 188 (2018) 268–275

R.R. Almeida et al.

90° pulse of 2.0 μs using magical spin-angle, and cross-polarization. The
chemical shifts were indirectly standardized through a glycine sample,

with carbonyl sign at 176.00 ppm in relation to TMS, which is the
primary standard.

Table 2
Chemical composition of the L. origanoides essential oil.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

2.6.3. Scanning Electron Microscopy (SEM)
The morphology of the nanogels was analyzed by a scanning electron microscope LEO EVO 40, being the samples analyzed before and
after the incorporation of the L. origanoides essential oil. For the analysis, the samples were coated with gold in a Balzers SCD 050 evaporator. In order to visualize the encapsulation form of the essential oil
by the nanogels, the samples were immersed in liquid nitrogen and
fractured, the same process was carried out for the pure nanogels.
2.6.4. Dynamic Light Scattering (DLS)
The measurements of particle size and Zeta Potential (ξ) were carried out using a Beckman Couter DelsaNano C Particle Analyzer at a
dispersion angle (θ) of 173°–25 °C. For analysis, 0.3 g of nanogels were
dispersed in 3.0 mL of 1% acetic acid (v/v) under agitation magnetic for
24 h.
2.6.5. Thermal stability analysis (TGA)
Thermogravimetric analyzes, TGA, were performed in a Shimadzu
equipment, model DTG – 60H, under N2 atmosphere. Samples were
heated from 35 °C to 600 °C with a heating rate of 10 °C min−1.
Chitosan, ferulic acid, nanogels in the different proportions of CS-FA,
essential oil of L. origanoides and CS-FA nanogels containing the encapsulated essential oil were analyzed.
Through the TGA curves of the nanogel (CS-FA) samples containing
encapsulated essential oil, it was also determined the contents of the
encapsulated L. origanoides essential oil, being the values of encapsulation efficiencies (EE) obtained according to Eq. (1).

EE =

mass of essential oil in nanogels
x 100
mass of essential oil

COMPOUNDS

CAI

TAI

%

α-thujene
α-pinene
sabinene
β-pinene
myrcene
α-phellandrene
α-terpinene
ρ-cimene
limonene
1–8-cineole
ɣ- terpinene
cis-sabinene hydrate
trans-sabinene hydrate
terpinen-4-ol
thymol methyl ether
thymol
carvacrol
carvacrol acetate
α-copaene
(E)-caryophyllene
α-trans-bergamotene
α-humulene
germacrene D
germacrene A
Monoterpene hydrocarbons

Oxygen hydrocarbons
Sesquiterpene hydrocarbons
Total

925
932
972
978
990
1005
1016
1024
1028
1030
1058
1067
1098
1178
1235
1292
1301
1373
1376
1420
1436
1454
1484
1506

924

932
969
974
988
1002
1016
1020
1024
1026
1054
1065
1098
1174
1232
1289
1298
1370
1374
1417
1432
1452
1484
1508

1.35
0.31
0.13
0.07
1.66
0.28

1.75
14.34
0.78
0.10
12.10
0.23
0.40
0.44
4.08
13.06
41.08
1.07
0.10
3.12
0.16
1.17
0.62
0.13
33.17
60.06
5.30
98.53

CAI = calculated arithmetic index, TAI = tabulated arithmetic index and (%) = concentration (ADAMS, 2007).

1269 cm−1 (carboxylic acid CeO stretching) as shown in Fig. 1B
(Panwar, Sharma, Kaloti, Dutt, & Pruthi, 2016; Woranuch & Yoksan,
2013).
The formation of the bond between chitosan CS and ferulic acid FA
(Fig. 1C) can be evidenced by the widening of the corresponding C]O

stretching of I amide band at 1650 cm−1. In addition, is possible to
observe in Fig. 1C the increase in the intensity and narrowing of the
absorption band in the 3443 cm−1 region, due to the OH groups of
ferulic acid and the disappearance of the NeH deformation vibrations
of chitosan II amide at 1594 cm−1 in the nanogel spectrum, being
overlapped by a 1550 cm−1 band corresponding to the vibration of the
aromatic ring C]C bond, these results are consistent with the results
observed in other studies (Beyki et al., 2014; Woranuch & Yoksan,
2013).

