PHASCI 3182 No.

of Pages 11, Model 5G

16 February 2015

European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

1

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

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6

3 Development of lamellar gel phase emulsion containing marigold oil

4 (Calendula officinalis) as a potential modern wound dressing
7 C.H. Okuma a,g,⇑, T.A.M. Andrade b, G.F. Caetano b, L.I. Finci c,d, N.R. Maciel a, J.F. Topan a, L.C. Cefali e,
8 A.C.M. Polizello f, T. Carlo g, A.P. Rogerio g,h, A.C.C. Spadaro f, V.L.B. Isaac e, M.A.C. Frade b, P.A. Rocha-Filho a
9 a
Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Brazil
10 b
Division of Dermatology, Department of Internal Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil
11 c
State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China
12 d
Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA
13 e
School of Pharmaceutical Sciences of Araraquara, Sao Paulo State University (UNESP), Brazil
14 f
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Brazil
15 g
Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
16 h
Federal University of Triangulo Mineiro, Uberaba, Brazil

17
18
a r t i c l e i n f o a b s t r a c t
2 3
3 0
21 Article history: Appropriate therapeutics for wound treatments can be achieved by studying the pathophysiology of 34
22 Received 25 November 2013 tissue repair. Here we develop formulations of lamellar gel phase (LGP) emulsions containing marigold 35
23 Received in revised form 28 December 2014 (Calendula officinalis) oil, evaluating their stability and activity on experimental wound healing in rats. 36
24 Accepted 28 January 2015
LGP emulsions were developed and evaluated based on a phase ternary diagram to select the best LGP 37
25 Available online xxxx
emulsion, having a good amount of anisotropic structure and stability. The selected LGP formulation 38
was analyzed according to the intrinsic and accelerated physical stability at different temperatures. In 39
26 Keywords:
addition, in vitro and in vivo studies were carried out on wound healing rats as a model. The LGP emulsion 40
27 Calendula officinalis oil
28 Lamellar gel phase emulsion
(15.0% marigold oil; 10.0% of blend surfactants and 75.0% of purified water [w/w/w]) demonstrated good 41
29 Liquid crystal stability and high viscosity, suggesting longer contact of the formulation with the wound. No cytotoxic 42
30 Stability tests activity (50–1000 lg/mL) was observed in marigold oil. In the wound healing rat model, the LGP 43
31 Wound healing (15 mg/mL) showed an increase in the leukocyte recruitment to the wound at least on days 2 and 7, 44
32 but reduced leukocyte recruitment after 14 and 21 days, as compared to the control. Additionally, colla- 45
gen production was reduced in the LGP emulsion on days 2 and 7 and further accelerated the process of 46
re-epithelialization of the wound itself. The methodology utilized in the present study has produced a 47
potentially useful formulation for a stable LGP emulsion-containing marigold, which was able to improve 48
the wound healing process. 49
Ó 2015 Elsevier B.V. All rights reserved. 50
51

52
53
54 1. Introduction the ability to retain adequate moisture in the bed of the wound 61
(Popovich et al., 2010). 62
55 Chronic wounds to the skin present a serious public health risk Calendula officinalis (Asteraceae) L., or marigold, is an herbal 63
56 and are painful, unsightly, and can require limb amputation if left plant that has been used to treat wounds since the 13th century 64
57 unattended. Adequate treatments are needed to increase the over- in Europe (Parente et al., 2012), and nowadays is used virtually 65
58 all patient quality of life. Products designed for the treatment of worldwide. This plant possesses several biological properties such 66
59 chronic wounds and/or skin ulcers require optimizing properties as antimicrobial (Faria et al., 2011), antimetastatic (Preethi et al., 67
60 such as the ease of application, increased patient comfort, and 2010), and antiparasitic activity (Szakiel et al., 2008). Its main 68
activity as an anti-inflammatory constituent (Parente et al., 2012) 69
which was observed in animal models (Preethi and Kuttan, 2009) 70
and clinical tests, justifies its utilization as a cosmetic and personal 71
⇑ Corresponding author at: Department of Pharmaceutical Sciences, School of care product for wound treatment (Grimme and Augustin, 1999). 72
Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Avenida do Café,
Secondary metabolites such as flavonoids, tannins, saponins, ter- 73
s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil. Tel.: +55 16 3602 4279; fax: +55 16
3602 4881.
penoids, coumarins and others (Santos et al., 2006; Schmidt 74

E-mail addresses: cindhana@hotmail.com, cindhana@fcfrp.usp.br (C.H. Okuma). et al., 2009) alone or in association are directly associated with 75

http://dx.doi.org/10.1016/j.ejps.2015.01.016
0928-0987/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Okuma, C.H., et al. Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a poten-
tial modern wound dressing. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.01.016
 PHASCI 3182 No. of Pages 11, Model 5G
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2 C.H. Okuma et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

