z
This study was financed in part by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior –CAPES (Finance Code 001)and Conselho Nacional
de Desenvolvimento Científico e Tecnológico - CNPq (grant number 453927/2014-
9). The authors would also like to thank these agencies for their research
fellowships.
Initially, a nanoemulsion containing essential oil of Aniba canelilla was produced by high pressure homogenizer. Hydroxyethylcellulose and chitosan hydrogels-thickened
nanoemulsion containing essential oil of A. canelilla were developed (HNE and CNE, respectively) by polymer addition. Formulations were characterized by their content of
NP and ME, droplet size, polydispersity index, zeta potential, transmission electron microscopy, rheological behavior and bioadhesion characteristics, and release studies
and skin permeation essays were also performed in Franz-type diffusion cell. To determine the irritant potential from the EOAC and derived formulations, the hen’s egg
chorioallantoic membrane test (HET-CAM) was performed on 10-day fertilized eggs. In order to assess the anti-inflammatory potential of the essential oil and formulations,
topical anti-inflammatory potential was investigated by croton oil-induced mice ear edema, and myeloperoxidase activity (MPO) and interleukins content (IL-1βand IL-6)
were also evaluated.
INTRODUCTION
ACKNOWLEDGEMENTS
CONCLUSION
Nanoemulsion-based hydrogels containing Aniba canelilla (H.B.K.) Mez
essential oil for topical use: in vivo anti-inflammatory efficacy
Tainá Kreutz1*, Letícia Grolli Lucca1, Simone Braga Carneiro2, Helder Ferreira Teixeira1, Renata Pereira Limberger1, Valdir Florêncio Veiga Jr.3, Bibiana Verlindo de Araújo1, Letícia Scherer Koester1
1Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, Santana, 2752, Porto Alegre, Rio Grande do Sul, Brazil
2Departamento de Química, Instituto de Ciências Exatas, Universidade Federal do Amazonas, Av. Gal. Rodrigo Octávio, Japiim, 6200, Manaus, Brazil
3Programa de Pós-Graduação em Química, Instituto Militar de Engenharia, Praça General Tibúrcio, Urca, 80, Rio de Janeiro, Brazil
*e-mail: tainakreutz@gmail.com
Keywords: Aniba canelilla essential oil, hydrogel-thickened nanoemulsion, topical anti-inflammatory activity
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Nanoemulsions are liquid mixture of two immiscible liquids (oil and water) stabilized by emulsifiers to which some advantages have been attributed, such as improving
stability, reducing volatility from essential oils, enhancing permeability and controlling release. However, nanoemulsion low viscosity is a restriction for topical administration.
In order to overcome this and to promote a suitable and pleasant topical application, hydrogels have been employed, since they are able to swell in aqueous media forming
a three-dimensional network. Hydrogel-thickened nanoemulsions may increase nanoemulsion viscosity and adhesiveness favoring its stability beyond suitability to topical
application. The essential oil of Aniba canelilla (H.B.K.) Mez (EOAC) has 1-nitro-2-phenylethane (NP) and methyleugenol (ME) as major compounds. Despite the folk
medicine practices and scientifically proven anti-inflammatory activity in treating topical disorders, there is no report of the development of a semisolid pharmaceutical
containing A. canelilla essential oil.
Hydroxyethylcellulose-hydrogel thickened nanoemulsion containing A. canelilla
essential oil seemed to be an interesting formulation since it showed a low irritant
potential in HET-CAM test and a promising anti-inflammatory response with reduced
MPO activity and interleukins content suggesting activity by decreasing edema,
lowering polymorphonuclear leukocytes migration and reducing synthesis/release of
inflammatory mediators mainly attributed to the essential oil compounds.
NE
CNE
HNE
Hydroxyethylcellulose
Chitosan
Fig. 1. Transmission
electron microscopy
analysis of NE (A, B,
C), HNE (D, E, F) and
CNE (G, H, I) at
magnification of 20 K
(A, D, G), 100 K (B,
E, H) and 300 K (C,
F, I).
