Wound Medicine 31 (2020) 100195

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Wound Medicine
journal homepage: www.elsevier.com/locate/wndm

Healing potential of Caiman yacare (Daudin, 1802) visceral fat oil T

a b a
Lucas Polizzeli Azevedo , Rosa Helena dos Santos Ferraz , Márcia Regina Lopes de Magalhães ,
Ana Paula Oliveiraa, Bruno Cogliatic, Larissa Maria Scalon Lemosa, Paola Cristina Brancod,
Demétrio de Abreu Sousae, José Roberto Machado Cunha da Silvaf,
Leandro Nogueira Pressinottia,*
a
Programa de Pós Graduação em Ciências Ambientais, Universidade do Estado de Mato Grosso, Cáceres, MT, Brazil
b
Faculdade de Medicina Veterinária, Universidade Federal de Mato Grosso, Cuiabá, MT, Brazil
c
Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil
d
Departamento de Farmacologia do Instituo de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil
e
Instituto Federal de Educação Ciência e Tecnologia de Mato Grosso, Cáceres, MT, Brazil
f
Laboratório de Histofisiologia Evolutiva, Departamento de Biologia Celular e Tecidual do Instituo de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP,
Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Caiman yacare visceral fat oil was extracted, analyzed, and tested for healing potential. In vivo assays were
Wound performed by excisional wounds in rats and treated topically daily with Caiman oil for 10 days. The positive and
In vivo negative controls were, respectively, with Dersani® (Essential Fatty Acids – EFA, reference substance) and saline
Scar solution (SS). The scattered area was measured daily, and at 3rd and 10th days the rats were euthanized and
samples were processed for histological analyses. The cytotoxicity was evaluated by the MTT assay in non-tumor
retinal pigment epithelium cells. The Caiman oil composition was 42.95 ± 1.03 % of saturated fatty acids and
43.74 ± 0.74 % of unsaturated ones. After 10 days of excisional wounds, the Caiman oil-treated animals present
a larger scarred area than the negative control ones. Caiman oil and EFA treated animals present significantly
more epidermal papillae than in SS-treated ones on day 10. Caiman oil treated animals, at 10th day, present a
relative increased area of collagen fibers, as well as an elevated number of fibroblasts and monomorphonuclear
cells in the wound region in comparison to both SS and EFA treated ones. The oil showed no significant cyto-
toxicity up to 500 μg/mL. Taken together, Caiman oil, extracted as a by-product of the C. yacare zootechnical
disposal, demonstrated wound healing properties equal to the commercial available solution, subsidizing its
ethnoknowledge.

1. Introduction often cited to treat a variety of diseases [12–15]. The ethnoknowledge

The healing process of skin lesions is divided into three phases:
inflammatory, proliferative, and remodeling [1]. The whole process is
concomitantly influenced by several factors, with chronic wounds being
a costly health problem [2] that requires efficient clinical intervention
[3].
Currently, coverings with different actions and active principles can
be used, however, so far there is no availability of products with pro-
mising effects, in all phases of the healing process [4–6]. In this context,
there is a need for in vivo research to develop alternative, less costly,
and more effective products for the treatment of skin wounds [7,8].
The value of wildlife in therapeutic practice permeates the routine
of many cultures [9–11], including oils extracted from crocodilian fat,
data have been the prelude to bio-prospective studies for pharmacolo-
gical discoveries, communing with the economic and biological areas,
promoted by the consumer market [16–18], with promising results in
the healing of skin burns from Crocodylus siamensis oil [19,20].
The Caiman yacare is a resilient crocodilian species living in the
Pantanal, Brazil, with an established production chain in the states of
Mato Grosso [21] and Mato Grosso do Sul. The alligator viscera fat is
discarded after slaughter according to current regulations, and we
verified the possibility of using this discarded fat as a healing agent.
In this context, Pantanal alligator visceral fat oil was extracted and
analyzed, in vivo assays were performed to observe the healing potential
in excisional wounds in Wistar rats, and in vitro cytotoxicity of the oil.

⁎
Corresponding author at: Av. São João, UNEMAT, sn, curso de Ciências Biológicas, Cavalhada, Cáceres, MT, 78200-000, Brazil.
E-mail address: microtomo@unemat.br (L.N. Pressinotti).

https://doi.org/10.1016/j.wndm.2020.100195
Received 7 April 2020; Received in revised form 5 June 2020; Accepted 26 June 2020
Available online 30 June 2020
2213-9095/ © 2020 Elsevier GmbH. All rights reserved.
L.P. Azevedo, et al. Wound Medicine 31 (2020) 100195

