Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a potential modern wound dressing

European Journal of Pharmaceutical Sciences 71 (2015) 62–72

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Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a potential modern wound dressing 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, 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 a

Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Brazil Division of Dermatology, Department of Internal Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China d Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA e School of Pharmaceutical Sciences of Araraquara, Sao Paulo State University (UNESP), Brazil f Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Brazil g Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA h Federal University of Triangulo Mineiro, Uberaba, Brazil b c

a r t i c l e

i n f o

Article history: Received 25 November 2013 Received in revised form 28 December 2014 Accepted 28 January 2015 Available online 12 February 2015 Keywords: Calendula officinalis oil Lamellar gel phase emulsion Liquid crystal Stability tests Wound healing

a b s t r a c t Appropriate therapeutics for wound treatments can be achieved by studying the pathophysiology of tissue repair. Here we develop formulations of lamellar gel phase (LGP) emulsions containing marigold (Calendula officinalis) oil, evaluating their stability and activity on experimental wound healing in rats. LGP emulsions were developed and evaluated based on a phase ternary diagram to select the best LGP emulsion, having a good amount of anisotropic structure and stability. The selected LGP formulation was analyzed according to the intrinsic and accelerated physical stability at different temperatures. In addition, in vitro and in vivo studies were carried out on wound healing rats as a model. The LGP emulsion (15.0% marigold oil; 10.0% of blend surfactants and 75.0% of purified water [w/w/w]) demonstrated good stability and high viscosity, suggesting longer contact of the formulation with the wound. No cytotoxic activity (50–1000 lg/mL) was observed in marigold oil. In the wound healing rat model, the LGP (15 mg/mL) showed an increase in the leukocyte recruitment to the wound at least on days 2 and 7, but reduced leukocyte recruitment after 14 and 21 days, as compared to the control. Additionally, collagen production was reduced in the LGP emulsion on days 2 and 7 and further accelerated the process of re-epithelialization of the wound itself. The methodology utilized in the present study has produced a potentially useful formulation for a stable LGP emulsion-containing marigold, which was able to improve the wound healing process. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Chronic wounds to the skin present a serious public health risk and are painful, unsightly, and can require limb amputation if left unattended. Adequate treatments are needed to increase the overall patient quality of life. Products designed for the treatment of chronic wounds and/or skin ulcers require optimizing properties such as the ease of application, increased patient comfort, and

⇑ Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Avenida do Café, s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil. Tel.: +55 16 3602 4279; fax: +55 16 3602 4881. E-mail addresses: [email protected], [email protected] (C.H. Okuma). http://dx.doi.org/10.1016/j.ejps.2015.01.016 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.

the ability to retain adequate moisture in the bed of the wound (Popovich et al., 2010). Calendula officinalis (Asteraceae) L., or marigold, is an herbal plant that has been used to treat wounds since the 13th century in Europe (Parente et al., 2012), and nowadays is used virtually worldwide. This plant possesses several biological properties such as antimicrobial (Faria et al., 2011), antimetastatic (Preethi et al., 2010), and antiparasitic activity (Szakiel et al., 2008). Its main activity as an anti-inflammatory constituent (Parente et al., 2012) which was observed in animal models (Preethi and Kuttan, 2009) and clinical tests, justifies its utilization as a cosmetic and personal care product for wound treatment (Grimme and Augustin, 1999). Secondary metabolites such as flavonoids, tannins, saponins, terpenoids, coumarins and others (Santos et al., 2006; Schmidt et al., 2009) alone or in association are directly associated with