(1)

3. Results and discussion
3.1. Essential oil
3.1.1. Characterization and identification of the L. origanoides essential oil
The constituents present in the L. origanoides essential oil and their
contents expressed in percentage are described in Table 2. The essential
oil presented as major constituent carvacrol (41.08%), followed by ρcymene (14.34%), thymol (13.06% %) and ɣ- terpinene (12.10%).
The oxygenated monoterpenes predominated, representing 60.06%
of the chemical composition of the essential oil, followed by the
monoterpene hydrocarbons (33.17%) and the sesquiterpene hydrocarbons (5.30%).

3.2.2. 13C Solid State Nuclear Magnetic Resonance (SSNMR)
The Fig. 2 shows the 13C SSNMR spectra of chitosan (CS), ferulic
acid (FA) and CS-FA nanogels. Signals related to chitosan and ferulic
acid are shown in Table 3. The observed signals are similar to those
reported by other authors (Phan, Flanagan, D'Arcy, & Gidley, 2017; Rui
et al., 2017).
The link formation between chitosan and ferulic acid (Fig. 2) can be
evidenced by the additional signals appearance at 109.70 and

151.16 ppm referring to the carbons (C]C) of the aromatic ring. In
addition, by comparison the 13C SSNMR spectrum of ferulic acid with
that of CS-FA nanogels (CS-FA) the carbonyl signal offset is observed,
which in the starting material appears with the chemical offset of
172.78 ppm and after the coupling reaction shifts to 163.86 ppm. When
comparing the 13C SSNMR spectrum of chitosan with that of CS-FA
nanogels (Fig. 2), it is also possible to observe the doubling of the signal
related to C2, which may be associated to the presence of a new conformation in the formation of the link between phenolic groups of
ferulic acid with the chitosan amino groups, which matches with other
authors observed results (Aljawish et al., 2012; Liu, Wen, Lu, Kan, &
Jin, 2014).

3.2. Nanogels
3.2.1. FT-IR measurement
The FT-IR spectra of chitosan, ferulic acid and chitosan linked to
ferulic acid nanogels are shown in Fig. 1A–C, respectively.
In the chitosan spectrum (Fig. 1A), can be observed 3413 cm−1
(stretching of the OeH bond, superimposed on the NeH stretching
band), 2893 cm−1 (stretching of the CeH bond), 1657 cm−1 (C]O I
amide), 1594 cm−1 (NeH deformation of II amide), 1093 cm−1
(stretching of the CeOeC bond of the glycosidic links) 897 cm−1
(stretching of the alcohols CeO bond) (Sousa, Silva Filho, & Airoldi,
2009; Woranuch & Yoksan, 2013).
The characteristic bands of ferulic acid (Fig. 1B) were observed in
3439 cm−1 (stretching of the OeH bond), 2947–3019 cm−1 (stretching
of the CeH bond), 1697 cm−1 (stretching of the C]O bond of carboxylic acid), 1510 cm−1 (C]C stretch of aromatic ring) and
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Carbohydrate Polymers 188 (2018) 268–275

R.R. Almeida et al.

throughout all the surface (Fig. 3E), which was not verified for the same
material without the essential oil presence (Fig. 3F). The presence of
these cavities indicates the essential oil possible incorporation into the
material surface, once that these cavities may be associated with essential oil droplets that have been incorporated by the polymer matrix.
Through the electromicrographs of the CF1EO sample fracture
surfaces (Fig. 3A and C) it is possible to observe internal structures with
uniformly distributed pores and of different sizes, differently from what
was observed in the fracture electromicrographs for the material
without the presence of encapsulated essential oil (Fig. 3B and D). The
CS-FA nanogel starting material had a uniform and dense internal
structure (Fig. 3B and D).
The presence of several pores inside the synthesized nanogel with
the highest amount of ferulic acid suggests that a high concentration of
essential oil was stably encapsulated in this material. However, the
formation of a few spherical cavities in the fracture surface of the nanogel containing essential oil of L. origanoides synthesized with the
lowest amount of ferulic acid of 0.095 g (CF4EO) (Fig. 3G and H) is
verified by SEM.
Considering the obtained results it is observed that the number and
size of the microporous holes inside the materials increased with the
increase of the ferulic acid concentration used in the synthesis, which
may be related to a greater presence of essential oil in the CF1EO
material.