76 these effects. Also, marigold oil (C. officinalis) could be used to treat using a rotor/stator homogenizer (Fisaton-Mod 713 D) at 600 rpm 135
77 thermal burn injury, acute dermatits (Pommier et al., 2004) during until the system reached room temperature (25 ± 5 °C). 136
78 irradiation for breast cancer and skin rashes (Parente et al., 2012).
79 C. officinalis extract is present in almost 200 cosmetic formulations 2.3. Emulsions characterization 137
80 and significant experimental wound healing studies or treatments
81 have been performed using only C. officinalis extract or cream con- 2.3.1. Macroscopic evaluation 138
82 taining the extract (Chandran and Kuttan, 2008). Samples were visually observed to determine organoleptic 139
83 The choice of formulation type for the treatment or care of the characteristics and identify instability processes (phase separation, 140
84 skin is important because it may affect the mode of active com- flocculation and creaming), at 24 h post-production. The formula- 141
85 pounds distribution in its surface. Lamellar gel phase (LGP) or liq- tions were visually evaluated and characterized as follows: (i) 142
86 uid crystals (Savic et al., 2007) formulations (emulsions) can be heavily modified (HM-presence of phase separation); (ii) moder- 143
87 obtained due to the bi-layer arrangement (separated by water lay- ately modified (MM-presence of flocculation process); (iii) slightly 144
88 ers) of the surfactant molecules in the interfacial film (Junginger, modified (SM-presence of creaming process); (iv) normal (N-no 145
89 1997). The LGP emulsions provide benefits such as enhanced sta- presence of instability processes/no change in appearance). 146
90 bility and incorporation of active components in the matrix of
91 (LGP) phase, water retention and the controlled release of active 2.3.2. Microscopic evaluation 147
92 ingredients (Kudla et al., 2010). LGP could thus be used for wound The microstructure (presence or absence of anisotropic struc- 148
93 healing purposes due to its adequate viscosity and because of the tures) of o/w emulsions was analyzed with an Olympus BX50 opti- 149
94 probability that it might increase the residence time of the formu- cal microscope (Olympus Optical Co., Ltd., Tokyo, Japan) in the 150
95 lation on the wound surface. bright field and under polarized light. Micrographs were made at 151
96 In the present study, we optimized the preparation of LGP a magnification of 200. 152
97 emulsions using the ternary and pseudo phase’s ternary diagram
98 with different proportions of C. officinalis oil, distilled water, and 2.3.3. Centrifugation 153
99 a mixed emulsifier (cetyl alcohol 2 polyoxyethylene and stearyl Each emulsion was weighted in graduated vials and centrifuged 154
100 alcohol 2 polyoxyethylene) whose HLB values were 6.0. This work at the Fanem model 206 R, Excelsa Baby II-440 W at three speeds: 155
101 is based on previous studies by Santos et al. (2006). The novel sys- 1500, 2500, and 3500 rpm (70, 440, and 863 G, respectively), 156
102 tem was designed with the aim of achieving an efficient lamellar standing for 15 min on each rotation. The procedure was conduct- 157
103 gel phase emulsion with wound healing activity. Santos et al. ed at room temperature (25 ± 2 °C). 158
104 (2006) did not use the ternary and pseudo phase’s ternary diagram
105 to select the best emulsion. In addition to this, the previous formu- 2.3.4. Thermal stress 159
106 lations using C. officinalis oil were not stable. Here, we have pre- Samples were submitted to water bath heating by Nova Techni- 160
107 sented a detailed physicochemical characterization (preliminary cal Ltd., Brazil model 281 NT). The temperature was increased in 161
108 tests) of samples to select the best LGP emulsion (i.e. good amount increments of 5 °C beginning at 40 ± 2 °C and each temperature 162
109 of anisotropic structure and stability). The selected LGP formula- was maintained for 30 min up until a temperature of 80 ± 2 °C 163
110 tion was analyzed according to the rheological behavior as well was achieved. 164
111 as the intrinsic and accelerated physical stability (pH, apparent vis-
112 cosity (g), and conductivity values) in different temperatures. In 2.4. Construction of ternary and pseudo phase’s ternary diagrams 165
113 addition, in vitro tests for cytotoxic activity toward L929 cells
114 and in vivo studies on experimental wound healing in rats were The pseudo-ternary phase diagrams of different concentrations 166
115 performed. Thus, the present study aimed to compare LGP emul- (v/v) oil, blend of surfactants (v/v) and water (v/v) were construct- 167
116 sions to simple emulsions (both containing C. officinalis oil) in the ed using the water titration method to obtain respective concen- 168
117 wound healing profile. tration ranges that could result in the area of lamellar gel phase 169
emulsions. The proportion of the surfactant mixture (Cethet-2/ 170
Steareth-20-0.97/0.03), which has been described in previous stud- 171
118 2. Material and methods ies (Santos et al., 2006) was the same for all formulations (Ceteth- 172
2:Steareth-20/0.93:0.07). 173
119 2.1. Material
2.5. Rheological measurements 174
120 C. officinalis (calendula oil or marigold oil) from flowers were
121 supplied by Beraca (Beraca Ingredients, Sao Paulo, Brazil). The Rheological properties of the LGP emulsions (n = 3) were exam- 175
122 information and specifications about the marigold oil can be found ined using a Haake rheometer (Model Rheostress RS-1, Germany). 176
123 at the European Pharmacopeia (CAS No.: 84776-23-8, 70892-20-5; The rheometer was based on a thermostatically controlled cone/- 177
124 EINECS No.: 283-949-5). The lipophilic (cetyl alcohol 2 poly- plate (C35/2° Ti) sensor with a 60 mm diameter and a 1° angle. 178
125 oxyethylen, named Ceteth-2, HLB = 5.3) and hydrophilic surfac- Rheowin 3.5 software was used to analyze the data. The assays 179
126 tants (stearyl alcohol 2 polyoxyethylene, named Steareth-20 performed were: (1) stress yield, using a shear stress of 0– 180
127 HLB = 15.4) were kindly provided by Oxiteno (Sao Paulo, Brazil). 100 Pa, for 180 s; (2) low limit curve (until emulsions star flowing); 181
(3) stress sweep analysis; (4) frequency sweep analysis; (5) vis- 182
cosity and thixotropic test. In the continuous shear analysis, 183
128 2.2. Preparation of lamellar gel phase emulsions/formulations (LGP) upward and downward flow curves for each formulation were 184
measured over shear rates ranging from 0.001 to 8900 s1. The 185
129 LGP emulsions were produced by the emulsion inversion phase shear rate was increased over an increment of 120 s, held at the 186
130 method (EPI), as previously described (Blonchard, 1970). Briefly, upper limit for 10 s, and then decreased over a period of 120 s. 187
131 aqueous (80%) and oily (10%) phases with 10% of a blend of surfac- The thixotropic value of the formulations can be calculated based 188
132 tants of Ceteth-2/Steareth-20 (0.97/0.07 v/v) were heated on the area of stress yield assay. The shear strain, the stress, and 189
133 separately until they reached 75.0 ± 5.0 °C. The aqueous phase the phase angle were determined from oscillating measurements. 190
134 was gently poured over the oily phase under continuous agitation The parameters obtained were the complex modulus, G⁄, and the 191

Please cite this article in press as: Okuma, C.H., et al. Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a poten-
tial modern wound dressing. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.01.016
 PHASCI 3182 No. of Pages 11, Model 5G
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C.H. Okuma et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx 3

192 phase angle d. The elastic modulus (G0 ), the viscous modulus (G00 ) After surgery, an intraperitoneal dose of 50 mg/kg body weight 253
193 and the dynamic viscosity (g) were calculated using the following of Dipyrone diluted in saline was administered. The rats were then 254
194 equations: divided into four distinct treatment groups (n = 20/group): (1) the 255
195 LGP group: treatment with lamellar gel phase formulation contain- 256
00
197 G ¼ G0 þ iG ð1Þ ing calendula oil; (2) the CAL group: treatment with a simple for- 257
198 mulation with calendula oil (no lamellar gel phase contained/ 258
200 G0 ¼ G cosðdÞ ð2Þ Appendix A); (3) the GEL group: treatment with only lamellar gel 259
201 phase emulsion without calendula oil; (4) the CONTROL group: 260
203 G00 ¼ G sinðdÞ ð3Þ no treatment (no lamellar gel phase emulsion, no calendula oil). 261
Wounds were treated daily with the four distinct groups as 262
204
206 00
g ¼ G =x ð4Þ described above. An occlusive gauze and adhesive tape dressing 263
(to avoid possible infections regarding to the environment) was 264
207 The angular frequency (x) ranged from 0.01 to 100 Hz. In each used in wounds of all group rats. The dressings were changed every 265
208 case, the dynamic rheological properties were determined with at day. 266
209 least three replicates of three independents formulations. Viscosity
210 and thixotropic measurements were performed for shear rates
211 between 0.001 and 8900 s1. The results are represented as mean
212 values (mean ± S.D., n = 3). 2.9. Evaluation of the potential wound healing effect of LGP emulsion 267
in the model of cutaneous wounds of the back of rats (excisional 268
213 2.6. Lamellar structure evaluation after water loss contractile wound) 269

