SUPPLEMENTARY MATERIAL

Effect of a topical formulation containing Calophyllum brasiliense Camb. extract on

cutaneous wound healing in rats

T.V.A. Lordania, M.A. Brenzanb, L.E.R. Corteza, C.R.F. Lordanic, P.A. Hondab, M. V.C.

Lonardonid and D.A.G. Corteza,b*

aPrograma de Pós-graduação em Promoção da Saúde, Centro Universitário de Maringá,

CESUMAR, Av. Guerdner 1610, Jd. Aclimação, 87050-900 Maringá, PR, Brasil.bPós-graduação em Ciências Farmacêuticas, Departamento de Farmácia e Farmacologia,

Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, PR, Brasil.cDepartamento de Nutrição, Faculdade Assis Gurgacz, Av. das Torres 500, 85806-095

Cascavel, PR, Brasil.dPós-Graduação em Biociências e Fisiopatologia, Universidade Estadual de Maringá, Av.

Colombo 5790, 87020-900 Maringá, PR, Brasil.* Corresponding author. Email: dagcortez@uem.br

Abstract

This study evaluated the wound healing effects of topical application of an emulsion containing

the HPLC standardized extract from C. brasiliense Cambess (Clusiaceae) leaves in rats. The

macroscopic analysis demonstrated that the wounds treated with the C. brasiliense emulsion

healed earlier than the wounds treated with emulsion base and Dersani®. The percentage of

wound healing in the group treated with the C. brasiliense emulsion was significantly higher

than in the other groups at 7 and 14 days. On day 14, the animals treated with the C. brasiliense

emulsion exhibited a 90.67% reduction of the wound areas. The histological evaluation revealed

that on day 21, the group treated with the C. brasiliense emulsion exhibited a significant increase

in fibroblasts compared with the other groups. Thus, the C. brasiliense emulsion had healing

properties in the topical treatment of wounds and accelerated the healing process.

Keywords: wound healing, topical treatment, Calophyllum brasiliense, crude extract, rat

1. Experimental

Plant material

Calophyllum brasiliense Cambess (Clusiaceae) leaves were collected in Cardoso Island,

São Paulo state, Brazil, in December 2010. A voucher specimen (SP 363818) was identified by

Prof. Dra. Maria Claudia M. Young and deposited and authenticated at the Herbarium of the

Botany Institute of São Paulo, São Paulo, Brazil. The leaves were dried at 35°C in a circulating

air oven and triturated in a knife mill (Usi-ram®).

Plant extraction

The powdered leaves (800 g) were extracted by exhaustive maceration in ethanol:water

(9:1) at room temperature, filtered, and concentrated under vacuum at 40°C to obtain an aqueous

extract and dark-green residue. The dark-green residue was dissolved with dichloromethane, and

the solvent was then completely evaporated at room temperature to yield a dichloromethane

extract (25.0 g) (Brenzan et al. 2007; 2008a, 2008b; Honda et al. 2010; Honda et al. 2011). The

bioactive (-) mammea A/BB coumarin was quantified in the crude dichloromethane extract by

high-performance liquid chromatography (HPLC) according to the methodology described by

Pires et al. (2014).

High-Performance Liquid Chromatography (HPLC) analysis

The extract from C. brasiliense leaves and the coumarin (-) mammea A/BB were

analyzed by HPLC-UV using HPLC-UV grade solvents and ultrapure water (Milli-Q system,

Millipore, Bedford, MA, USA) and a Waters 1525 liquid chromatograph equipped with a binary

pump (LC-10 AD), automatic injection valve 135 (Rheodyne) with a 20 μL loop, CTO-10AVP

thermostat-controlled oven compartment (Shimadzu) and a 2489 UV/visible detector (Waters)

controlled by Breeze 2 software (OmniSolv EM Science, Gibbstown, NJ, USA). A MetaSil ODS

column (5 μm; 150 × 4.6 mm) maintained at 25ºC was used in the chromatographic analysis. The

separation was conducted in a gradient system, using a mixture of acetonitrile and ultrapure

water 55:45 v/v to 80:20 (0-10 min), 80:20 v/v to 100% (10-20 min), 100% acetonitrile (20-25

min) and 55:45 v/v (26-30 min) as the mobile phase, at a flow rate of 0.6 mL/min. Detection was

performed at 336 nm and the running time was 30 min. The sample injection volume was 20 μL.

The extract from C. brasilense leaves (3 mg/mL) and compound (-) mammea A/BB (1 mg/mL)

were dissolved in methanol, filtered through a membrane filter (Millipore, Brazil). Compound (-)

mammea A/BB was quantified in the extract from C. brasilense leaves by HPLC-UV using as

the standard the (-) mammea A/BB previously isolated and identified from the C. brasiliense

leaves (Brenzan et al. 2008a, 2008b, 2012; Tiuman et al. 2013). The calibration curve was

prepared using (-) mammea A/BB standard at concentrations ranging from 15.63 to 250 µg/mL

in methanol. Three determinations were carried out for each sample. For the determination of the

contents of (-) mammea A/BB in the extract, the regression equation y = 61589x + 52298 was

used. The statistical analyses of the data were performed using Statistica 8.0 software (Statsoft

Inc, Tulsa, OK, USA) (Pires et al. 2014).

