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.
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 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: lguimaraes@ufsj.edu.br (L.G de Lima Guimarães) https://doi.org/10.1016/j.carbpol.2018.01.103 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 °C for 24 h The nanogel was centrifuged at 3000 rpm for 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 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 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 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 °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 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 °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 μ L diluted in hexane (1: 100 v/v) The column temperature was programmed from 60 °C to 240 °C at a rate of °C min−1, after going, to a heating rate of 10 °C min−1–300 °C, remaining at that temperature for 10 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 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 mm The spectra were obtained at a rotation frequency of 10000 Hz, with relaxation time of 3.5 s and a Table 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, 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 Chemical composition of the L origanoides essential oil 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) 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 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 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 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 270 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 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 Despite the reduction in the stability of the particles the values for all samples still indicate good stability Fig 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 271 Carbohydrate Polymers 188 (2018) 268–275 R.R Almeida et al Fig 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 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 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 272 Carbohydrate Polymers 188 (2018) 268–275 R.R Almeida et al Table RMN data for 13 Table 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 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) 273 Carbohydrate Polymers 188 (2018) 268–275 R.R Almeida et al Fig 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 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 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 25 247 89 379 – – 11 42 473 598 – 42 145 209 – 73 277 390 – 11 2 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 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 1021/jf0208482 de Oliveira, E F., Paula, H C B., & Paula de, R M (2014) Alginate/cashew gum nanoparticles for essential oil encapsulation Colloids and Surfaces B: Biointerfaces, 113, 146–151 http://dx.doi.org/10.1016/j.colsurfb.2013.08.038 Hu, Q., & Luo, Y (2016) Polyphenol-chitosan conjugates: Synthesis, characterization, and applications Carbohydrate Polymers, 151, 624–639 http://dx.doi.org/10.1016/j carbpol.2016.05.109 Li, Y., Maciel, D., Rodrigues, J., Shi, X., & Tomás, H (2015) Biodegradable polymer nanogels for drug/nucleic acid delivery Chemical Reviews, 115(16), 8564–8608 http://dx.doi.org/10.1021/cr500131f Liu, J., Wen, X., Lu, J., Kan, J., & Jin, C (2014) Free radical mediated grafting of chitosan with caffeic and ferulic acids: Structures and antioxidant activity International Journal of Biological Macromolecules, 65, 97–106 http://dx.doi.org/10.1016/j ijbiomac.2014.01.021 Oliveira, D R., Leitão, G G., Fernandes, P D., & Leitão, S G (2014) Ethnopharmacological studies of Lippia origanoides Brazilian Journal of Pharmacognosy, 24(2), 206–214 http://dx.doi.org/10.1016/j.bjp.2014.03.001 Panwar, R., Sharma, A K., Kaloti, M., Dutt, D., & Pruthi, V (2016) Characterization and anticancer potential of ferulic acid-loaded chitosan nanoparticles against ME-180 human cervical cancer cell lines Applied Nanoscience, 6(6), 803–813 http://dx.doi org/10.1007/s13204-015-0502-y Paula, H., Oliveira, E., Carneiro, M., & de Paula, R (2016) Matrix effect on the spray drying nanoencapsulation of lippia sidoides essential oil in chitosan-native gum blends Planta Medica, 83(5), 392–397 http://dx.doi.org/10.1055/s-0042-107470 Phan, A D T., Flanagan, B M., D’Arcy, B R., & Gidley, M J (2017) Binding selectivity of dietary polyphenols to different plant cell wall components: Quantification and mechanism Food Chemistry, 233, 216–227 http://dx.doi.org/10.1016/j.foodchem 2017.04.115 Arteche Pujana, M., Pérez-Álvarez, L., Cesteros Iturbe, L C., & Issa, K (2013) Biodegradable chitosan nanogels crosslinked with genipin Carbohydrate Polymers, 94(2), 836–842 http://dx.doi.org/10.1016/j.carbpol.2013.01.082 Ramimoghadam, D., Bagheri, S., & Abd Hamid, S B (2014) Stable monodisperse nanomagnetic colloidal suspensions: An overview Colloids and Surfaces B: Biointerfaces, 