214 The sample was then spread to obtain a uniform layer The wounds were assessed 2, 7, 14 and 21 days post-surgery. 270
215 approximately 0.2 mm thick. Water evaporation was measured Following assessment, 5 rats per group, per day of follow-up, were 271
216 by a scale equipped with infrared light heating the sample at euthanized by inhalation of CO2. The wound-healing rate (WHR) of 272
217 70 °C. Every 10% of weight loss a photomicrograph was taken different groups was evaluated by clinical and photographic 273
218 under polarized light and a new slide was prepared for further test- assessment of the wounds, and the evolution of the wounded areas 274
219 ing. Images of the samples were obtained at distinct stages of was performed using ImageJ software. To determinate the wound 275
220 dehydration using an Olympus microscope (Model BX 50) healing rate (WHR), the formula [(Ai  Af)/Ai] was used. The values 276
221 equipped with a polarizer. greater than zero represent a decrease in the wounded area, 277
whereas values less than zero represent an increase in the wound- 278
ed area and WHR = 1.0 represents full re-epithelialization (Caetano 279
222 2.7. In vitro assay
et al., 2009). The initial area (Ai) corresponds to the area of the 280
wound the day of surgery and the final area (Af) corresponds to 281
223 2.7.1. Cell viability assay of C. officinalis oil
the final evaluation day. Ultimately, all wound biopsies were col- 282
224 Cytotoxicity was measured using an apoptosis and a necrosis
lected for subsequent histological analysis. 283
225 assay. L929 cells (1  105 cells/mL) were treated with calendula
226 oil (50–1000 lg/mL) for 24 h. Cells were centrifuged and incubated
227 in 100 lL of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM
228 CaCl2) containing conjugated Annexin V FITC (FITC-Annexin Apop-
2.10. Histological analysis 284
229 tosis Detection Kit I V, PharmigenTM BD) and Propidium Iodide
230 (1 lg/mL) for 15 min (dark) at room temperature. Flow cytometry
The tissues excised from wound sites were fixed in 10% (v/v) 285
231 was performed with an acquisition of 10,000 cells per sample. Each
formalin solution, dehydrated through a graded series of alcohol 286
232 experiment was performed in triplicate. Data was analyzed by BD
(50–100% (v/v)), cleared in xylene, and embedded in paraffin. Serial 287
233 FACSDIVA software (BD Biosciences).
sections of 3.0 lm thickness were made using a microtome, and 288
stained with hematoxylin and eosin (evaluation level of inflamma- 289
234 2.8. In vivo wound healing model tory infiltrate). The slides were then stained with Gomory’s tri- 290
chrome (evaluation of collagenesis), and examined with an 291
235 2.8.1. Animals optical microscope using the Leica Application Suite Version 3.2.0 292
236 Male Wister rats (average weight of 190–210 g) were used in software. For each wounded skin sample of each animal, 10 sec- 293
237 this study. All experiments were performed with approval of the tions were taken and examined (400 magnifications). 294
238 University of Sao Paulo Animal Ethics Committees (CEUA/Protocol The quantification of collagenesis was made using the ‘‘Colour 295
239 No. 09.1.544.53.1/05/12/2011). Deconvolution’’ plug-in of the ImageJ software, where the three 296
colors of trichrome were deconvoluted. Only the percentage of 297
240 2.8.2. Wounds and treatments the total area of blue color (collagen) of the image was determined. 298
241 On the day of surgery (day 0), 80 rats were pre-anaesthetized The results were reported as the average distribution of collagen 299
242 (intramuscularly) and anaesthetized with 10% Xylazine (DopaserÒ) per treatment (Andrade et al., 2011 and Carvalho et al., 2006). 300
243 at a dose of 10 mg/kg body weight and Ketamine (20% DopalenÒ) at
244 a dose of 10 mg/kg body weight, respectively. After shaving their
245 backs, two surgical excisions were made in different areas with a
246 histological punch of 1.5 cm in diameter (Stiefel Laboratories, Sligo, 2.11. Statistical analysis 301
247 Ireland), reaching the dermo-epidermal junction. The surgical exci-
248 sions performed by the histological punch is a full-thickness Data were expressed as the mean value ± SEM. All studies were 302
249 wound used to investigate contractile wound repair and it is the analyzed using the One-way ANOVA analysis with a = 5% and the 303
250 most common wound type used by other investigators. The wound Bonferroni. To identify the statistically significant differences 304
251 can be harvested at any time during the healing process (Reid et al., between all groups the GraphPad Prism 5.0 software (San Diego, 305
252 2004). CA) was used. 306

Please cite this article in press as: Okuma, C.H., et al. Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a poten-
tial modern wound dressing. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.01.016
 PHASCI 3182 No. of Pages 11, Model 5G
16 February 2015

4 C.H. Okuma et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx

307 3. Results and discussion After determining the region of interest (LGP), we developed six 340
additional formulations (A–F) varying the components with 5% 341
308 3.1. Pre-formulation of LGP emulsions and development of ternary and increments (w/w/w) from the formulation number 36 (Table 1 342
309 pseudo phase’s ternary diagram and Fig. 1). 343