Preparation of the topical formulation containing the extract from C. brasiliense leaves

A non-ionic emulsion containing the extract from C. brasiliense leaves at a concentration

of 10% (w/w) was prepared according to described in the patent for this topical formulation

(Honda et al. 2010, 2011). To prepare the emulsion was used the HPLC standardized extract that

contained 28.04 ± 0.9 µg of (-) mammea A/BB per mg of extract. The emulsion formulation was

prepared by adding an aqueous phase in oil phase under constant agitation. To prepare the

emulsion formulation, we used 6 % cetostearyl alcohol (w/w), 5 % octyldodecanol (w/w), 7 %

mineral oil (w/w), 12 % ethoxylated alcohol stearyl (w/w), 18 % glycerin monoestarate (w/w),

10 % propylene glycol (w/w), and purified water in sufficient quantity. The standardized extract

from C. brasiliense leaves was weighed and added to the emulsion in small amounts until a final

concentration of 10% (w/w) was reached under constant stirring. The pH of the formulation was

5.5. The formulations, with and without the addition of the extract, were termed as the emulsion

of the C. brasiliense extract and emulsion base control, respectively. The formulations were

prepared a few days before beginning the experiments, stored in closed flasks, and protected

from light and heat.

Experimental animals

Forty-five adult male albino Wistar rats (Rattus norvegicus), weighing 200 ± 5 g, were

obtained from the Animal Facility of the Assis Gurgacz Faculty. The animals were randomly

divided into three groups (n = 15 per group). These groups were further randomly divided into

three subgroups according to the observation period (7, 14 and 21 days after surgery; n = 5 per

subgroup). The animals were individually housed in polyethylene cages under controlled

environmental conditions (23 ± 2C, 50-70% relative humidity, 12 h/12 h light/dark cycle) and

were provided water and food ad libitum. The animals were allowed a 7 day acclimation period

before the start of the experiments. The experimental protocol was approved by the Animal

Ethics Committee of the Assis Gurgacz Faculty (protocol no. 032/2012).

Experimentally induced excision wounds

An excision wound was inflicted in each rat according to the methods described by

Panchatcharam et al. (2006) with modifications. Prior to skin wound induction, the animals were

intraperitoneally anesthetized with 10 mg/kg xylazine and 75 mg/kg ketamine hydrochloride

(Fiocruz, 2005).

The skin was manually depilated using a stainless steel blade and disinfected with 70%

alcohol. To demarcate the surgical incision area, a transparent 2 2 cm sheet was used with a

total area of 4 cm2. The boundaries were demarcated with black ink on the skin. Prior to

performing the surgical incision, an antiseptic alcoholic solution of 2% chlorhexidine was

applied. The surgical incisions were made on the back, close to the cervical area of each animal.

The resection of the skin was performed using a scalpel blade in the demarcated area. Afterward,

the incision was deepened to expose the dorsal muscle fascia (Figure S1). Hemostasis was

performed using sterile compression gauze. Wound areas were measured immediately using a

digital caliper (Pantec®Brazil). The wounds of the animals were topically treated according to the

experimental groups.

Topical wound treatment

Soon after making the excision wounds, the animals were individually housed in cages

and received their respective treatments. The cutaneous wounds were topically treated according

to the experimental groups. Group A: The wounds were treated with 0.2 ml of the non-ionic

emulsion containing the extract of C. brasiliense at 10%, equivalent to 0.32 g of the C.

brasiliense extract. Group B: The wounds in the emulsion base control group were treated with

0.2 ml of the non-ionic emulsion base without the addition of the extract, equivalent to 0.3 g of

the excipients of the base. Group C: The wounds in the Dersani® group were treated with 0.2 ml

of Dersani®, sufficient to cover the entire wound. Dersani® is indicated for any type of cutaneous

lesion as a bactericide during the different phases of the healing process (Maldebaum et al.

2003).

The treatment was repeated daily. The animals did not receive postoperative dressings

and continued to receive this treatment until the date set for the measurements (7, 14, and 21

days after the first treatment). The experimental protocol was approved by the Animal Ethics

Committee of the Assis Gurgacz Faculty (protocol no. 032/2012).

Estimation of wound healing (wound closure)

Measurements were performed in all of the animals on day 0 (i.e., the day of surgery) and

7, 14 and 21 days after the first treatment. For this, the animals were intraperitoneally

anesthetized with 40 mg/kg thiopental and immobilized. The evaluation of the healing process

was performed using a digital caliper (Pantec®,Brazil) by measuring the length and width of the

wound, which were measured as the linear distance between the edges of the lesion (Garros et al.

2006). The length refers to the direction of the wound going from the head to tail of the animal.

