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Pharmaceutical Development and Technology

ISSN: 1083-7450 (Print) 1097-9867 (Online) Journal homepage: http://www.tandfonline.com/loi/iphd20

Anti-inflammatory effects of eugenol

nanoemulsion as a topical delivery system

Fariba Esmaeili, Saeid Rajabnejhad, Ali Reza Partoazar, Shahram Ejtemaei

Mehr, Reza Faridi-Majidi, Mousa Sahebgharani, Leila Syedmoradi,
Mohammad Reza Rajabnejhad & Amir Amani

To cite this article: Fariba Esmaeili, Saeid Rajabnejhad, Ali Reza Partoazar, Shahram Ejtemaei
Mehr, Reza Faridi-Majidi, Mousa Sahebgharani, Leila Syedmoradi, Mohammad Reza
Rajabnejhad & Amir Amani (2015): Anti-inflammatory effects of eugenol nanoemulsion as a
topical delivery system, Pharmaceutical Development and Technology

To link to this article: http://dx.doi.org/10.3109/10837450.2015.1078353

Published online: 12 Sep 2015.

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ISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–7

! 2015 Taylor & Francis. DOI: 10.3109/10837450.2015.1078353


Anti-inflammatory effects of eugenol nanoemulsion as a topical delivery

Fariba Esmaeili1, Saeid Rajabnejhad2, Ali Reza Partoazar3, Shahram Ejtemaei Mehr3, Reza Faridi-Majidi1,
Mousa Sahebgharani4, Leila Syedmoradi1, Mohammad Reza Rajabnejhad5, and Amir Amani1,6
Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran,
School of Pharmacy, University of Camerino, Camerino, Italy, 3Experimental Medicine Research Center, Tehran University of Medical Sciences,
Tehran, Iran, 4Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, 5Research Institute for Islamic
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and Complementary Medicine, Iran University of Medical Sciences, Tehran, Iran, and 6Medical Biomaterials Research Center (MBRC), Tehran
University of Medical Sciences, Tehran, Iran

Abstract Keywords
Eugenol is the main constituent of clove oil with anti-inflammatory properties. In this work, for Anti-inflammatory, eugenol, nanoemulsion,
the first time, O/W nanoemulsion of eugenol was designed for the evaluation of anti- piroxicam
inflammatory effects as a topical delivery system. Topical formulations containing 1%, 2% and
4% of eugenol as well as a nanoemulsion system containing 4% eugenol and 0.5% piroxicam History
were prepared. Further to physicochemical examinations, such as determination of particle size,
polydispersity index, zeta potential and physical stability, anti-inflammatory activity was Received 19 May 2015
examined in carrageenan-induced paw edema in rats. The optimum formulation was found to Revised 12 July 2015
contain 2% eugenol (oil phase), 14% Tween 20 (surfactant) and 14% isopropyl alcohol (co- Accepted 17 July 2015
surfactant) in water. Nanoemulsion with polydispersity index of 0.3 and median droplet Published online 10 September 2015
diameter of 24.4 nm (d50) was obtained. Animal studies revealed that the nanoemulsions
exhibited significantly improved anti-inflammatory activity after 1.5 h, compared with marketed
piroxicam gel. Additionally, it was shown that increasing the concentration of eugenol did not
show higher inhibition of inflammation. Also, the nanoemulsion having piroxicam showed less
anti-inflammatory properties compared with the nanoemulsion without piroxicam.