310 A ternary phase diagram was constructed to represent the for-

311 mulations with the percentage of C. officinalis oil, distilled water,
3.2. Emulsions characterization 344
312 and surfactants (Ceteth-2 and Steareth-20). The weight ratio of
313 surfactants (Ceteth-2/Steareth-20 = 0.93/0.07) was chosen to
3.2.1. Macroscopic analysis 345
314 achieve an HLB value of 6.0. These surfactants were chosen due
The emulsion number 36 and its derivations were subjected to 346
315 to their low toxicity with the skin and low incompatibility regard-
additional tests, such as centrifugation, thermal stress analysis, and 347
316 ing other excipients (Santos and da Rocha-Filho, 2007). In addition,
presence or absence of anisotropic structures (AS) to further scru- 348
317 it has been suggested that the anisotropic structures presented by
tinizes their stability by macroscopic evaluation. The formulations 349
318 the lamellar gel phase emulsions are due to the non-ionic surfac-
with emulsifier concentrations of more than 10% (A and B formu- 350
319 tants Ceteth-2/Steareth-20 (Santos et al., 2005).
lations) were stable, but ruled out for further studies due to their 351
320 The first pseudo-diagram was varied to 10% (w/w/w) of each
propensity to irritate skin. The remaining formulations (36, C–F) 352
321 component to obtain 36 formulations (Appendix B) depicted as
were subjected to thermal stress 24 h post-production (Table 2). 353
322 an equilateral triangle (Fig. 1). After being equilibrated, samples
Formulations D and E coalesced after the second cycle of centrifu- 354
323 were assessed visually according to the aspect of the formulation
gation, indicating low separation energy and were subsequently 355
324 at 24 h post-production: (1) separation phase (PS); (2) wax aspect
rejected. This phase separation may have been due to the low con- 356
325 (WA); and (3) lamellar gel phase (LGP/presence or absence of
centration (5% w/w/w) of surfactants. Sample number 36, C, and F 357
326 anisotropic structures detected by optical microscope magnifica-
remained stable up to 50 °C with no apparent changes. However, 358
327 tion of 200). The phase separation was associated with high oil
these emulsions became unstable at temperatures above 50 °C, 359
328 content and low surfactant and water concentrations. The formula-
displaying creaming and complete phase separation, indicating 360
329 tions of the wax phase demonstrated instability as a consequence
that the systems stability is temperature dependent. These emul- 361
330 of high surfactant concentration and low distilled water concentra-
sions were considered to be stable emulsions, because they 362
331 tion. Nevertheless, formulations of the LGP area were selected for
reached higher temperatures in the thermal test than others. 363
332 further optimization due to its enhanced physical stability,
Moreover, the pH values of these emulsions were evaluated and 364
333 anisotropic structures, opaque characteristics, low apparent vis-
the mean ± SEM was 5.4 ± 0.5 after 24 h post-production and com- 365
334 cosity, white color, and neutral odor and properties required for
patible with the human physiology. 366
335 medicinal and cosmetic applicability (Figure not shown). Next,
The instability of the emulsions demonstrated by the centrifu- 367
336 we determined that the best formulation was the number 36 con-
gation and thermal stress tests, might be a consequence of the 368
337 sisting of 10% marigold oil; 10% of the blend of surfactants and 80%
destabilization of the energy barrier, which indicates the maxi- 369
338 of purified water (w/w/w). This formulation was selected to further
mum energy of repulsion among the dispersed droplets in the 370
339 continue our studies.
emulsion (Friberg et al., 1988). When energy is supplied to the sys- 371

Fig. 1. Ternary diagram and pseudo ternary phase diagram were prepared from marigold oil/mixture of surfactants (Ceteth-2/Steareth-20)/water (w/w/w). PS = phase
separation, WA = wax aspect and LGP = lamellar gel phase.

Please cite this article in press as: Okuma, C.H., et al. Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a poten-
tial modern wound dressing. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.01.016
 PHASCI 3182 No. of Pages 11, Model 5G
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C.H. Okuma et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx 5

Table 1
Pseudo phase ternary diagram consisting of the composition of complementary formulations.

(HLB = 6.0) Weight ratio of Ceteth-2/Steareth-20 = 0.93/0.07

Samples Oil Mixture of surfactants Water Oil Ceteth-2 Steareth-20 Water
30.0 g (%) (%) (%) (g) 0.93 0.07 (g)
A 10 15 75 3.0 4.185 0.315 22.5
B 5 15 80 1.5 4.185 0.315 24.0
C 5 10 85 1.5 2.790 0.210 25.5
D 10 5 85 3.0 1.395 0.105 25.5
E 15 5 80 4.5 1.395 0.105 24.0
F 15 10 75 4.5 2.790 0.210 22.5

Table 2
Results of intrinsic tests for LGP emulsions.

Emulsions Centrifugation test (rpm) Stress test (°C) pH value A.S.

2500 3000 50 55
36 N N SM MM 5.72 ± 0.02 +++
C N N SN MM 5.70 ± 0.02 ++
D SM MM AM AM 5.82 ± 0.02 ++
E MM SM MM AM 5.84 ± 0.02 ++
F N N N SM 5.74 ± 0.02 +++

A.S. = Anisotropic Structures/(+) small amount of A.S.; (++) moderate amount of A.S.; (+++) higher amount of A.S. N = no changes; SM = slightly modified; MM = moderate
modified and AM = acutely modified.

372 tem, the ‘‘Brownian motion’’ potentially leads to creaming, floccu- The results from these assays combined with the yield stress 411
373 lation, and coalescence (Kong et al., 2001). test provides ideal conditions for the stress sweep test and fre- 412
374 Our investigation identified that the sample number 36 and for- quence sweep test. The stress sweep test defines the interval of 413
375 mulation F demonstrated better stability and greater anisotropic tension required to induce linearity in the emulsion. The analysis 414
376 structures than the previous formulations developed Santos et al. demonstrated that the emulsions were linear from 0.1 to 10 Pa 415
377 (2006), for this reason, these formulations were selected to further (Fig. 2B). The date obtained from the stress sweep test suggested 416
378 continue our studies. that a tension of 1 Pa in both formulations can be used for the fre- 417
quency sweep test and creep and recovery test. Both formulations 418
379 3.2.2. Rheological measurements showed a linearity range in all compounds (G0 , G00 and g) in a 419
380 The rheological analysis evaluated both the physical–chemical frequency range of approximately 1–10 Hz demonstrated by the 420
381 nature of the emulsions and potential instabilities. In addition, frequency sweep analysis (Fig. 2D). However, emulsion 36 present- 421
382 the rheological behavior is influenced by some basic parameters ed a slightly higher deformation than emulsion F due to its lower 422
383 such as the continuous phase rheology and the physical nature of viscosity. Moreover, the general frequency confirmed the presence 423
384 the dispersed phase (size, concentration, and deformability) of a gel-like structure at this condition with a G0 –G00 versus fre- 424
385 (Barnes, 1994). Rheological analyses performed on samples 36 quency curve characterized by a remarkable predominance of elas- 425
386 and F demonstrated non-Newtonian behavior with flow curves in ticity over the viscous behavior (G0 > G00 ) and the G0 modulus being 426
387 the form of anti-clockwise hysteresis loops (Fig. 2A). Both formula- parallel to the frequency axis. The compound (n), G0 and G00 values 427
388 tions (36 and F) displayed shear-thinning behavior, due to the of sample 36 were smaller than F, which indicates that formulation 428
389 apparent decrease in viscosity with increasing shear rate. Also, 36 demonstrated more deformation than formulation F. This is due 429
390 the formulations displayed hysteresis, which is characteristic of to the fact that the more deformation that is presented the less the 430
391 thixotropic emulsions. Emulsion 36 showed a higher hysteresis values of these compounds n, G0 and G00 showed, for the same shear 431
392 value (118.8 Pa/s) than formulation F (19.38 Pa/s). Emulsion F stress at a specific time (Dolz et al., 2008). Samples 36 and F both 432
393 was predicted to show a lower hysteresis value due to its lower demonstrated viscoelastic properties. However, sample F (Fig. 3) 433
394 water ratio. The rheologies of emulsions 36 and F were measured was selected for further investigation due to its adequate viscosity 434
395 by the dynamic flow of water between the interlamellar space and because of the probability that it might increase the residence 435
396 and the dispersed phase. The capacity of keeping water between time of the formulation on the wound surface (desirable for wound 436
397 the lamellas and also the rheological properties of these emulsions healing purposes). 437
398 (36 and F) are determined by both the lipophilic carbon chain and
399 the amount of surfactant ethoxylation (Gregolin et al., 2010 and 3.2.2.1. Apparent viscosity (g)/thixotropy. Apparent viscosity analy- 438
400 Ribeiro et al., 2004). It is possible to predict the residence time of sis as a function of shear rate is predictive of physical instability 439
401 the emulsion on the skin surface by measuring the viscosity of and can be correlated with molecular behavior (Masmoudi et al., 440
402 the formulation (Boateng et al., 2008). In wound healing, one of 2005). The apparent viscosity of formulation F was determined as 441
403 the requirements for treatments is that the compounds remain a function of shear rate (100.02/s) from the apex of the loop 442
404 on the wound surface for extended periods of time (Matthews (500 s1) during the flow curve determination. Thixotropy 443
405 et al., 2008). Formulation F, with its higher viscosity, was predicted and apparent viscosity (g) values were analyzed over a period 444
406 to remain on the target area for longer periods of time than formu- of 9 months exposure to specific conditions; 25.0 ± 2.0 °C, 445
407 lation 36. As depicted in the Flow limit curve (Fig. 2B), the formu- 5.0 ± 2.0 °C and 40.0 ± 2.0 °C with a relative humidity of 75% (Figs. 4 446
408 lations 36 and F were further subjected to shear stress range of and Supplementary 1A). Graphical analysis (2000 Rheo software 447
409 0.1–10 Pa. Samples 36 deformed in a range of 0.04499 Pa in V2. 8) demonstrated that samples stored at 5.0 ± 2.0 °C and con- 448
410 4.05 s while formulation F changed in 0.04614 Pa in 3.95 s. trolled room temperature (25 ± 2.0 °C) displayed characteristics 449