The width was measured from side to side. After the measurements, the square area of the lesion

was calculated.

During the measurements, the wounds were photographed using a Sony Cyber shot DSC-

W320 camera to macroscopically analyze wound healing.

The wound closure rate is expressed as a percentage of the wound area compared with

postoperative day 0 as described previously (Zahra et al. 2011; Hajiaghaalipour et al. 2013) i.e.,

the change in wound size is expressed as a percentage of the original wound size.

Histological evaluation of the wounds

For histological evaluation, a fragment of the wound was removed in all of the groups (a

0.5 cm edge on each side of the injury). The skin fragments were immediately fixed in 10%

buffered formalin, dehydrated and embedded in paraffin. Afterward, the animals were sacrificed

by decapitation. Tissue blocks were cut at 4 μm thickness using a microtome (Leica

Microsystems, Germany), stained with hematoxylin-eosin and Masson’s trichome, and examined

using an Olympus BX60 light microscope. Three slides were prepared per animal.

The histological analysis evaluated acute inflammation, chronic inflammation, wound

healing, fibroblast proliferation, collagen deposition and neovascularization. The intensity of

these histological parameters was classified using a 0-4 numerical scale as described previously

(Hunt & Mueller 1994; Hajiaghaalipour et al. 2013): 0 (absence), 1 (occasional presence), 2

(light scattering), 3 (abundant), and 4 (confluence of cells or fibers).

Statistical analysis

The experimental results are expressed as the mean ± standard deviation of five animals

in each group. The statistical analysis was performed using Statistica® 7.0 software. Analysis of

variance (ANOVA) was used for comparisons between groups. Tukey’s test was used for

comparisons between means. The intergroup analysis was performed using the nonparametric

Kruskal-Wallis test. Values of p < 0.05 were considered statistically significant.

Table S1. Effect of an emulsion containing the extract from C. brasiliense leaves on excision

wound healing in rats 7, 14 and 21days, after surgery.

Treatment groups

Percentage of wound healing (mean ±SD) on days

after surgery

Day 7 Day 14 Day 21

Emulsion of C. brasiliense extract 26.67 ± 2.08a,b 90.67 ± 4.71a,b 92.0 ± 4.72

Emulsion base control 9.64 ± 4.73 66.33 ± 2.08 79.8 ± 4.73

Dersani® 8.25 ± 7.18 62.4 ± 6.7 82.67 ± 3.51

Mean values (n=5) followed by different letters (a and b) in a column are significantly difference

(p <0.05). a Different from the control group, b Different from the Dersani® group.

Table S2. The median histopathologic scores of wound healing were determined in the emulsion of C. brasiliense extract, emulsion base control

and Dersani® groups on day 21 by using a 0 to 4 numerical scale. The scores were 0 for absence, 1 for occasional presence, 2 for light scattering, 3

for abundance, and 4 for confluence of cells or fibres.

Data are

expressed as mean ± standard deviation (n=5). Different letters (a and b) in a column indicate statistically significant difference (p < 0.05).

Groups

Acute

inflammation

Chronic

inflammation

Fibroblast

proliferation Neovascularization Epithelization

Emulsion of C. brasiliense extract 1.0 ± 0.0a 1.6 ± 0.55a 3.2 ± 0.45a 1.0 ± 0.0a 3.2 ± 0.45a

Emulsion base control 1.0 ± 0.0a 0.6 ± 0.55b 1.0 ± 0.0b 0.82 ± 0.55a 2.4 ± 0.55a

Dersani® 0.8 ± 0.45a 1.6 ± 0.55a 1.4 ± 0.55b 1.8 ± 0.44b 3.0 ± 0.0a

Figure S1. Excision skin wound (4 cm2) on the day of surgery before treatment. Where can see

the exposure of the dorsal muscle fascia.

Figure S2. Chromatograms of the standard (-) mammea A/BB (1) (Rt = 21.8 min) (A) and

extract from C. brasiliense leaves (B). Chromatographic conditions: MetaSil ODS column;

mobile phase: acetonitrile-water 55:45 v/v to 80:20 (0-10 min), 80:20 v/v to 100% (10-20 min),

100% acetonitrile (20-25 min) and 55:45 v/v (26-30 min); flow rate 0.6 mL/min; temperature

25°C; detection: 336 nm. (1) (-) mammea A/BB

Figure S3. Macroscopic appearance of the wounds 7, 14 and 21 days after surgery. (A-C) Group

A: Topical treatment with emulsion of the C. brasiliense extract. (D-F) Group B: Treatment with

emulsion base only. (G-I) Group C: Treatment with Dersani®.

Figure S4. Histology of wound area stained with hematoxylin and eosin on day 21 after surgery.

A) Group A: Topical treatment with emulsion of the C. brasiliense extract. (B) Group C:

Treatment with Dersani®. (C) Group B: Treatment with emulsion base only. The arrows

indicated the fibroblasts. Magnification: 40 x.

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