Introduction activities has been reported for this compound, including

Nanoemulsions are transparent or translucent dispersions of oil anti-microbial8, anti-fungal9, anti-viral10, anti-oxidative11 and
and water that are stabilized by surfactant and co-surfactant anti-tumor12 properties. Additionally, its analgesic, local anes-
molecules. Moreover, nanoemulsions have a droplet size of less thetic and anti-inflammatory activities have been well-docu-
than 100 nm that make them resist against aggregation or mented in the literature7,13,14.
creaming processes1,2. When using topically, nanoemulsions Eugenol, as a natural product with anti-inflammatory effects,
have shown promising over conventional topical formulations has gained a great deal of attention in topical applications.
(e.g. gels and emulsions) dues to enhance transdermal perme- Previous studies have demonstrated that eugenol has anti-inflam-
ation1. For instance, a nanoemulsion preparation containing matory properties comparable with some NSAIDs, such as
aspirin enhanced the anti-inflammatory activity of aspirin in a etodolac and indomethacin15. Molecular studies for anti-inflam-
CD-1 mouse model of induced inflammation, compared with a matory activity of eugenol show COX-2 inhibition without
suspension3. In another study, acaprylic acid-based nanoemulsion affecting COX-1 in mice macrophage cell culture16,17.
gel could reduce the skin barrier effects and increase the Additionally, eugenol has anti-nociceptive activity. Thus, its
transdermal permeation and penetration of meloxicam4. topical applications along with preparations of lidocaine/prilo-
Similarly, the capability of nanoemulsion formulations to improve caine may have synergistic effects in reducing the pain18. In
permeation rate has also been demonstrated for ketoprofen5. topical applications, eugenol has shown to enhance the perme-
The main component of clove oil is eugenol, a phenolic ation of active ingredients too19,20. While at low concentrations,
compound6, which is colorless or a light yellowish fluid7. Eugenol analgesic and anti-inflammatory effects are observed, at high
has been employed in dental cares and as a flavoring agent in concentrations, production of free radicals which cause tissue
cosmetics and food products. A variety of pharmacological damage has been reported from eugenol21. Eugenol has been
safely applied topically up to 5%, while an 8% concentration of
the drug makes local irritation22.
Address for correspondence: Dr. Amir Amani, Department of Medical
Considering the potential of using nanoemulsions as penetra-
Nanotechnology, School of Advanced Technologies in Medicine, Tehran tion enhancers, using a nanoemulsion-based preparation could be
University of Medical Sciences, Tehran, Iran. E-mail: appropriate topical vehicle for eugenol. Currently, nanoemulsions
aamani@sina.tums.ac.ir of eugenol have been reported in the literature, with antioxidant
2 F. Esmaeili et al. Pharm Dev Technol, Early Online: 1–7