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tial modern wound dressing. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.01.016
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Fig. 2. Rheograms of formulations 36 and F: (A) Flow curve (r [Pa]  c [1/s]); (B) Flow limit curve (c [1/s]  r [Pa]); (C) Creep and recovery test (c [1/s]  T [s]); (D) Stress
Sweep analysis (G0 [Pa] G00 [Pa]  r [Pa]) and (E) Frequency sweep analysis (G0 [Pa]G00 [Pa]  F [Hz]).

Fig. 3. Photomicrograph of anisotropic structures of emulsion F developed from a mixture of surfactants: Ceteth 2/Steareth 20 (HLB = 6.0)/Calendula officinalis oil/H2O stored
at 25 ± 2 °C. (A) Normal light, (B) polarized light, after 24 h of preparation.

450 consistent with the presence of thixotropy in all times of follow-up until the 15th day following by increasing the apparent viscosity 462
451 (90 days). However, samples stored at 40.0 ± 2.0 °C reduced the until the 90th day (Fig. S1A). It may be due to the ethoxylated 463
452 area of hysteresis and proved to reduce the thixotropy of the for- non-ionic surfactants present in the sample, which can cause 464
453 mulations, causing reduced stability and spreadability (Fig. 4). It structural changes during storage (Eccleston, 1990). Moreover, 465
454 is inferred that an increase in temperature may have altered the the decrease in repulsive forces and an increase in the attractive 466
455 surfactant pairs, leading to conformational modifications such as forces caused by higher temperature may store water in the inter- 467
456 macroscopic changes and the reduced spreadability (Lindman face of the anisotropic structure a period greater than that expect- 468
457 and Karlstrom, 2009). Samples stored at 25 ± 2 °C displayed a con- ed contributing to the increase of viscosity of the product (Lindman 469
458 stant apparent viscosity until the end of the analyses (90th day) and Karlstrom, 2009). The apparent viscosity and stability of the 470
459 while the formulations stored at 5.0 ± 2.0 °C increased until the LGP formulations are relatively larger than the simple emulsions 471
460 end of the analyses (90th day) (see Supplementary Fig. 1a). The for- due to the reorganization of the lamellar microstructures around 472
461 mulation stored at 40.0 ± 2.0 °C showed a small initial instability the droplets and the penetration of free water in the interlayer 473

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Fig. 4. Rheogram of shear stress values showing variation of emulsion F as a function of shear rate during accelerated stability test for 0 (A), 7 (B), 14 (C), 30 (D), 60 (E) and 90
(F) days. Ceteth 2/Steareth 20 (HLB = 6.0)/Calendula officinalis oil/H2O (w/w/w). (⁄) p < 0.05.

474 space of structures in the gel phase, with a consequent increase in the skin, the mechanism of action of the final product, and the 493
475 viscosity of the system (Eccleston et al., 2000; Muller-Goymann, maintenance of skin hydration (Santos et al., 2006; Friberg, 2007). 494
476 2004). We found that even after 70% of water loss and under extreme stor- 495
477 Furthermore, we also had the pH and electrical conductivity age conditions, anisotropic structures could still be observed 496
478 values of the formulation F determined. The formulation F had (Fig. 5G). This indicated that the lamellar phase formed after the 497
479 stable pH values at 25.0 ± 2.0 °C and 5.0 ± 2.0 °C and displayed production of the emulsions resisted the decrease of water in the 498
480 decreasing pH values over time at 40.0 ± 2.0 °C (see Supplementary system. Lamellar phases are organized according to their orienta- 499
481 Fig. S1B). However, the pH values remained at around 4.5–6.0, tion of the surfactant molecules that allows part of an external 500
482 which is an acceptable value for a non-skin irritating compound. water phase to become adsorbed, further changing its state and 501
483 In regard to the electrical conductivity test, the samples stored at preventing the water from evaporating (due to the intense interac- 502
484 40.0 ± 2.0 °C showed a decreased electrical conductivity only in tion between the water molecules and the polar groups of the non- 503
485 the initial 14 days and thereafter stabilized (Supplementary ionic surfactants by hydrogen bonds) (Santos and da Rocha-Filho, 504
486 Fig. S1C). The other samples stored at 5.0 ± 2.0 °C and 2007; Gao et al., 2003). In this case, water molecules need higher 505
487 25.0 ± 2.0 °C demonstrated stable electrical conductivity values quantities of energy to evaporate, which keeps forming the 506
488 and the absence of electrolytes. anisotropic structures during its hydrated state, assuming lamellar 507
organization. 508

489 3.3. Microscopic study of the behavior of LGP formulation upon

490 evaporation of water 3.4. Effect of C. officinalis oil on apoptosis and necrosis 509

491 Evaporation of water can alter a formulation’s intrinsic proper- Cytotoxic effects were analyzed using FITC-annexin V and PI 510
492 ties, such as the behavior of the formulation after application to double staining in L929 cells in flow citometry. We demonstrated 511

Fig. 5. Microphotographs of formulation F with the anisotropic structures after evaporation. (A) 10% (w/w) of water loss. (B) 20% (w/w) of water loss. (C) 30% (w/w) of water
loss. (D) 40% (w/w) of water loss. (E) 50% (w/w) of water loss. (F) 60% (w/w) of water loss. (G) 70% (w/w) of water loss. (H) 80% (w/w) of water loss. Magnification: 200.