3.2.4. Determination of particle size and zeta potential
Table 4 presents the results of mean particle size and zeta potential
of CS-FA and CS-FA containing encapsulated essential oil (CFEO).
It can be observed for the average particle size (CS-FA) that these

did not show a tendency in relation to the increase of the ferulic acid
content used in the synthesis, presenting random values between
865.8 ± 233.9 nm and 4285.1 ± 1241.5 nm (Table 4). A probable
explanation is that nanogels with higher particle size CF2
(4285.1 ± 1241.5 nm) and CF4 (2369.7 ± 587.4) have a lower proportion of chitosan linked to ferulic acid, since that, at low pH, free
amine groups are protonated, provoking electrostatic repulsion between the polymer chains and thus leading to larger sizes of nanoparticles (Abreu, Oliveira, Paula, & de Paula, 2012; Szymańska &
Winnicka, 2015).
Due to the presence of protonated amine groups on the surface of
CS-FA nanogels, these presented a positive surface charge, with zeta
potential values varying between +55.34 mV and +43.97 mV
(Table 4). The data suggest that the nanogels (CF2 and CF4) presented a
higher proportion of protonated amine groups on their surface and are
more stable in relation to the others, once that zeta potentials, in
magnitude, greater than 30 mV, are considered stable. In this case, repulsive forces act to prevent particle aggregation (Zhao, Zhang, & Feng,
2016).
The essential oil of L. origanoides encapsulation by means of the
nanogels did not significantly affect the medium particle size for the
CF1EO sample (Table 4) compared to the free nanogel (Table 4).
However, the addition of essential oil to the composition of the nanogels CF2 and CF3 resulted in a larger medium particle size, whereas for
nanogel CF4 the medium size was reduced (Table 4). The results indicate that the interactions between the essential oil and nanogel influence the structure of these materials.
In the same form the pure CS-FA nanogels, and the CS-FA chitosan
nanogels containing essential oil had a positive surface charge and the
CF2EO and CF3EO samples were the ones that presented highest
medium particle size (Table 4).
The results of zeta potentials also indicate that by encapsulating the
essential oil of L. origanoides the particles become less stable, this can be
observed by comparing the zeta potentials presented in Table 4. Despite
the reduction in the stability of the particles the values for all samples
still indicate good stability.

Fig. 1. FT-IR spectra of (A) chitosan, (B) ferulic acid and (C) CS-FA nanogel.

3.2.3. Scanning Electron Microscopy (SEM)
By the electromicrographs it can be seen that the incorporation of
the essential oil into the polymer matrix led to nanogels structure
changes (Fig. 3). This can be verified by comparing the CF1EO and
CF4EO morphologies (Fig. 3A, C, E and G) with that of the CS-FA nanogel starting material (Fig. 3B, D and F).
Fig. 3A, C and E show the electromicrographs of the nanogel synthesized with the highest amount of ferulic acid (0.760 g) containing
essential oil (CF1EO). It is possible to observe that this nanogel had
presented defined spherical cavities and of varied sizes, distributed

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Carbohydrate Polymers 188 (2018) 268–275

R.R. Almeida et al.

Fig. 2. SSNMR

13

C spectra of CS-FA nanogel, chitosan (CS) and ferulic acid (FA).

thermal events related to the decomposition of the compound.
Similar to pure chitosan, chitosan linked to ferulic acid nanogels
(CS-FA) presented three thermal events (Fig. 4C). The second thermal
event related to the nanogels decomposition (Fig. 4C) occurred between
243 °C and 357 °C with mass loss between 36% and 40% for all samples.
The reduction in the decomposition range in nanogel samples in relation to pure chitosan Table 5 may be related to the decrease in chitosan

crystallinity after structural modification with ferulic acid, and due to
strong intermolecular interactions between the polymer chains (Panwar
et al., 2016; Szymańska & Winnicka, 2015).

