SUPPLEMENTARY MATERIAL Effect of a topical formulation

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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*
a
Programa de Pós-graduação em Promoção da Saúde, Centro Universitário de Maringá,
CESUMAR, Av. Guerdner 1610, Jd. Aclimação, 87050-900 Maringá, PR, Brasil.
b
Pós-graduação em Ciências Farmacêuticas, Departamento de Farmácia e Farmacologia,
Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, PR, Brasil.
c
Departamento de Nutrição, Faculdade Assis Gurgacz, Av. das Torres 500, 85806-095
Cascavel, PR, Brasil.
d
Pós-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 ± 2C, 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 DSCW320 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.
Percentage of wound healing (mean ±SD) on days
after surgery
Treatment groups
Day 7
Day 14
Day 21
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
Emulsion of C. brasiliense extract
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.
Acute
Chronic
Fibroblast
inflammation
inflammation
proliferation
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
Groups
Neovascularization
Epithelization
Data
expressed as mean ± standard deviation (n=5). Different letters (a and b) in a column indicate statistically significant difference (p < 0.05).
are
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.
References
Brenzan MA, Nakamura CV, Dias-Filho BP, Ueda-Nakamura T, Young MCM, Cortez DAG.
2007. Antileishmanial activity of crude extract and coumarin from Calophyllum brasiliense
leaves against Leishmania amazonensis. Parasitol Res. 101:715-22.
Brenzan MA, Ferreira ICP, Lonardoni MVC, Honda PA, Rodriguez- Filho E, Nakamura CV,
Cortez, DAG. 2008a. Activity of extracts and coumarins from leaves of Calophyllum
brasiliense Camb. on Leishmania braziliensis. Pharm Biol. 46:1-7.
Brenzan MA, Nakamura CV, Dias-Filho BP, Ueda-Nakamura T, Young MCM, Côrrea AG,
Alvim-Júnior J, Santos AO, Cortez DAG. 2008b. Structure–activity relationship of (−)
mammea A/BB derivatives against Leishmania amazonensis. Biomed Pharmacother.
62(6):651–658.
Brenzan MA, Santos AO, Nakamura CV, Dias-Filho BP, Ueda-Nakamura T, Young MCM,
Côrrea AG, Alvim-Júnior J, Morgado-Díaz JA, Cortez DAG. 2012. Effects of (−) mammea
A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial
membrane of Leishmania amazonensis. Phytomedicine. 19:223–230.
Fundação Oswaldo Cruz (Fiocruz). 2005. Curso de Manipulação de Animais de Laboratório.
Centro de Pesquisas Gonçalo Luiz. Ministério da Saúde. Bahia.
Garros IC, Campos ACL, Tâmbara EM, Tenório SB, Torres OJM, Agulham MA, Araújo ACF,
Sains-Isolan PMB, Oliveira EM, Arruda ECM. 2006. Extrato de Passiflora edulisna
cicatrização de feridas cutâneas abertas em ratos: estudo morfológico e histológico. Acta Cir
Bras. 21(3):55-65.
Hajiaghaalipour F, Kanthimathi MS, Abdulla MA, Sanusi J. 2013. The effect of Camellia
sinensis on wound healing potential in an animal model. Evid Bas Complement Alter Med.
2013:1-7.
Honda PA, Ferreira ICP, Cortez DAG, Amado CAB, Silveira TGV, Brenzan MA, Lonardoni
MVC. 2010. Efficacy of components from leaves of Calophyllum brasiliense against
Leishmania (Leishmania) amazonensis. Phytomedicine. 17:333–38.
Honda PA, Brenzan MA. Ferreira ICP, Lonardoni MVC, Nakamura CV, Dias-Filho BP, Cortez
DAG. 2011. Topical formulation used for treating leishmaniasis (cutaneous leishmaniasis
with cutaneous lesions) in animal and humans, contains hexane or dichloromethane extract
of Calophyllum brasiliense with vehicles, diluents and additives Brazil patent BR200904042A2.
Hunt T, Mueller R. 1994. Wound healing: Current Surgical Diagnosis and Treatment. 10th ed.,
Appleton and Lange: Paramus, NJ, USA. p. 80–93.
Mandelbaum SH, Di Santis EP, Mandelbaum MHS. 2003. Cicatrização, conceitos atuais e
recursos auxiliares – Parte I. An Bras Dermatol. 78(5):525-542.
Panchatcharam M, Miriyala S, Gayathri VS, Suguna L. 2006. Curcumin improves wound
healing by modulating collagen and decreasing reactive oxygen species. Mol Cel Biochem.
290:87–96.
Pires CTA, Brenzan MA, Scodro RBL, Cortez DAG, Lopes LDG, Siqueira VLD, Cardoso RF.
2014. Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense
Cambess (Clusiaceae). Mem Inst Oswaldo Cruz. 109(3):324-329.
Tiuman TS, Brenzan MA, Ueda-Nakamura T, Dias-Filho BP, Cortez DAG, Nakamura CV.
2012. Intramuscular and topical treatment of cutaneous leishmaniasis lesions in mice infected
with Leishmania amazonensis using coumarin (−) mammea A/BB. Phytomedicine.
19(3):1196-1199.
Zahra AA, Kadir FA, Mahmood AA, Alhadi AA, Suzy SM, Sabri SZ, Latif II, Ketuly KA.
2011. Acute toxicity study and wound healing potential of Gynura procumbens leaf extract in
rats. J Med Plants Res. 5(12):2551–2558.
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