C.H. Okuma et al. / European Journal of Pharmaceutical Sciences 71 (2015) 62–72

these effects. Also, marigold oil (C. officinalis) could be used to treat thermal burn injury, acute dermatits (Pommier et al., 2004) during irradiation for breast cancer and skin rashes (Parente et al., 2012). C. officinalis extract is present in almost 200 cosmetic formulations and significant experimental wound healing studies or treatments have been performed using only C. officinalis extract or cream containing the extract (Chandran and Kuttan, 2008). The choice of formulation type for the treatment or care of the skin is important because it may affect the mode of active compounds distribution in its surface. Lamellar gel phase (LGP) or liquid crystals formulations (emulsions) can be obtained due to the bi-layer arrangement (separated by water layers) of the surfactant molecules in the interfacial film. The LGP emulsions provide benefits such as enhanced stability and incorporation of active components in the matrix of (LGP) phase, water retention and the controlled release of active ingredients (Kudla et al., 2010). LGP could thus be used for wound healing purposes due to its adequate viscosity and because of the probability that it might increase the residence time of the formulation on the wound surface. In the present study, we optimized the preparation of LGP emulsions using the ternary and pseudo phase’s ternary diagram with different proportions of C. officinalis oil, distilled water, and a mixed emulsifier (cetyl alcohol 2 polyoxyethylene and stearyl alcohol 2 polyoxyethylene) whose HLB values were 6.0. This work is based on previous studies by Santos et al. (2006). The novel system was designed with the aim of achieving an efficient lamellar gel phase emulsion with wound healing activity. Santos et al. (2006) did not use the ternary and pseudo phase’s ternary diagram to select the best emulsion. In addition to this, the previous formulations using C. officinalis oil were not stable. Here, we have presented a detailed physicochemical characterization (preliminary tests) of samples to select the best LGP emulsion (i.e. good amount of anisotropic structure and stability). The selected LGP formulation was analyzed according to the rheological behavior as well as the intrinsic and accelerated physical stability (pH, apparent viscosity (g), and conductivity values) in different temperatures. In addition, in vitro tests for cytotoxic activity toward L929 cells and in vivo studies on experimental wound healing in rats were performed. Thus, the present study aimed to compare LGP emulsions to simple emulsions (both containing C. officinalis oil) in the wound healing profile.

2. Material and methods

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using a rotor/stator homogenizer (Fisaton-Mod 713 D) at 600 rpm until the system reached room temperature (25 ± 5 °C). 2.3. Emulsions characterization 2.3.1. Macroscopic evaluation Samples were visually observed to determine organoleptic characteristics and identify instability processes (phase separation, flocculation and creaming), at 24 h post-production. The formulations were visually evaluated and characterized as follows: (i) heavily modified (HM-presence of phase separation); (ii) moderately modified (MM-presence of flocculation process); (iii) slightly modified (SM-presence of creaming process); (iv) normal (N-no presence of instability processes/no change in appearance). 2.3.2. Microscopic evaluation The microstructure (presence or absence of anisotropic structures) of o/w emulsions was analyzed with an Olympus BX50 optical microscope (Olympus Optical Co., Ltd., Tokyo, Japan) in the bright field and under polarized light. Micrographs were made at a magnification of 200. 2.3.3. Centrifugation Each emulsion was weighted in graduated vials and centrifuged at the Fanem model 206 R, Excelsa Baby II-440 W at three speeds: 1500, 2500, and 3500 rpm (70, 440, and 863 G, respectively), standing for 15 min on each rotation. The procedure was conducted at room temperature (25 ± 2 °C). 2.3.4. Thermal stress Samples were submitted to water bath heating by Nova Technical Ltd., Brazil model 281 NT). The temperature was increased in increments of 5 °C beginning at 40 ± 2 °C and each temperature was maintained for 30 min up until a temperature of 80 ± 2 °C was achieved. 2.4. Construction of ternary and pseudo phase’s ternary diagrams The pseudo-ternary phase diagrams of different concentrations (v/v) oil, blend of surfactants (v/v) and water (v/v) were constructed using the water titration method to obtain respective concentration ranges that could result in the area of lamellar gel phase emulsions. The proportion of the surfactant mixture (Cethet-2/St eareth-20-0.97/0.03), which has been described in previous studies (Santos, 2006) was the same for all formulations (Ceteth-2:Steare th-20/0.93:0.07).

2.1. Material 2.5. Rheological measurements C. officinalis (calendula oil or marigold oil) from flowers were supplied by Beraca (Beraca Ingredients, Sao Paulo, Brazil). The information and specifications about the marigold oil can be found at the European Pharmacopeia (CAS No.: 84776-23-8, 70892-20-5; EINECS No.: 283-949-5). The lipophilic (cetyl alcohol 2 polyoxyethylen, named Ceteth-2, HLB = 5.3) and hydrophilic surfactants (stearyl alcohol 2 polyoxyethylene, named Steareth-20 HLB = 15.4) were kindly provided by Oxiteno (Sao Paulo, Brazil).