3.2.5. Thermogravimetric analysis (TGA)
Fig. 4A–D show the TGA curves for chitosan, ferulic acid, nanogels
(CS-FA) and nanogels of CS-FA nanogels containing encapsulated essential oil, respectively. All the mass loss processes shown in Fig. 4A, B
and D are described in Table 5.
As is possible to verify, the thermogravimetric profile of chitosan
(Fig. 4A) presents three characteristic thermal events corresponding to
the polymer decomposition, being the experimental results according to
the literature (Thangavel, Ramachandran, & Muthuvijayan, 2016).
While the ferulic acid degradation profile (Fig. 4B) evidences two

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R.R. Almeida et al.

Table 3
RMN data for

13

Table 4
Medium size values and nanogels zeta potential.

C for chitosan and ferulic acid.

(13C) Chitosan

(13C) Ferulic acid

Sample

Mean particle size (nm)

Zeta potencial (mV)

CF1
CF2
CF3
CF4
CF1EO
CF2EO
CF3EO
CF4EO

1125.3 ± 321.2
4285.7 ± 1241.5
865.8 ± 233.9
2369.7 ± 587.4
1033.0 ± 296,2
5060.1 ± 1462,3
1021.6 ± 270,7
1057.3 ± 277,5

+50.86

+55.34
+43.97
+53.80
+33.76
+52.98
+31.27
+53.42

(δc, ppm)

Assignment

(δc, ppm)

Assignment

23.11
57.08
59.93
74.91
81.99
104.50
173.57

C8 (-NHCOCH3)
C2 (NH2CH-)
C6 (-CH2OH)
C5/C3(HOCH3CHeO)
C4 (-CHeO)
C1(-CHeO) anomeric

C7 (-NHCOCH3)

56.23
108.55
111.69
113.95
125.60
144.77
147.94
172.78

C10 (eOCH3)
C8 (]CHCOOH)
C5 (OCeHC]CH) aromatic
C2 (OCeHC]C) aromatic
C1/C6 (C]C) aromatic
C7 (CeCH]CH)
C3/C4 (eCOe) aromatic
C9 (eCOOH)

CF1 = 0.760 g; CF2 = 0.380 g; CF3 = 0.190 g and CF4 = 0.095 g of ferulic acid used in
synthesis of the CS-FA nanogels.

Fig. 3. Electromicrographs: (A) cross section, approximation 130x (CF1EO); (B) cross section, approximation 130x (CS-FA); (C) cross section, approximation 370x (CF1EO); (D) cross
section, approximation 370x (CS-FA); (E) surface, approximation 370x (CF1EO); (F) surface, approximation 370x (CS-FA); (G) cross section, approximation 130x (CF4EO); (H) cross
section, approximation 370x (CF4EO).

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R.R. Almeida et al.

Fig. 4. TGA Curves of (A) chitosan, (B) ferulic acid, (C) chitosan and CS-FA nanogels and (D) essential oil and nanogels of CS-FA containing encapsulated essential oil.

that the interaction between the nanogel and the essential oil influenced its volatilization. The Tmax variation may be associated to the
encapsulation of the essential oil by the nanogels, once that it was
observed through the SEM analysis that most of the essential oil is
stably trapped inside the nanogel, making its volatilization difficult.
The presence of essential oil in the samples (CF2EO and CF4EO)
displaced the second thermal events related to the CS-FA degradation
for higher temperatures, while the samples (CF1EO and CF3EO) started
to present a lower Tonset, which can be verified in Table 5. In spite of the
variation in the degradation temperature of the samples containing
essential oil in relation to the CS-FA nanogels (243 and 357 °C), they
presented Tmax around 299 °C, similar to the CS-FA. The results indicate
that the nanogels interaction with the essential oil does not present
much influence on the thermal stability of the CS-FA.
By means of the TGA curves of the CS-FA nanogels samples (CS-FA)
containing encapsulated essential oil (Fig. 4D), according to Eq. (1), the
values related to the encapsulation efficiencies (EE) were calculated.
The CS-FA nanogel synthesized using the highest proportion of ferulic
acid in relation to chitosan (CF1EO) presented the best EE (20%), once
1.0 g of this nanogel was able to encapsulate 0.20 g of essential oil.
For the sample CF1OE the L. origanoides essential oil was encapsulated in 1.0 g of nanogel with an efficiency of 20.0%. However, for
the other samples CF2EO, CF3EO and CF4EO very similar values of EE
were obtained of 12.0, 10.0 and 13.4%, respectively. Considering the
EE found for the different nanogels, it is possible to observe that the
increase of ferulic acid in the polymer matrix significantly affected the

materials loading capacity.
Similar studies were carried out in order to determine the EE of the
essential oil of L. origanoides (presenting the thymol as major constituent) by different polymers, however, using UV–vis spectrophotometry as a methodology. de Oliveira, Paula, and Paula (2014)
using alginate nanoparticles (ALG) with cashew gum (CG), evaluated
the efficiency of the nanoparticles obtaining results ranging from 21%