and antimicrobial activities23, and with healing properties24. not less than 48 h. Samples remaining unchanged after cycles
However, no work so far has reported the use of nanoemulsions as were considered as stable26.
carriers of eugenol for anti-inflammatory purposes. In this study,
the effect of eugenol nanoemulsion on inhibition of inflammation Characterization of the most stable nanoemulsion
has been evaluated in vivo as a topical delivery system.
Particle size, polydispersity index and zeta potential
Furthermore, to examine the possible synergistic effects, pirox-
icam was loaded into a nanoemulsion system and the anti-
inflammatory effects were compared. The mean droplet size and polydispersity of the most stable
nanoemulsion were measured by dynamic light scattering (DLS)
Materials and methods using Scatteroscope-I (K-ONE Inc., Korea). The z-average mean
Materials and zeta potential of the sample were measured without any
Carrageenan type IV (lambda), carbopol 934 and isopropyl
alcohol were obtained from Sigma-Aldrich (Germany). Eugenol Morphology studies
and Tween 20 were purchased from Merck Chemicals (Germany).
The marketed piroxicam gel (0.5%) was purchased from Razak For morphological assessment, one drop was placed on a carbon-
Pharmaceutical Industries (Tehran, Iran). coated copper grid (200 mesh) and the excess amount was
removed with a piece of filter paper. A drop of aqueous solution
Construction of pseudo-ternary phase diagrams of phosphotungistic acid27 was added onto the grid and staining
was allowed for 5 min at room temperature. The prepared sample
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In order to illustrate and identify the structure of nanoemulsions, was then evaluated by transmission electronic microscopy using a
pseudo-ternary phase diagrams were constructed at ambient LEO 906 (Zeiss, Germany) instrument at 200 KV.
temperature. Eugenol was chosen as the oil phase and Tween 20
and isopropyl alcohol were used as surfactant and co-surfactant, Viscosity measurements
respectively. Surfactant and co-surfactant were mixed at specific
volume ratios of 1:1, 1:2 and 2:1, then, vortexed for 1 min to Viscosity was measured using a Physica MCR 300 with an
obtain the surfactant/co-surfactant (Smix) mixture. The mixture of embedded double Couette cylindrical system rheometer (Anton
eugenol and Smix was prepared at various volume ratios of 1:1 to Paar GmbH, Graz, Austria). The nanoemulsion (20 mL, adjusted
1:9, and 0.5:7, 1:14, 1:9, 1:12, 1:16, 1:18, 1:22, 1:26, 1:28, 2:1 to 25 ± 0.1  C) was poured into the cylinder. The analyses were
and 2:21 (v/v). The mixtures were titrated with distilled water done in triplicate.
under magnetic stirring. Resulted samples were evaluated visually
for transparency and phase separation. The nanoemulsion region Refractive index
was displayed graphically using the Origin Pro version b9.2.272 The refractive index was measured by an Abbe Mark II
software (OriginLab, Northampton, MA). Refractometer (Reichert Scientific Instrument, BuGalo, NY) by
placing one drop of the formulation on the slide at 25  C. The
Preparation of nanoemulsions
analyses were done in triplicate.
Based on the pseudo-ternary diagrams, four formulations were
prepared by stirring different amounts of surfactant and co- Determination of pH
surfactant. Subsequently, oil and water phases were added,
The pH values were determined directly in the samples using a
respectively. The formulations were stirred until a transparent
calibrated potentiometer (Inolab pH 720, WTW, Germany), at
solution was obtained at room temperature (Table 1). Piroxicam
ambient temperature. The measurements were performed in
was accurately weighed and dissolved in the oil phase, prior to
addition of oil phase to the preparations (i.e. sample 4% + pirox-
icam in Table 1).
Animal studies
Thermodynamic stability studies To prepare a nanoemulsion-based topical gel, 50 mg of Carbopol
934 was added to the most stable nanoemulsion with constant
Nanoemulsions were constituted at a certain concentration of oil
stirring. Sodium hydroxide 1 N was then added under gentle
phase, surfactant/co-surfactant mixture and aqueous phase which
stirring and the pH was adjusted to 7.0. The gel was allowed to
are thermodynamically stable systems without phase separation,
stand overnight to remove entrapped air.
creaming or cracking. Also, the thermodynamic stability of
Male Albino rats weighting 200 ± 30 g were used for the
nanoemulsions distinguishes from macroemulsions through phase
evaluation of the anti-inflammatory activities. The animals were
separation which are kinetically stable systems25. The prepared
housed six per cage in the standardized conditions under constant
formulations were subjected to two different thermodynamic
temperature (25 ± 2  C) and a 12-h light/dark cycle and had free
stability tests to evaluate their physical stability.
access to water and food before experimentation. The animal
Centrifugation test
Table 1. Size and the composition of five prepared nanoemulsion
In order to estimate physical stability, the nanoemulsion formu- systems.
lations were centrifuged at 2700 rpm for 5 min at 4  C, followed
by a 30 000 rpm centrifugation for 5 min at 30  C. The samples Eugenol Smix Piroxicam Water Size
were then observed for signs of phase separation, creaming or Samples (v/v) (v/v) (w/v) (v/v) (d50, nm)
1% 1% 28% 71% 16.3
2% 2% 28% 70% 24.4
Freeze–thaw cycles 3% 3% 28% 69% 39.3
4% 4% 56% 40% 48.5
Three freeze–thaw cycles were performed for the formulations
4% + Piroxicam 4% 56% 0.5% 40% 8180
between 21 and +25  C with storage time at each temperature
DOI: 10.3109/10837450.2015.1078353 Anti-inflammatory effects of topical eugenol nanoemulsion 3

procedures were performed in accordance with the Ethics Group 6: Pretreated with marketed piroxicam gel (Px gel).
Committee of Tehran University of Medical Sciences, and
followed the recommendations for the proper care and use of Statistical analysis
laboratory animals.
The obtained results were displayed as mean (SD). The outcomes
The anti-inflammatory activities were examined by carra-
were statistically analyzed using Student’s t-test for unpaired data
geenan-induced paw edema test, according to the method of
or by one-way analysis of variance (ANOVA). p values 50.05
Winter et al.28. The rats were anesthetized with ketamine–
were considered as statistically significant.
xylazine and as a pre-treatment, 100 mg of a treatment sample was
applied topically to the left hind paw of the rats. The site of
Results and discussion
application was bandaged lightly. After 2 h, the dressing was
removed and the gel remaining on the surface of the skin was Construction of pseudo-ternary phase diagrams and
wiped off with a piece of cotton. The left hind paw was injected nanoemulsion formulation
with 0.1 mL of 1% freshly carrageenan solution in saline into sub-
The nanoemulsion regions were constructed with phase diagrams.
plantar region. The right hind paw was injected with the same
The various volume ratios of eugenol and Smix are displayed with
amount of carrageenan solution as a control (i.e. with no pre-
phase diagrams in Figure 1. The translucent nanoemulsion region
treatment). Paw volume was measured before injection (time
was presented in phase diagrams. As shown in Figure 1, a small
zero) and 0.5, 1, 1.5, 2 and 2.5 h after the administration of
area in each diagram is associated with nanoemulsion. Based on
carrageenan using a digital caliper.
visual inspection, all the remaining area is contributed to emulsion.
The treatment samples were divided into six groups, each
The diagrams indicate that with an increase in the volume ratio of
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group administered to six animals, as follows:

Smix, the nanoemulsion region becomes slightly larger.
Group 1: Pretreated with 2% nanoemulsion without eugenol
(sample 0%).
Characterization of nanoemulsion
Group 2: Pretreated with 1% eugenol nanoemulsion (sample 1%).
Group 3: Pretreated with 2% eugenol nanoemulsion (sample 2%). Non-steroidal anti-inflammatory drugs (NSAIDs), such as
Group 4: Pretreated with 4% eugenol nanoemulsion (sample 4%). piroxicam, are being widely used as clinical suppressors of
Group 5: Pretreated with 4% eugenol nanoemulsion having 0.5% inflammation. In present study, eugenol was applied topically in
piroxicam (sample 4% + Px). the form of nanoemulsion as an alternative to topical NSAIDs.

Figure 1. Pseudo-ternary phase diagrams of eugenol nanoemulsions, indicating O/W nanoemulsion region of eugenol (oil), Tween 20 (surfactant) with
isopropyl alcohol (co-surfactant) at Smix 1:1, 1:2, and 2:1.
4 F. Esmaeili et al. Pharm Dev Technol, Early Online: 1–7

The carrageenan-induced rat paw inflammatory response was containing different ratios of surfactants and co-surfactant, the
used as a well-known animal model test to evaluate the anti- optimum formulation was selected based on visual observation
inflammatory activity of the preparations16. of the samples prepared freshly or gone under accelerated
Tables 2 and 3 brief the results of screening different stability studies. Having mentioned that high concentrations of
ingredient concentrations to obtain the optimum preparation in surfactants commonly end up in skin irritation29,30. The
terms of its stability. After examining various formulations optimum formulation included 2% eugenol, 14% Tween20,

Table 2. Stability screening of nanoemulsion formulations.

Percentage v/v of different components in formulations Visual inspection

Smix ratio Based on the
(S:CoS) S. no. Oil Smix Water duration Inference
1:1 1 0.5 7 92.5 0 Failed
2 1 9 90 0a Failed
3 1 12 87 0a Failed
4 1 14 85 0a Failed
5 1 16 83 0a Failed
6 1 18 81 0a Failed
7 1 20 77 36 months Passed
8 1 24 75 18 months Passed
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9 1 26 73 18 months Passed
10 1 28 71 30months Passed
11 1 30 69 30 months Passed
12 1 32 67 30 months Passed
13 1 34 65 30 months Passed
14 1 38 61 30 months Passed
15 2 14 84 0a Failed
16 2 18 80 0a Failed
17 2 28 70 48 months Passed
18 2 30 68 36 months Passed
19 2 32 66 36 months Passed
20 2 34 64 36 months Passed
21 2 36 62 36 months Passed
22 2 40 58 36 months Passed
23 3 27 70 0a Failed
24 3 28 69 36 months Passed
25 3 30 67 18 months Passed
26 3 32 65 18 months Passed
27 3 34 63 18 months Passed
28 3 36 61 18 months Passed
29 4 28 68 18 months Passed
30 4 34 62 18 months Passed
31 4 36 60 18 months Passed
32 4 56 40 18 months Passed
33 5 28 67 0a Failed
1:2 34 1 15 84 18 months Passed
35 1 18 81 18 months Passed
36 1 21 78 18 months Passed
37 1 24 75 18 months Passed
38 1 30 69 18 months Passed
39 1 36 63 18 months Passed
40 2 21 77 0a Failed
41 2 30 68 18 months Passed
42 2 36 62 18 months Passed
43 3 24 73 0a Failed
44 3 30 67 18 months Passed
45 3 36 61 18 months Passed
2:1 46 1 18 81 0a Failed
47 1 21 78 18 months Passed
48 1 24 75 18 months Passed
49 1 27 72 18 months Passed
50 2 18 80 0a Failed
51 2 21 77 18 months Passed
52 2 30 68 18 months Passed
53 2 36 62 18 months Passed
54 3 21 76 18 months Passed
55 3 24 73 18 months Passed
56 3 27 70 18 months Passed
57 4 21 75 0a Failed
58 5 21 74 0a Failed