2.2. Preparation of lamellar gel phase emulsions/formulations (LGP) LGP emulsions were produced by the emulsion inversion phase method (EPI), as previously described (Blonchard, 1970). Briefly, aqueous (80%) and oily (10%) phases with 10% of a blend of surfactants of Ceteth-2/Steareth-20 (0.97/0.07 v/v) were heated separately until they reached 75.0 ± 5.0 °C. The aqueous phase was gently poured over the oily phase under continuous agitation

Rheological properties of the LGP emulsions (n = 3) were examined using a Haake rheometer (Model Rheostress RS-1, Germany). The rheometer was based on a thermostatically controlled cone/plate (C35/2° Ti) sensor with a 60 mm diameter and a 1° angle. Rheowin 3.5 software was used to analyze the data. The assays performed were: (1) stress yield, using a shear stress of 0– 100 Pa, for 180 s; (2) low limit curve (until emulsions star flowing); (3) stress sweep analysis; (4) frequency sweep analysis; (5) viscosity and thixotropic test. In the continuous shear analysis, upward and downward flow curves for each formulation were measured over shear rates ranging from 0.001 to 8900 s1. The shear rate was increased over an increment of 120 s, held at the upper limit for 10 s, and then decreased over a period of 120 s. The thixotropic value of the formulations can be calculated based on the area of stress yield assay. The shear strain, the stress, and the phase angle were determined from oscillating measurements. The parameters obtained were the complex modulus, G⁄, and the

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phase angle d. The elastic modulus (G0 ), the viscous modulus (G00 ) and the dynamic viscosity (g) were calculated using the following equations:

G ¼ G0 þ iG

00

ð1Þ

G0 ¼ G cosðdÞ

ð2Þ

G00 ¼ G sinðdÞ

ð3Þ

g ¼ G00 =x

ð4Þ

The angular frequency (x) ranged from 0.01 to 100 Hz. In each case, the dynamic rheological properties were determined with at least three replicates of three independents formulations. Viscosity and thixotropic measurements were performed for shear rates between 0.001 and 8900 s1. The results are represented as mean values (mean ± S.D., n = 3). 2.6. Lamellar structure evaluation after water loss The sample was then spread to obtain a uniform layer approximately 0.2 mm thick. Water evaporation was measured by a scale equipped with infrared light heating the sample at 70 °C. Every 10% of weight loss a photomicrograph was taken under polarized light and a new slide was prepared for further testing. Images of the samples were obtained at distinct stages of dehydration using an Olympus microscope (Model BX 50) equipped with a polarizer. 2.7. In vitro assay 2.7.1. Cell viability assay of C. officinalis oil Cytotoxicity was measured using an apoptosis and a necrosis assay. L929 cells (1  105 cells/mL) were treated with calendula oil (50–1000 lg/mL) for 24 h. Cells were centrifuged and incubated in 100 lL of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) containing conjugated Annexin V FITC (FITC-Annexin Apoptosis Detection Kit I V, PharmigenTM BD) and Propidium Iodide (1 lg/mL) for 15 min (dark) at room temperature. Flow cytometry was performed with an acquisition of 10,000 cells per sample. Each experiment was performed in triplicate. Data was analyzed by BD FACSDIVA software (BD Biosciences). 2.8. In vivo wound healing model 2.8.1. Animals Male Wister rats (average weight of 190–210 g) were used in this study. All experiments were performed with approval of the University of Sao Paulo Animal Ethics Committees (CEUA/Protocol No. 09.1.544.53.1/05/12/2011). 2.8.2. Wounds and treatments On the day of surgery (day 0), 80 rats were pre-anaesthetized (intramuscularly) and anaesthetized with 10% Xylazine (DopaserÒ) at a dose of 10 mg/kg body weight and Ketamine (20% DopalenÒ) at a dose of 10 mg/kg body weight, respectively. After shaving their backs, two surgical excisions were made in different areas with a histological punch of 1.5 cm in diameter (Stiefel Laboratories, Sligo, Ireland), reaching the dermo-epidermal junction. The surgical excisions performed by the histological punch is a full-thickness wound used to investigate contractile wound repair and it is the most common wound type used by other investigators. The wound can be harvested at any time during the healing process (Reid et al., 2004).