Table 5
TGA data for chitosan (CS), ferulic acid (FA) and CS-FA nanogels containing essential oil.
Sample

Event

Tonset (°C)

Tendset (°C)

Tmax (°C)

Mass
Loss
(%)

Explanation

CS

1
2

25

247

89
379

–
–

11
42

3

473

598

–

42

1

145

209

–

73

2

277

390

–

11

1
2
1
2
1
2
1
2

21
240
23
260
21
239
23
256

239

437
260
466
239
443
260
482

151

22

115

12

122

10

127

13

Loss of water
Polymer
decomposition
Polymer
degradation and
loss of inorganic

material
Compound
decomposition
Compound
decomposition
Essential oil
volatilization
Essential oil
volatilization
Essential oil
volatilization
Essential oil
volatilization

FA

CF1EO
CF2EO
CF3EO
CF4EO

CF1EO = 0.760 g; CF2EO = 0.380 g; CF3EO = 0.190 g and CF4EO = 0.095 g of ferulic
acid used in synthesis of the CS-FA nanogels.

In the thermogravimetric curves of nanogel samples containing
encapsulated essential oil (CF1EO, CF2EO, CF3EO and CF4EO)
(Fig. 4D) a new mass loss event is observed, which are related to the
essential oil volatilization. All of the mass loss processes shown in
Fig. 4D are described in Table 5.
For all samples (Table 5) Tmax were verified, that is, the temperature

which the rate of mass loss is maximum, related to the essential oil at
higher temperatures, than at the pure essential oil (107 °C). Inferring
274


Carbohydrate Polymers 188 (2018) 268–275

R.R. Almeida et al.

in the proportion (3:1) of (ALG: CG) to 48% (1:1) of (ALG: CG). However, Paula, Oliveira, Carneiro, and de Paula (2016) using nanoparticles
based in chitosan (CS) with cashew gum (CG), chichar gum (ChG) and
angico gum (AG) obtained 33% EE in the ratio of (1:2.5) of (CS: CG), of
15% (5:1) (CS: ChG) and 25% (5:1) (CS: AG). In this study, the highest
EE of essential oil of 62% and 59% were obtained for the samples
synthesized at the highest concentrations of CG and ChG, respectively.
Front of the efficiencies of encapsulation of the L. origanoides essential oil obtained by other matrices in previous works, it is inferred
that the results obtained in this work are satisfactory. It should be noted
that different methodologies were used to evaluate the encapsulation
efficiencies.

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4. Conclusion
In this study, nanogels of chitosan linked to ferulic acid was synthesized, and the chemical interaction was confirmed by the FTIR and
13
C SSNMR analyzes. The nanogels showed great capacity of encapsulation imprisoning of the essential oil of L. origanoides in its interior, being observed the formation of well defined pores or spherical,
influencing, in this way, in its volatilization. On the other hand, it was
possible to verify that the ferulic acid increase in the polymer matrix
significantly affects this capacity. The best results regarding particle
size, encapsulation efficiency and essential oil stability were obtained
for the synthesized nanogel with the highest amount of ferulic acid. In
this sense, the nanogel presents relevant characteristics and a high
potential for application in the protection of the essential oil of L. origanoides, considering the capacity that this nanogel presented in encapsulating the active substances.
Declarations of interest
None.
Acknowledgment
To the Laboratory of Electron Microscopy and Ultrastructural
Analysis (LME), at the Federal University of Lavras, Lavras (UFLA)
Minas Gerais State, Brazil. This work was supported by the grant from

the Rede Mineira de Química (RQ-MG) and Coordination for the
Improvement of Higher Education Personnel (CAPES).
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