133, 388–411 http://dx.doi.org/10.1016/j.colsurfb.2015.02.003 Raut, J S., & Karuppayil, S M (2014) A status review on the medicinal properties of essential oils Industrial Crops and Products, 62, 250–264 http://dx.doi.org/10.1016/ j.indcrop.2014.05.055 Ribeiro, A F., Andrade, E H A., Salimena, F R G., & Maia, J G S (2014) Circadian and seasonal study of the cinnamate chemotype from Lippia origanoides Kunth Biochemical Systematics and Ecology, 55, 249–259 http://dx.doi.org/10.1016/j.bse 2014.03.014 Rui, L., Xie, M., Hu, B., Zhou, L., Saeeduddin, M., & Zeng, X (2017) Enhanced solubility and antioxidant activity of chlorogenic acid-chitosan conjugates due to the conjugation of chitosan with chlorogenic acid Carbohydrate Polymers, 170, 206–216 http://dx.doi.org/10.1016/j.carbpol.2017.04.076 Soares, B V., Cardoso, A C F., Campos, R R., Gonỗalves, B B., Santos, G G., Chaves, F C M., et al (2017) Antiparasitic, physiological and histological effects of the essential oil of Lippia origanoides (Verbenaceae) in native freshwater fish Colossoma macropomum Aquaculture, 469, 72–78 http://dx.doi.org/10.1016/j.aquaculture 2016.12.001 Sousa, K S., Silva Filho, E C., & Airoldi, C (2009) Ethylenesulfide as a useful agent for incorporation into the biopolymer chitosan in a solvent-free reaction for use in cation removal Carbohydrate Research, 344(13), 1716–1723 http://dx.doi.org/10.1016/j carres.2009.05.028 Szymańska, E., & Winnicka, K (2015) Stability of Chitosan—a challenge for pharmaceutical and biomedical applications Marine Drugs, 13(4), 1819–1846 http://dx.doi org/10.3390/md13041819 Thangavel, P., Ramachandran, B., & Muthuvijayan, V (2016) Fabrication of chitosan/ gallic acid 3D microporous scaffold for tissue engineering applications Journal of Biomedical Materials Research Part B: Applied Biomaterials, 104(4), 750–760 http://dx doi.org/10.1002/jbm.b.33603 Tiwari, A., & Tiwari, A (2013) Nanomaterials in drug delivery, imaging, and tissue engineering http://dx.doi.org/10.1002/9781118644591 Woranuch, S., & Yoksan, R (2013) Preparation, characterization and antioxidant property of water-soluble ferulic acid grafted chitosan Carbohydrate Polymers, 96(2), 495–502 http://dx.doi.org/10.1016/j.carbpol.2013.04.006 Younes, I., & Rinaudo, M (2015) Chitin and chitosan preparation from marine sources Structure, properties and applications Marine Drugs, 13(3), 1133–1174 http://dx doi.org/10.3390/md13031133 Zhang, H., Zhai, Y., Wang, J., & Zhai, G (2016) New progress and prospects: The application of nanogel in drug delivery Materials Science & Engineering C, Materials for Biological Applications, 60, 560–568 http://dx.doi.org/10.1016/j.msec.2015.11.041 Zhao, Y., Zhang, Z., & Feng, W (2016) Toxicology of nanomaterials Weinheim: WileyVCH (Chapter 1) Zhaveh, S., Mohsenifar, A., Beiki, M., Khalili, S T., Abdollahi, A., Rahmani-Cherati, T., et al (2015) Encapsulation of Cuminum cyminum essential oils in chitosan-caffeic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus Industrial Crops and Products, 69, 251–256 http://dx.doi.org/10.1016/j.indcrop 2015.02.028 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) References Abreu, F O M S., Oliveira, E F., Paula, H C B., & de Paula, R C M (2012) Chitosan/ cashew gum nanogels for essential oil encapsulation Carbohydrate Polymers, 89(4), 1277–1282 http://dx.doi.org/10.1016/j.carbpol.2012.04.048 Adams, R P (2007) Identification of essential oil components by gas chromatography/mass spectrometry (4th ed.) Illinois, USA: Allured Publishing Corp [pp 456] Agnihotri, S A., Mallikarjuna, N N., & Aminabhavi, T M (2004) Recent advances on chitosan-based micro- and nanoparticles in drug delivery Journal of Controlled Release, 100(1), 5–28 http://dx.doi.org/10.1016/j.jconrel.2004.08.010 Aljawish, A., Chevalot, I., Piffaut, B., Rondeau-Mouro, C., Girardin, M., Jasniewski, J., et al (2012) Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions Carbohydrate Polymers, 87(1), 537–544 http://dx.doi.org/10.1016/j.carbpol.2011.08.016 Asbahani, A., El, M., Badri, K., Sala, W., Addi, M., Casabianca, E H A., et al (2015) Essential oils: From extraction to encapsulation International Journal of Pharmaceutics, 483(1–2), 220–243 http://dx.doi.org/10.1016/j.ijpharm.2014.12 069 Beyki, M., Zhaveh, S., Khalili, S T., Rahmani-Cherati, T., Abollahi, A., Bayat, M., et al (2014) Encapsulation of Mentha piperita essential oils in chitosan-cinnamic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus Industrial Crops and Products, 54, 310–319 http://dx.doi.org/10.1016/j.indcrop.2014.01.033 Chen, X.-G., Lee, C M., & Park, H.-J (2003) O/W emulsification for the self-Aggregation and nanoparticle formation of linoleic AcidModified chitosan in the aqueous system Journal of Agricultural and Food Chemistry, 51(10), 3135–3139 http://dx.doi.org/10 275 ... 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)... 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... 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