After surgery, an intraperitoneal dose of 50 mg/kg body weight of Dipyrone diluted in saline was administered. The rats were then divided into four distinct treatment groups (n = 20/group): (1) the LGP group: treatment with lamellar gel phase formulation containing calendula oil; (2) the CAL group: treatment with a simple formulation with calendula oil (no lamellar gel phase contained/Appendix A); (3) the GEL group: treatment with only lamellar gel phase emulsion without calendula oil; (4) the CONTROL group: no treatment (no lamellar gel phase emulsion, no calendula oil). Wounds were treated daily with the four distinct groups as described above. An occlusive gauze and adhesive tape dressing (to avoid possible infections regarding to the environment) was used in wounds of all group rats. The dressings were changed every day.

2.9. Evaluation of the potential wound healing effect of LGP emulsion in the model of cutaneous wounds of the back of rats (excisional contractile wound) The wounds were assessed 2, 7, 14 and 21 days post-surgery. Following assessment, 5 rats per group, per day of follow-up, were euthanized by inhalation of CO2. The wound-healing rate (WHR) of different groups was evaluated by clinical and photographic assessment of the wounds, and the evolution of the wounded areas was performed using ImageJ software. To determinate the wound healing rate (WHR), the formula [(Ai  Af)/Ai] was used. The values greater than zero represent a decrease in the wounded area, whereas values less than zero represent an increase in the wounded area and WHR = 1.0 represents full re-epithelialization (Caetano et al., 2009). The initial area (Ai) corresponds to the area of the wound the day of surgery and the final area (Af) corresponds to the final evaluation day. Ultimately, all wound biopsies were collected for subsequent histological analysis.

2.10. Histological analysis The tissues excised from wound sites were fixed in 10% (v/v) formalin solution, dehydrated through a graded series of alcohol (50–100% (v/v)), cleared in xylene, and embedded in paraffin. Serial sections of 3.0 lm thickness were made using a microtome, and stained with hematoxylin and eosin (evaluation level of inflammatory infiltrate). The slides were then stained with Gomory’s trichrome (evaluation of collagenesis), and examined with an optical microscope using the Leica Application Suite Version 3.2.0 software. For each wounded skin sample of each animal, 10 sections were taken and examined (400 magnifications). The quantification of collagenesis was made using the ‘‘Colour Deconvolution’’ plug-in of the ImageJ software, where the three colors of trichrome were deconvoluted. Only the percentage of the total area of blue color (collagen) of the image was determined. The results were reported as the average distribution of collagen per treatment (Andrade et al., 2011 and Carvalho et al., 2006).

2.11. Statistical analysis Data were expressed as the mean value ± SEM. All studies were analyzed using the One-way ANOVA analysis with a = 5% and the Bonferroni. To identify the statistically significant differences between all groups the GraphPad Prism 5.0 software (San Diego, CA) was used.

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3. Results and discussion 3.1. Pre-formulation of LGP emulsions and development of ternary and pseudo phase’s ternary diagram A ternary phase diagram was constructed to represent the formulations with the percentage of C. officinalis oil, distilled water, and surfactants (Ceteth-2 and Steareth-20). The weight ratio of surfactants (Ceteth-2/Steareth-20 = 0.93/0.07) was chosen to achieve an HLB value of 6.0. These surfactants were chosen due to their low toxicity with the skin and low incompatibility regarding other excipients (Santos and da Rocha-Filho, 2007). In addition, it has been suggested that the anisotropic structures presented by the lamellar gel phase emulsions are due to the non-ionic surfactants Ceteth-2/Steareth-20 (Santos et al., 2005). The first pseudo-diagram was varied to 10% (w/w/w) of each component to obtain 36 formulations (Appendix B) depicted as an equilateral triangle (Fig. 1). After being equilibrated, samples were assessed visually according to the aspect of the formulation at 24 h post-production: (1) separation phase (PS); (2) wax aspect (WA); and (3) lamellar gel phase (LGP/presence or absence of anisotropic structures detected by optical microscope magnification of 200). The phase separation was associated with high oil content and low surfactant and water concentrations. The formulations of the wax phase demonstrated instability as a consequence of high surfactant concentration and low distilled water concentration. Nevertheless, formulations of the LGP area were selected for further optimization due to its enhanced physical stability, anisotropic structures, opaque characteristics, low apparent viscosity, white color, and neutral odor and properties required for medicinal and cosmetic applicability (Figure not shown). Next, we determined that the best formulation was the number 36 consisting of 10% marigold oil; 10% of the blend of surfactants and 80% of purified water (w/w/w). This formulation was selected to further continue our studies.