Fig. 2. Rheological profile showing ascendant and
descendant curves (A) and viscosity profile (B) of
hydrogels containing or not nanoemulsion (n = 3).
SUBTITLE: EOAC: essential oil of Aniba canelilla; NP: 1-nitro-2-phenylethane; ME: methyleugenol; BM: blank micellar solution; NE:
nanoemulsion containing A. canelilla essential oil;HH: blank hydroxyethylcellulose hydrogel without nanoemulsion or blank micellar solution;
HBM: hydroxyethylcellulose hydrogel-thickened blank micellar solution; HNE: hydroxyethylcellulose hydrogel-thickened nanoemulsion
containing A. canelilla essential oil;CH:blank chitosan hydrogel without nanoemulsion or blank micellar solution; CBM: chitosan hydrogel-
thickened blank micellar solution; CNE: chitosan hydrogel-thickened nanoemulsion containing A. canelilla essential oil;MPO: myeloperoxidase
activity; IL:interleukins.
Fig. 3. Force of detachment (A) and work of
adhesion (B) for NE, HNE and CNE measured by
strength test in porcine skin after 60 sof contact ( n
= 6). Results analyzed by one-way ANOVA followed
by Tukey’s post hoc test. Statistically different (*p ≤
0.05,***p ≤ 0.001).
Fig. 4. Release profile of NP (A) and ME (B)
from EOAC, NE, HNE and CNE (n = 3).
Fig. 5. Cumulative amount permeated (µg.cm-2)of NP (A) and
ME (B) on stratum corneum (SC), epidermis (EP), dermis (D) and
receptor fluid (RF) from applied amounts of EOAC, NE, HNE and
CNE (n = 6). Significant statistical difference was performed by
ANOVA followed by Tukey’s post hoc test. Asterisk (*) means
statistical difference (p ≤0.05)while hash (#) means no statistical
difference (p ≥0.05).
Fig. 6. Sequence of photographs illustrating the effects of different substances applied
on the chorioallantoic membrane over a 5-min period. (A) NaCl 0.9% w/v; (B) NaOH 0.1
M; (C) Sodium lauryl sulfate 1% w/v; (D) Propylene glycol; (E) EOAC : Propylene glycol
(1:10 v/v); (F) Olive oil; (G) EOAC : Olive oil (1:10 v/v); (H) BM; (I) NE; (J) HBM; (K)
HNE;(L) CBM; (M) CNE.
Fig. 7. Croton oil induced-
mouse ear edema measured
by ear weight (mg). Results
are expressed as mean ±
SEM (n= 6-7) and statistical
difference between treatment
and negative control was
obtained by ANOVA followed
by Student-Newman-Keuls
post hoc test (*p ≤0.05,***p ≤
0.001). Edema inhibition
percentages were placed
inside the bars.
Fig. 8. MPO activity after 24 hof induction by croton oil in mice
(OD/biopsy). Results are expressed as mean ±SEM (n= 4-6) and
statistical difference between treatment and negative control was
obtained by ANOVA followed by Student-Newman-Keuls post hoc test
(**p ≤0.01,***p ≤0.001).
Fig. 9. IL-1βcontent (pg/mg) (A) and IL-6 content (pg/mg) (B) on mouse ear. Results are expressed as mean ±SEM. (n= 5-7) and statistical
difference between treatment and negative control was obtained by ANOVA followed by Student-Newman-Keuls post hoc test (*p ≤0.05,**p ≤
0.01,***p ≤0.001).
EOAC
NP
ME
Aniba canelilla
Table 1. Characterization of A. canelilla nanoemulsion and derived
hydrogels at day 1after preparation (n = 3).
NE HNE CNE
Droplet size (nm)
154.96
±
3.95
147.96
±
2.61
249.54 ±14.66
Polidispersity Index
0.23 ±0.02 0.16 ±0.01 0.34 ±0.03
Zeta potential (mV)
-35.97 ±
2.71
-33.80 ±1.70 44.80 ±2.58
NP content (%) 98.69 ±4.76
105.76
±
8.60
87.52 ±9.76
ME content (%) 95.24 ±3.36 98.31 ±7.93 93.92 ±3.67