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day for all groups. On the 7th day, all groups showed an improve- 530
ment in wound re-epithelialization relative to the 2nd day, but no 531
difference between the groups was observed (p > 0.05). Finally, all 532
groups enhanced the reepithelialization after the 14th day, but 533
unlike the GEL and CONTROL groups, the LGP group was complete- 534
ly re-epithelialized on 14th day. Both the GEL and the CONTROL 535
groups showed wounds that were not totally re-epithelialized on 536
the 14th and 21st day as depicted in Fig. 7. The wound increasing 537
in size characterizes the inflammatory phase of wound healing. 538
This is mostly attributed to the high activity of proteases and sub- 539
sequent tissue degradation that results as a consequence of com- 540
pounds being released from inflammatory cells (like the reactive 541
Fig. 6. The flow cytometric analysis of apoptosis in L929 cells using FITC-annexin V oxygen and nitrogen species from oxidative stress) during phago- 542
and PI double staining. Quadrant analysis of the gated cells in FL-1 versus FL-2
channels was from 10,000 events. Annexin V+/PI (lower right quadrant) areas are
cytosis of the pathogens and senescent cells (Guo and Dipietro, 543
depicted by early apoptotic cells, and Annexin V+/PI+ (upper right quadrant) areas 2010 and Schreml et al., 2010). 544
are depicted by late apoptotic or necrotic cells. The scientific literature shows numerous studies describing the 545
wound re-epithelialization effect by calendula oil (Faria et al., 546
2011; Preethi et al., 2010; Szakiel et al., 2008; Schmidt et al., 547

512 that neither early apoptosis (Annexin V+/PI) nor late apoptosis 2009; Chandran and Kuttan, 2008; Pommier et al., 2004). The 548

513 (Annexin V+/PI+) was modified by formulation F containing C. advancement made by our study was the particular association 549

514 officinalis oil (50–1000 lg/mL) as compared to the control or the between the calendula oil and the lamellar gel phase emulsion (- 550

515 vehicle (Fig. 6). The marigold extract of C. officinalis was shown formulation F). This association maintained the wound healing 551

516 not to be cytotoxic for L929 and HepG2 cells at concentrations less process profile of the CAL group after the 7th day. To understand 552

517 than or equal to of 15 mg/mL. Thus, based on these results, C. how the associated products might act to enhance the wound re- 553

518 officinalis oil should be classified as a non-cytotoxic oil, and could epithelialization process, we performed a wound histological ana- 554

519 be used as a stimulator of the wound healing process. lysis (Fig. 8). The wounds of the LGP group demonstrated higher 555
inflammatory infiltrate than the CAL group on the 2nd and 7th 556
day and both of these groups presented more inflammatory infil- 557
520 3.5. In vivo wound healing studies
trate than the GEL and the CONTROL groups. After the 14th day, 558
all groups showed decreases of in levels of inflammatory infiltrate. 559
521 Following the in vitro assay, the effect of the LGP emulsion (for-
By microscopic histological analysis on the 7th day, the LGP group 560
522 mulation F) on the experimental wound healing model (in vivo)
showed new blood vessels that sustained growth up to the 21st 561
523 was evaluated. Large, full-thickness wounds performed on the
day, which was different from the other groups (Fig. 8). 562
524 backs of healthy rats were treated and monitored for 21 days.
Fibroblasts are the type of cell that produce collagen, the major 563
525 Re-epithelialization, a highly important event in restoration of
structural component of granulation tissue that compounds the 564
526 skin barrier function, was evaluated by measuring the wound heal-
extracellular matrix. Proceeding the 14th day of the follow-up, 565
527 ing rate (Fig. 7). The LGP group showed less re-epithelialization
when the inflammatory infiltrate was reduced (shown in Fig. 7), 566
528 than the GEL and the CONTROL groups on the 2nd day (p < 0.05).
intense fibroblastic proliferation was observed in all groups. The 567
529 Furthermore, several wounds had increased in size on the 2nd

Fig. 7. (A) The evolution of wound healing rates (WHR) at 2, 7, 14 and 21 days, according to the treatment: LGP group: lamellar gel phase emulsion (formulation F) with
calendula oil; CAL group: treatment with a simple formulation with calendula oil (no lamellar gel phase contained); GEL group: only lamellar gel phase emulsion; CONTROL
group: no lamellar gel phase emulsion and no calendula oil. (B) Clinical follow-up wounds were assessed at 0, 2, 7, 14 and 21 days, respectively, according to the treatment.

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Fig. 8. Histological analyses from paraffin-wound sample stained by hematoxylin and eosin staining (400 magnification). The analyses were demonstrated on day 0, 2, 7, 14
and 21 for rats treated daily with LGP, CAL and GEL at dose of 15 mg/mL through occlusive dressing with gauze and adhesive tape. The same methods were performed on the
CONTROL group, but no product was applied.

568 LGP group displayed significantly more collagen than the CONTROL tion of collagen to new tissue formation, as observed in the 585
569 groups on the 2nd day (p < 0.005) in the beginning of the healing collagenesis evaluation, when compared to the CAL group. If the 586
570 process (Fig. 9). Moreover, in the CAL group more collagen was inflammatory stimulus is indeed persistent, high concentrations 587
571 observed compared to the other groups, especially on the 2nd of proteolytic enzymes are produced, which stimulates oxidative 588
572 day (p < 0.05). On the 7th day, the CAL group still presented higher stress, impairing the healing process (Schreml et al., 2010; 589
573 collagenesis than the GEL and the CONTROL groups (p < 0.05), Schäfer and Werner, 2008). However, from the 14th day onward, 590
574 albeit similar to LGP (p > 0.05). On the 14th day, the LGP and the there was an important reduction of inflammatory infiltrate. 591
575 CAL groups presented higher collagenesis than the GEL group Furthermore, the wounds treated with LGP emulsion showed 592
576 (p < 0.05) and this stimulus continued until the 21st day considerable humidity on the 2nd and the 7th day, when compared 593
577 (p < 0.05), when both groups had higher collagenesis than the macroscopically to the wounds of the other groups (Figure not 594
578 CONTROL group (Fig. 9). shown). In general, it has been proposed that the hydration of 595
579 The intense inflammatory infiltrate observed at the beginning of the wound stimulates epithelialization, granulation, tissue forma- 596
580 the healing process (2nd day) of the LGP group might produce high tion, angiogenesis, fibroblast migration, collagen synthesis, and 597
581 levels of proteolytic enzymes (such as metalloproteinases and col- remodeling of injured tissue, thus reducing the possible trauma 598
582 lagenases), that further hinder re-epithelialization, breaking the during changes of the dressing (Kumar et al., 2008). This suggested 599
583 surrounding tissue that causes wounds to increase in size. Further- that the LGP emulsion (in addition to the higher inflammatory 600
584 more, the enzymes also might aid in the prevention of the produc- infiltrate observed) might have influenced the maintenance of 601

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Fig. 9. Histological analyses (collagenesis). The evolution of the percentage area of collagen located on samples stained by Gomori thrycome on days, 0, 2, 7 14 and 21,
according to the treatment: LGP, CAL, GEL and CONTROL.