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After determining the region of interest (LGP), we developed six additional formulations (A–F) varying the components with 5% increments (w/w/w) from the formulation number 36 (Table 1 and Fig. 1).

3.2. Emulsions characterization 3.2.1. Macroscopic analysis The emulsion number 36 and its derivations were subjected to additional tests, such as centrifugation, thermal stress analysis, and presence or absence of anisotropic structures (AS) to further scrutinizes their stability by macroscopic evaluation. The formulations with emulsifier concentrations of more than 10% (A and B formulations) were stable, but ruled out for further studies due to their propensity to irritate skin. The remaining formulations (36, C–F) were subjected to thermal stress 24 h post-production (Table 2). Formulations D and E coalesced after the second cycle of centrifugation, indicating low separation energy and were subsequently rejected. This phase separation may have been due to the low concentration (5% w/w/w) of surfactants. Sample number 36, C, and F remained stable up to 50 °C with no apparent changes. However, these emulsions became unstable at temperatures above 50 °C, displaying creaming and complete phase separation, indicating that the systems stability is temperature dependent. These emulsions were considered to be stable emulsions, because they reached higher temperatures in the thermal test than others. Moreover, the pH values of these emulsions were evaluated and the mean ± SEM was 5.4 ± 0.5 after 24 h post-production and compatible with the human physiology. The instability of the emulsions demonstrated by the centrifugation and thermal stress tests, might be a consequence of the destabilization of the energy barrier, which indicates the maximum energy of repulsion among the dispersed droplets in the emulsion (Friberg et al., 1988). When energy is supplied to the

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.

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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 30.0 g (%) (%)

Water (%)

Oil (g)

Ceteth-2 0.93

Steareth-20 0.07

Water (g)

A B C D E F

75 80 85 85 80 75

3.0 1.5 1.5 3.0 4.5 4.5

4.185 4.185 2.790 1.395 1.395 2.790

0.315 0.315 0.210 0.105 0.105 0.210

22.5 24.0 25.5 25.5 24.0 22.5

10 5 5 10 15 15

15 15 10 5 5 10

Table 2 Results of intrinsic tests for LGP emulsions. Emulsions

36 C D E F

Centrifugation test (rpm)

Stress test (°C)

2500

3000

50

55

N N SM MM N

N N MM SM N

SM SN AM MM N

MM MM AM AM SM

pH value

A.S.

5.72 ± 0.02 5.70 ± 0.02 5.82 ± 0.02 5.84 ± 0.02 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.

system, the ‘‘Brownian motion’’ potentially leads to creaming, flocculation, and coalescence (Kong et al., 2001). Our investigation identified that the sample number 36 and formulation F demonstrated better stability and greater anisotropic structures than the previous formulations developed Santos et al. (2006), for this reason, these formulations were selected to further continue our studies. 3.2.2. Rheological measurements The rheological analysis evaluated both the physical–chemical nature of the emulsions and potential instabilities. In addition, the rheological behavior is influenced by some basic parameters such as the continuous phase rheology and the physical nature of the dispersed phase (size, concentration, and deformability) (Barnes, 1994). Rheological analyses performed on samples 36 and F demonstrated non-Newtonian behavior with flow curves in the form of anti-clockwise hysteresis loops (Fig. 2A). Both formulations (36 and F) displayed shear-thinning behavior, due to the apparent decrease in viscosity with increasing shear rate. Also, the formulations displayed hysteresis, which is characteristic of thixotropic emulsions. Emulsion 36 showed a higher hysteresis value (118.8 Pa/s) than formulation F (19.38 Pa/s). Emulsion F was predicted to show a lower hysteresis value due to its lower water ratio. The rheologies of emulsions 36 and F were measured by the dynamic flow of water between the interlamellar space and the dispersed phase. The capacity of keeping water between the lamellas and also the rheological properties of these emulsions (36 and F) are determined by both the lipophilic carbon chain and the amount of surfactant ethoxylation (Gregolin et al., 2010 and Ribeiro et al., 2004). It is possible to predict the residence time of the emulsion on the skin surface by measuring the viscosity of the formulation (Boateng et al., 2008). In wound healing, one of the requirements for treatments is that the compounds remain on the wound surface for extended periods of time (Matthews et al., 2008). Formulation F, with its higher viscosity, was predicted to remain on the target area for longer periods of time than formulation 36. As depicted in the Flow limit curve (Fig. 2B), the formulations 36 and F were further subjected to shear stress range of 0.1–10 Pa. Samples 36 deformed in a range of 0.04499 Pa in 4.05 s while formulation F changed in 0.04614 Pa in 3.95 s.