Oil used: eugenol, surfactant used: Tween-20, co-surfactant used: isopropyl alcohol, external phase: distilled water.
Using 0 means observation of phase separation at the time of preparation.
DOI: 10.3109/10837450.2015.1078353 Anti-inflammatory effects of topical eugenol nanoemulsion 5

14% isopropyl alcohol and water. The DLS studies showed a eugenol nanoemulsion has comparatively higher viscosity than
size of 24.4 nm with a polydispersity index and zeta potential of the 2% preparation. Considering the fact that the 2% preparation
0.33 mV. Nearly spherical particles were obtained from TEM was more stable than the 1% (samples 10 and 17 in Table 2), it
studies with size in range of 30–80 nm, in agreement with the is arguable that in 1% preparation, an extra amount of surfactant
DLS findings (Figure 2). The 4% + Px sample was able to carry and co-surfactant is present in the formulation with respect to
the drug for up to 8 h after preparation, a time length enough the oil. These extra molecules do not enter the structure of
for our in vivo studies, but, undoubtedly, not enough for a final nanoemulsion droplets and are in fact dispersed throughout the
product (see sample 14 in Table 3). continuous medium (i.e. water), thus, increase the viscosity of
Table 4 and Figure 3 brief the refractive index, pH and the preparation.
viscosity results of the four nanoemulsion samples studied in The refractive index of the nanoemulsions was from 1.28 to
vivo. The viscosity of formulations was obtained in the range of 1.36, with all values in the range expected for transparent
0.11–105 Pa s, relatively low values which in turn is associated isotropic nanoemulsions. Also, all the pH values obtained (4.63–
with easy handling and smooth administration of the formula- 5.09) were consistent with pH values acceptable for dermal
tions. In addition, the size of the drop and low viscosity of formulations (i.e. 4.0–7.0)31.
nanoemulsion may affect its performance due to increasing
percutaneous uptake of nanoemulsion. From Table 4, 1%
Anti-inflammatory activity of eugenol nanoemulsion
Figure 4 shows the inflammatory results obtained from the six
treatment groups. As the details show, in control group (0%), the
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Table 3. Piroxicam-loaded eugenol nanoemulsions. paw edema has increased during the study period, as reported
previously7. This pattern is more or less observable in the group
% Oil % Smix % Water % Piroxicam of marketed gel (Px Gel). Additionally, comparing the results of
S. no (v/v) (v/v) (v/v) (w/v) Result Px Gel with the blank group, no significant change in the
1 2 28 70 0.5 Not dissolved inflammation may be observed, a finding which has already been
2 3 42 55 0.5 Unstable reported32.
3 3 47 50 0.5 Unstable Figure 4 shows the inflammatory data from the six studied
4 3 51 46 0.5 Unstable groups. The statistical analyses are briefed in Table 5. From the
5 3 55 42 0.5 Unstable
details, the treatment groups having eugenol (i.e. 1%, 2%, 4% and
6 3.5 49 57.5 0.5 Unstable
7 3.5 50.5 46 0.5 Unstable 4% + Px) show a different pattern in edema measured compared
8 3.7 38.89 57.41 0.5 Precipitated with the control group (i.e. 0%). A minimum edema is observed at
9 4 28 68 0.5 Not dissolved 1.5 h for all the samples. This shows that the eugenol has played
10 4 36 60 0.5 Unstable an important role in the inhibition of inflammation. However, its
11 4 42 54 0.5 Unstable effect becomes less visible after 2 h, showing shorter bio-
12 4 46 50 0.5 Unstable logical life for eugenol compared with carrageenan. k
13 4 52 44 0.5 Unstable
14 4 56 40 0.5 Stable till 8 h Carrageenan has been reported to maintain its maximum activity
15 5 52.5 42.5 0.5 Unstable up to 3 h33, while the maximum activity of eugenol appears to be
16 6 40 54 0.5 Precipitated at 1.5 h here in.
17 6 38 56 0.5 Precipitated Furthermore, comparison of the edema results in the three
18 6 42 52 0.5 Unstable concentrations of eugenol shows that the inflammation is not
19 6 44 50 0.5 Unstable significantly affected by the concentration of eugenol, espe-
20 6 48 46 0.5 Unstable
21 6 63 31 0.5 Dissolved
cially in case of 1% and 2% treatment groups. Looking more
22 7 63 30 0.5 Precipitated closely to the results, samples 1% and 2% appear to be similar
in terms of their anti-inflammatory activities. While, the 4%
Sample number 14 is the only formulation that was stable for 8 h (in group appears to be slightly (but not significantly) less effective
bold). at time 1.5 h, but more effective at times 0.5, 2 and 2.5 h. This
could be showing a type of improved biological half-life in the
sample subject to further studies. In general, however, all the
three samples may be considered as more potent compared
with the 4% + Px sample. By taking into account the stability
studies, the sample 2% is suggested as the optimum sample in
our work.
On the other hand, it is interesting to note that when piroxicam
has been introduced into the nanoemulsion, the anti-inflammatory
effects of eugenol become negligible. To examine the possible
reasons for this phenomenon, size of the 4% sample with and
without piroxicam was studied. The very high difference between
the samples (i.e. 48.5 nm versus 8180 nm) could be a reason for
the sample not passing through the skin barriers. Additionally,
stability of the sample with and without the drug is considerably
different (i.e. 18 months versus 8 h). When the carrier is passing
the skin barriers, the less stable preparation may undergo
structural changes which in turn make the diffusion more
difficult. Therefore, less transfer is expected from the drug
molecules which make the preparation less effective in vivo.
Further in vitro or in vivo skin permeation studies may help in this
Figure 2. TEM of 2% eugenol nanoemulsion (16 700). issue.
6 F. Esmaeili et al. Pharm Dev Technol, Early Online: 1–7