602 moisture in the wound, maintaining the properties of calendula oil Appendix A. Supplementary material 635
603 (which is an important factor in autolytic debridement)
604 (Mandelbaum et al., 2003) and enhanced the total wound re- Supplementary data associated with this article can be found, in 636
605 epithelialization. the online version, at http://dx.doi.org/10.1016/j.ejps.2015.01.016. 637
606 Although the proposed wound healing animal model does not
607 take chronic wounds into consideration, the data presented herein
608 demonstrate an implication to treat chronic ulcers in clinical prac- References 638
609 tices. In general, there is a need for a significant inflammatory sti-
610 mulus to remove the ulcers of a senescent status, providing Andrade, T.A., Lyer, A., Das, P.K., Foss, N.T., Garcia, S.B., Coutinho-Netto, J., Jordão Jr., 639
A.A., Frade, M.A., 2011. The inflammatory stimulus of a natural latex 640
611 granulation tissue (angiogenesis, fibroplasia, and collagenesis) 641
biomembrane improves healing in mice. Braz. J. Med. Biol. Res. 44 (10),
612 and promoting re-epithelialization, as previously observed by 1036–1047. 642
613 Caetano et al. (2014). Barnes, H.A., 1994. Rheology of emulsions – a review. Colloid Surf. A. Physicochem. 643
Eng. Asp. 91, 89–95. 644
Blonchard, P., 1970. Measurement of the hydrophilic–lipophilic balance of an 645
emulsifying agente by a method based on phase inversion. The concept of 646
614 4. Conclusions ‘‘hydrophilic–lipophilic balance, inversion limit’’ and its applications. Ann. 647
Pharm. Fr. 28 (9), 553–560. 648
Boateng, J.S., Matthews, K.H., Stevens, H.N.E., Eccleston, G.M., 2008. Wound healing 649
615 The LGP emulsion (15.0% marigold oil; 10.0% of blend surfac-
dressings and drug delivery systems: a review. J. Pharm. Sci. 97 (8), 2892–2923. 650
616 tants and 75.0% of purified water [w/w/w]) has demonstrated Caetano, G.F., Frade, M.A.C., Andrade, T.A.M., Leite, M.N., Bueno, C.Z., Moraes, A.M., 651
617 greater stability and its low spread ability suggests longer contact Ribeiro-Paes, J.T. 2014. Chitosan-alginate membranes accelerate wound 652
healing. J. Biomed. Mater. Res. Part B (in press). 653
618 with wounds. The C. officinalis oil was not detrimental and also was
Caetano, K.S., Frade, M.A.C., Minatel, D.G., Santana, L.A., Enwemeka, C.S., 2009. 654
619 non-cytotoxic as assayed in L929 cells. The LGP emulsion was Phototherapy improves healing of chronic venous ulcers. Photomed. Laser Surg. 655
620 relatively stable and promoted better quality wound healing in a 27 (1), 111–118. 656
621 rat skin wound model than the other groups. LGP emulsion seemed Carvalho, P.T., Mazzer, N., dos Reis, F.A., Belchior, A.C., Silva, I.S., 2006. Analysis of 657
the influence of low-power HeNe laser on the healing of skin wounds in diabetic 658
622 to modulate the inflammatory phase of wound healing. Taken and non-diabetic rats. Acta Cir. Bras. 21 (3), 177–183. 659
623 together these data suggests that the novel lamellar gel phase Chandran, P.K., Kuttan, R., 2008. Effect of Calendula officinalis flower extract on acute 660
624 emulsion (formulation F) we have developed with C. officinalis oil phase proteins, antioxidant defense mechanism and granuloma formation 661
during thermal Burns. J. Clin. Biochem. Nutr. 43 (2), 58–64. 662
625 shows high potential in improved wound-healing applications. 663
Dolz, M., Hernandez, M.J., Delegido, J., 2008. Creep and recovery experimental
investigation of low oil content food emulsions. Food Hydrocolloids 22 (3), 421– 664
427. 665
626 Conflict of interest Eccleston, G.M., Behan-Martin, M.K., Jones, G.R., Towns-Andrews, E., 2000. 666
Synchrotron X-ray investigations into the lamellar gel phase formed in 667
pharmaceutical creams prepared with cetrimide and fatty alcohols. Int. J. 668
627 The authors declare that they have no competing interests Pharm. 203 (1–2), 127–139. 669
Eccleston, G.M., 1990. Multiple-phase oil-in-water emulsions. J. Soc. Cosmet. Chem. 670
41 (1), 1–22. 671
Faria, R.L., Cardoso, L.M., Akisue, G., Pereira, C.A., Junqueira, J.C., Jorge, A.O., Santos 672
628 5. Uncited references 673
Júnior, P.V., 2011. Antimicrobial activity of Calendula officinalis, Camellia sinensis
and chlorhexidine against the adherence of microorganisms to sutures after 674
629 Preethi et al. (2009) and Santos (2006). extraction of unerupted third molars. J. Appl. Oral Sci. 19 (5), 476–482. 675
Friberg, S.E., 2007. Evaporation from an ionic liquid emulsion. J. Colloid Interface Sci. 676
307 (2), 494–499. 677
Friberg, S.E., Goldsmith, L., Suhaimi, H., Rhein, L.D., 1988. Surfactants and the 678
630 Acknowledgments 679
stratum–corneum lipids. Colloid Surf. 30, 1–12.
Gao, T., Tien, J.M., Choi, Y.H., 2003. Sunscreen formulas with multilayer lamella 680
631 This work was supported by CAPES (Coordenação de Aper- structure. Cosmet Toilet. 118 (10), 41–52. 681
Gregolin, M.T., Chiari, B.G., Ribeiro, H.M., Isaac, V.L.B., 2010. Rheological 682
632 feiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacio-
characterization of hydrophylic gels. J. Dispers. Sci. Technol. 31, 820–825. 683
633 nal de Desenvolvimento Científico e Tecnológico) and FAPESP Grimme, H., Augustin, M., 1999. Phytotherapy in chronic dermatoses and wounds: 684
634 (Fundação de Amparo à Pesquisa do Estado de São Paulo). what is the evidence? Forsch. Komplementarmed. 6, 5–8. 685