The results from these assays combined with the yield stress test provides ideal conditions for the stress sweep test and frequence sweep test. The stress sweep test defines the interval of tension required to induce linearity in the emulsion. The analysis demonstrated that the emulsions were linear from 0.1 to 10 Pa (Fig. 2B). The date obtained from the stress sweep test suggested that a tension of 1 Pa in both formulations can be used for the frequency sweep test and creep and recovery test. Both formulations showed a linearity range in all compounds (G0 , G00 and g) in a frequency range of approximately 1–10 Hz demonstrated by the frequency sweep analysis (Fig. 2D). However, emulsion 36 presented a slightly higher deformation than emulsion F due to its lower viscosity. Moreover, the general frequency confirmed the presence of a gel-like structure at this condition with a G0 –G00 versus frequency curve characterized by a remarkable predominance of elasticity over the viscous behavior (G0 > G00 ) and the G0 modulus being parallel to the frequency axis. The compound (n), G0 and G00 values of sample 36 were smaller than F, which indicates that formulation 36 demonstrated more deformation than formulation F. This is due to the fact that the more deformation that is presented the less the values of these compounds n, G0 and G00 showed, for the same shear stress at a specific time (Dolz et al., 2008). Samples 36 and F both demonstrated viscoelastic properties. However, sample F (Fig. 3) was selected for further investigation due to its adequate viscosity and because of the probability that it might increase the residence time of the formulation on the wound surface (desirable for wound healing purposes). 3.2.2.1. Apparent viscosity (g)/thixotropy. Apparent viscosity analysis as a function of shear rate is predictive of physical instability and can be correlated with molecular behavior (Masmoudi et al., 2005). The apparent viscosity of formulation F was determined as a function of shear rate (100.02/s) from the apex of the loop (500 s1) during the flow curve determination. Thixotropy and apparent viscosity (g) values were analyzed over a period of 9 months exposure to specific conditions; 25.0 ± 2.0 °C, 5.0 ± 2.0 °C and 40.0 ± 2.0 °C with a relative humidity of 75% (Figs. 4 and Supplementary 1A). Graphical analysis (2000 Rheo software V2. 8) demonstrated that samples stored at 5.0 ± 2.0 °C and controlled room temperature (25 ± 2.0 °C) displayed

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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.

characteristics consistent with the presence of thixotropy in all times of follow-up (90 days). However, samples stored at 40.0 ± 2.0 °C reduced the area of hysteresis and proved to reduce the thixotropy of the formulations, causing reduced stability and spreadability (Fig. 4). It is inferred that an increase in temperature may have altered the surfactant pairs, leading to conformational modifications such as macroscopic changes and the reduced spreadability (Lindman and Karlstrom, 2009). Samples stored at 25 ± 2 °C displayed a constant apparent viscosity until the end of the analyses (90th day) while the formulations stored at 5.0 ± 2.0 °C increased until the end of the analyses (90th day) (see Supplementary Fig. 1a). The formulation stored at

40.0 ± 2.0 °C showed a small initial instability until the 15th day following by increasing the apparent viscosity until the 90th day (Fig. S1A). It may be due to the ethoxylated non-ionic surfactants present in the sample, which can cause structural changes during storage (Eccleston, 1990). Moreover, the decrease in repulsive forces and an increase in the attractive forces caused by higher temperature may store water in the interface of the anisotropic structure a period greater than that expected contributing to the increase of viscosity of the product (Lindman and Karlstrom, 2009). The apparent viscosity and stability of the LGP formulations are relatively larger than the simple emulsions due to the reorganization of the lamellar microstructures around the droplets and

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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.