Figure 3. Shear stress versus shear rate results

for the 1%, 2%, 3% and 4% eugenol
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Table 4. Size, reflective index, pH and viscosity of four prepared nanoemulsions.

Samples Size (d50, nm) pH Reflective index Viscosity (Pa s)

1% 16.3 4.76 ± 0.02 1.35 ± 0.003 0.105 ± 0.02
2% 24.4 4.63 ± 0.04 1.35 ± 0.001 0.07 ± 0.03
3% 39.3 4.67 ± 0.02 1.36 ± 0.002 0.11 ± 0.06
4% 48.5 5.09 ± 0.02 1.28 ± 0.002 0.24 ± 0.05

Figure 4. The results of rat paw edema in the six treatment groups at time intervals of 0.5, 1, 1.5, 2 and 2.5 h. *A minimum edema is observed at 1.5 h
for all the samples.

Table 5. Statistical comparison between groups on carrageenan-induced Conclusion

paw edema in 1–2 h.
Our results demonstrated that a nanoemulsion preparation of 1%,
Time (h) Groups 0% 4% + Px Px gel 2% or 4% of eugenol possesses improved anti-inflammatory
1 2% – p50.05 p50.05
effects compared with piroxicam commercial gel on carrageenan-
1 4% – – p50.05 induced paw edema in rats. It was also found that increasing the
1.5 1%, 2%, 4% p50.05 p50.05 p50.05 concentration of eugenol from 1% to 4% does not make higher
2 2%, 4% – – p50.05 inhibition of inflammation. Furthermore, comparing the nanoe-
mulsion with and without piroxicam suggested that piroxicam
DOI: 10.3109/10837450.2015.1078353 Anti-inflammatory effects of topical eugenol nanoemulsion 7

decreases the anti-inflammatory effect of eugenol in the prepar- 15. Öztürk A, Özbek H. The anti-inflammatory activity of Eugenia
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This research has been supported by Tehran University of Medical
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Sciences & health Services grant No. 91-01-87-17072.
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