Please cite this article in press as: Okuma, C.H., et al. Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a poten-
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 PHASCI 3182 No. of Pages 11, Model 5G
16 February 2015

C.H. Okuma et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx 11

686 Guo, S., Dipietro, L.A., 2010. Factor affecting wound healing. J. Dent. Res. 89 (3), 219– Preethi, K.C., Kuttan, R., 2009. Wound healing activity of flower extract of Calendula 724
687 229. officinalis. J. Basic Clin. Physiol. Pharmacol. 20 (1), 73–79. 725
688 Kong, L., Beattie, J.K., Hunter, R.J., 2001. Effects of nonionic surfactant and sodium Preethi, K.C., Kuttan, G., Kuttan, R., 2009. Anti-inflammatory activity of flower 726
689 dodecyl sulfate layers on electroacoustics of hexadecane/water emulsions. extract of Calendula officinalis Linn. and its possible mechanism of action. Indian 727
690 Colloid Polym. Sci. 279, 678–687. J. Exp. Biol. 47 (2), 113–120. 728
691 Kudla, P., Sokolowski, T., Blümich, B., Wittern, K.P., 2010. Phase behavior of liquid- Reid, R.R., Said, H.K., Mogford, J.E., Mustoe, T.A., 2004. The future of wound healing: 729
692 crystalline emulsion systems. J. Colloid Interface Sci. 349 (2), 554–559. pursuing surgical models in transgenic and knockout mice. J. Am. Coll. Surg. 199 730
693 Kumar, M.S., Kirubanandan, S., Sripriya, R., Sehgal, P.K., 2008. Triphala promotes (4), 578–585. 731
694 healing of infected full-thickness dermal wound. J. Surg. Res. 144 (1), 94–101. Ribeiro, H.M., Morais, J.A., Eccleston, G.M., 2004. Structure and rheology of semisolid 732
695 Lindman, B., Karlstrom, G., 2009. Nonionic polymers and surfactants: temperature o/w creams containing cetyl alcohol/non-ionic surfactant mixed emulsifier and 733
696 anomalies revisited. Compte Rendu Chim. 12 (1), 121–128. different polymers. Int. J. Cosmet. Sci. 26 (2), 47–59. 734
697 Mandelbaum, S.H., Di Santis, E.P., Mandelbaum, M.H.S., 2003. Cicatrização: Santos, O.D., da Rocha-Filho, P.A., 2007. Influence of surfactant on the thermal 735
698 conceitos atuais e recursos auxiliares – Parte II. An. Bras. Dermatol. 78 (4), behavior of marigold oil emulsions with liquid crystal phases. Drug Dev. Ind. 736
699 393–410. Pharm. 33 (5), 543–549. 737
700 Masmoudi, H., Le Dreu, Y., Piccerelle, P., Kister, J., 2005. The evaluation of cosmetic Santos, O.D.H., 2006. Desenvolvimento e Avaliação das Propriedades Físico – 738
701 and pharmaceutical emulsions aging process using classical techniques and a Químicas e Atividade Cosmética in vivo de Emulsões de Óleo de Calendula 739
702 new method: FTIR. Int. J. Pharm. 289 (1–2), 117–131. officinalis com Cristal Líquido. Doctoral Thesis. p. 130. 740
703 Matthews, K.H., Stevens, H.N.E., Auffret, A.D., Humphrey, M.J., Eccleston, G.M., 2008. Santos, O.D.H., Camargo, M.F.P., Andrade, F.F., Rocha-Rocha, P.A., 2006. Study of 741
704 Formulation, stability and thermal analysis of lyophilised wound healing wafers liquid-crystalline phase changes during evaporation in vegetable oil emulsions. 742
705 containing an insoluble MMP-3 inhibitor and a non-ionic surfactant. Int. J. J. Dispers. Sci. Technol. 27 (7), 997–1001. 743
706 Pharm. 356 (1–2), 110–120. Santos, O.D.H., Miotto, J.V., Morais, J.M., Rocha-Filho, P.A., 2005. Attainment of 744
707 Muller-Goymann, C.C., 2004. Physicochemical characterization of colloidal drug emulsions with liquid crystal from marigold oil using the required HLB method. 745
708 delivery systems such as reverse micelles, vesicles, liquid crystals and J. Dispers. Sci. Technol. 26 (2), 243–249. 746
709 nanoparticles for topical administration. Eur. J. Pharm. Biopharm. 58 (2), 343– Schäfer, M., Werner, S., 2008. Oxidative stress in normal and impaired wound 747
710 356. repair. Pharmacol. Res. 58 (2), 165–171. 748
711 Parente, L.M., Lino Júnior, R.deS., Tresvenzol, L.M., Vinaud, M.C., de Paula, J.R., Paulo, Schmidt, C., Fronza, M., Goettert, M., Geller, F., Luik, S., Flores, E.M.M., Bittencourt, 749
712 N.M., 2012. Wound healing and anti-inflammatory effect in animal models of C.F., Zanetti, G.D., Heinzmann, B.M., Laufer, S., Merfort, I., 2009. Biological 750
713 Calendula officinalis L. growing in Brazil. Evid. Complement. Altern. Med., studies on Brazilian plants used in wound healing. J. Ethnopharmacol. 122 (3), 751
714 375671 523–532. 752
715 Pommier, P., Gomez, F., Sunyach, M.P., D’Hombres, A., Carrie, C., Montbarbon, X., Schreml, S., Szeimies, R.M., Prantl, L., Landthaler, M., Babilas, P., 2010. Wound 753
716 2004. Phase III randomized trial of Calendula officinalis compared with healing in the 21st century. J. Am. Acad. Dermatol. 63 (5), 866–881. 754
717 trolamine for the prevention of acute dermatitis during irradiation for breast Szakiel, A., Ruszkowski, D., Grudniak, A., Kurek, A., Wolska, K.I., Doligalska, M., 755
718 cancer. J. Clin. Oncol. 22 (8), 1447–1453. Janiszowska, W., 2008. Antibacterial and antiparasitic activity of oleanolic acid 756
719 Popovich, K., Tohm, P., Hurd, T., 2010. Skin and wound care excellence: integrating and its glycosides isolated from marigold (Calendula officinalis). Planta Med. 74 757
720 best-practice evidence. Healthc. Q. 13, 42–46. (14), 1709–1715. 758
721 Preethi, K.C., Siveen, K.S., Kuttan, R., Kuttan, G., 2010. Inhibition of metastasis of 759
722 B16F-10 melanoma cells in C57BL/6 mice by an extract of Calendula officinalis L.
723 flowers. Asian Pac. J. Cancer Prev. 11 (6), 1773–1779.

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