the penetration of free water in the interlayer space of structures in the gel phase, with a consequent increase in viscosity of the system (Eccleston et al., 2000; Muller-Goymann, 2004). Furthermore, we also had the pH and electrical conductivity values of the formulation F determined. The formulation F had stable pH values at 25.0 ± 2.0 °C and 5.0 ± 2.0 °C and displayed decreasing pH values over time at 40.0 ± 2.0 °C (see Supplementary Fig. S1B). However, the pH values remained at around 4.5–6.0, which is an acceptable value for a non-skin irritating compound. In regard to the electrical conductivity test, the samples stored at 40.0 ± 2.0 °C showed a decreased electrical conductivity only in the initial 14 days and thereafter stabilized (Supplementary Fig. S1C). The other samples stored at 5.0 ± 2.0 °C and 25.0 ± 2.0 °C demonstrated stable electrical conductivity values and the absence of electrolytes.

skin, the mechanism of action of the final product, and the maintenance of skin hydration (Santos et al., 2006; Friberg, 2007). We found that even after 70% of water loss and under extreme storage conditions, anisotropic structures could still be observed (Fig. 5G). This indicated that the lamellar phase formed after the production of the emulsions resisted the decrease of water in the system. Lamellar phases are organized according to their orientation of the surfactant molecules that allows part of an external water phase to become adsorbed, further changing its state and preventing the water from evaporating (due to the intense interaction between the water molecules and the polar groups of the non-ionic surfactants by hydrogen bonds) (Santos and da Rocha-Filho, 2007; Gao et al., 2003). In this case, water molecules need higher quantities of energy to evaporate, which keeps forming the anisotropic structures during its hydrated state, assuming lamellar organization.

3.3. Microscopic study of the behavior of LGP formulation upon evaporation of water

3.4. Effect of C. officinalis oil on apoptosis and necrosis

Evaporation of water can alter a formulation’s intrinsic properties, such as the behavior of the formulation after application to the

Cytotoxic effects were analyzed using FITC-annexin V and PI double staining in L929 cells in flow citometry. We demonstrated

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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Fig. 6. The flow cytometric analysis of apoptosis in L929 cells using FITC-annexin V 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 depicted by early apoptotic cells, and Annexin V+/PI+ (upper right quadrant) areas are depicted by late apoptotic or necrotic cells.

that neither early apoptosis (Annexin V+/PI) nor late apoptosis (Annexin V+/PI+) was modified by formulation F containing C. officinalis oil (50–1000 lg/mL) as compared to the control or the vehicle (Fig. 6). The marigold extract of C. officinalis was shown not to be cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL. Thus, based on these results, C. officinalis oil should be classified as a non-cytotoxic oil, and could be used as a stimulator of the wound healing process. 3.5. In vivo wound healing studies Following the in vitro assay, the effect of the LGP emulsion (formulation F) on the experimental wound healing model (in vivo) was evaluated. Large, full-thickness wounds performed on the backs of healthy rats were treated and monitored for 21 days. Re-epithelialization, a highly important event in restoration of skin barrier function, was evaluated by measuring the wound healing rate (Fig. 7). The LGP group showed less re-epithelialization than the GEL and the CONTROL groups on the 2nd day (p < 0.05). Furthermore, several wounds had increased in size on the 2nd

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day for all groups. On the 7th day, all groups showed an improvement in wound re-epithelialization relative to the 2nd day, but no difference between the groups was observed (p > 0.05). Finally, all groups enhanced the reepithelialization after the 14th day, but unlike the GEL and CONTROL groups, the LGP group was completely re-epithelialized on 14th day. Both the GEL and the CONTROL groups showed wounds that were not totally re-epithelialized on the 14th and 21st day as depicted in Fig. 7. The wound increasing in size characterizes the inflammatory phase of wound healing. This is mostly attributed to the high activity of proteases and subsequent tissue degradation that results as a consequence of compounds being released from inflammatory cells (like the reactive oxygen and nitrogen species from oxidative stress) during phagocytosis of the pathogens and senescent cells (Guo and Dipietro, 2010 and Schreml et al., 2010). The scientific literature shows numerous studies describing the wound re-epithelialization effect by calendula oil (Faria et al., 2011; Preethi et al., 2010; Szakiel et al., 2008; Schmidt et al., 2009; Chandran and Kuttan, 2008; Pommier et al., 2004). The advancement made by our study was the particular association between the calendula oil and the lamellar gel phase emulsion (formulation F). This association maintained the wound healing process profile of the CAL group after the 7th day. To understand how the associated products might act to enhance the wound re-epithelialization process, we performed a wound histological analysis (Fig. 8). The wounds of the LGP group demonstrated higher inflammatory infiltrate than the CAL group on the 2nd and 7th day and both of these groups presented more inflammatory infiltrate than the GEL and the CONTROL groups. After the 14th day, all groups showed decreases of in levels of inflammatory infiltrate. By microscopic histological analysis on the 7th day, the LGP group showed new blood vessels that sustained growth up to the 21st day, which was different from the other groups (Fig. 8). Fibroblasts are the type of cell that produce collagen, the major structural component of granulation tissue that compounds the extracellular matrix. Proceeding the 14th day of the follow-up, when the inflammatory infiltrate was reduced (shown in Fig. 7), intense fibroblastic proliferation was observed in all groups. The

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.

LGP group displayed significantly more collagen than the CONTROL groups on the 2nd day (p < 0.005) in the beginning of the healing process (Fig. 9). Moreover, in the CAL group more collagen was observed compared to the other groups, especially on the 2nd day (p < 0.05). On the 7th day, the CAL group still presented higher collagenesis than the GEL and the CONTROL groups (p < 0.05), albeit similar to LGP (p > 0.05). On the 14th day, the LGP and the CAL groups presented higher collagenesis than the GEL group (p < 0.05) and this stimulus continued until the 21st day (p < 0.05), when both groups had higher collagenesis than the CONTROL group (Fig. 9). The intense inflammatory infiltrate observed at the beginning of the healing process (2nd day) of the LGP group might produce high levels of proteolytic enzymes (such as metalloproteinases and collagenases), that further hinder re-epithelialization, breaking the surrounding tissue that causes wounds to increase in size. Furthermore, the enzymes also might aid in the prevention of the

production of collagen to new tissue formation, as observed in the collagenesis evaluation, when compared to the CAL group. If the inflammatory stimulus is indeed persistent, high concentrations of proteolytic enzymes are produced, which stimulates oxidative stress, impairing the healing process (Schreml et al., 2010; Schäfer and Werner, 2008). However, from the 14th day onward, there was an important reduction of inflammatory infiltrate. Furthermore, the wounds treated with LGP emulsion showed considerable humidity on the 2nd and the 7th day, when compared macroscopically to the wounds of the other groups (Figure not shown). In general, it has been proposed that the hydration of the wound stimulates epithelialization, granulation, tissue formation, angiogenesis, fibroblast migration, collagen synthesis, and remodeling of injured tissue, thus reducing the possible trauma during changes of the dressing (Kumar et al., 2008). This suggested that the LGP emulsion (in addition to the higher inflammatory

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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.

infiltrate observed) might have influenced the maintenance of moisture in the wound, maintaining the properties of calendula oil (which is an important factor in autolytic debridement) (Mandelbaum et al., 2003) and enhanced the total wound re-epithelialization. Although the proposed wound healing animal model does not take chronic wounds into consideration, the data presented herein demonstrate an implication to treat chronic ulcers in clinical practices. In general, there is a need for a significant inflammatory stimulus to remove the ulcers of a senescent status, providing granulation tissue (angiogenesis, fibroplasia, and collagenesis) and promoting re-epithelialization, as previously observed by Caetano et al. (2014). 4. Conclusions The LGP emulsion (15.0% marigold oil; 10.0% of blend surfactants and 75.0% of purified water [w/w/w]) has demonstrated greater stability and its low spread ability suggests longer contact with wounds. The C. officinalis oil was not detrimental and also was non-cytotoxic as assayed in L929 cells. The LGP emulsion was relatively stable and promoted better quality wound healing in a rat skin wound model than the other groups. LGP emulsion seemed to modulate the inflammatory phase of wound healing. Taken together these data suggests that the novel lamellar gel phase emulsion (formulation F) we have developed with C. officinalis oil shows high potential in improved wound-healing applications. Conflict of interest The authors declare that they have no competing interests Acknowledgments This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil), CNPq and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo – Brazil). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2015.01.016.

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