2. Methods Table 1
Average fatty acid profile values of C. yacare oil, expressed in g/100 g (%) and
2.1. Experimental design standard deviation, measured in triplicates.
Fatty acid Nomenclature Average g/100 g
The healing experiment was controlled and randomized to measure
the action of oil extracted from C. yacare visceral fat on in vivo excision Saturated 42.95 ± 1.03
C 14:0 Myristic acid 2.29 ± 0.18
wounds in Wistar mice. Positive and negative controls were performed,
C 16:0 Palmitic acid 21.61 ± 0.57
respectively, with Dersani® (EFA, reference substance) and saline so- C 17:0 Heptadecanoic acid 0.95 ± 0.08
lution (SS). The experiment was conducted with the permission of the C 18:0 Stearic acid 18.50 ± 0.30
Chico Mendes Institute for Biodiversity Conservation (ICMBio), under Unsaturated 43.74 ± 0.74
Monounsaturated 34.65 ± 0.39
number 58681−1, it also followed the ethical principles on animal
C 16:1 Palmitoleic acid 2.33 ± 0.08
experimentation adopted by the National Council for Animal C 18:1n9c Oleic acid 32.31 ± 0.46
Experimentation Control (CONCEA), approved by the Animal Use C 18:1n9t Elaidic acid 0.26 ± 0.08
Ethics Committee of the Federal University of Mato Grosso (CEUA/ Polyunsaturated 9.09 ± 0.42
UFMT), under nº. 23108.110383/2015-15. C 18:2n6c Linoleic acid 5.94 ± 0.38
C 20:4n6 Arachidonic acid 3.15 ± 0.21

2.2. Collection and extraction of C. yacare

The collected visceral fat is a caudolateral encapsulated adipose
body to the right of the liver, henceforth referred to as corpus adiposum.
The viscera used belonged to alligators aged over two and a half years.
From a sample of 854.9 g of corpus adiposum, oil was extracted by the
Soxhlet method, refluxed for six continuous hours in hexane extractor
solvent at 65 °C for five continuous days, followed by concentration by
evaporation of solvent to 65 °C by rotary evaporator coupled to the
vacuum system [22–25].
2.3. Characterization of C. yacare oil
Chromatographic analysis of the oil for identification and quantifi-
cation of fatty acids was performed after its conversion to methyl esters.
Samples were analyzed by gas chromatography (GC) on SHIMADZU®
GC-14B equipment with flame ionization detector and HP-20 (car-
bowax 20 M), capillary column (25 m x 032 mm x 0,3 μm). The initial
column temperature was 40 °C, maintained for one minute, increased to
150 °C by 55 °C/min, and finished at 220 °C by 1.7 °C/min.
Temperatures were 200 °C in the injector and 220 °C in the detector.
Nitrogen was used as a carrier gas at a flow rate of 1,0 mL/min; split
injection (1:20) of 1.0 μL of the test sample. Fatty acid methyl ester
standards (Supelco, USA) were used to identify the methyl esters of the
sample, the quantification was done by the area normalization method,
and the results were expressed as an area percentage of each fatty acid
peak relative to its total area.
2.4. Animal management
For in vivo evaluation were used Rattus norvegicus Albinus (n = 48)
of Wistar strain, male, adults, and with body mass between 150 g and
180 g. The rats were individually housed in metallic metabolic cages at
room temperature between 22 and 24 °C, with drinking water and in-
dustrial feed (Labina®) ad libitum, submitted to a 12 -h light-dark cycle
and a 3-day acclimatization period.
The animals were randomly distributed in three groups of 16 rats,
regarding the treatments: saline solution 0,9% (SS), essential fatty acids
(EFA - Dersani®), and Caiman yacare oil (CyO). Subsequently, the ani-
mals from each group were randomly divided into two subgroups (8
animals), each subgroup being euthanized at the 3rd and 10th days
after the beginning of the experiment.
2.5. Experimental protocol
The rats were subjected to an 8 -h fasting diet. The pre-anesthetic
medication was administered with subcutaneous morphine 1 mg/kg,
anesthetic induction and maintenance with 100 % isoflurane, via a
mask, diluted in 100 % oxygen, 1 L/min in a universal vaporizer. After
anesthesia, each animal underwent trichotomy in the dorsal region,
where two circular lesions were made in the dorsal midline, one cranial
and one caudal until exposure of the muscular fascia.
Before topical application to the wounds, the solutions for each
treatment (CyO, SS and EFA) were heated (65 °C) in a water bath to
ensure equal temperature between treatments. Immediately after the
CyO fat liquefaction, 100 μL of each solution was gently poured over
the wound. The temperature decreases rapidly during the procedure, as
evidenced by thermal comfort after contact with the oil in the back of
the hand and the small solidification solvated in the wall of the mi-
cropipette tip. The first application occurred immediately after exci-
sion, covering the bottom and edges of the lesion, continuing the daily
topical treatment until euthanasia.
2.6. Macroscopic analysis
The lesions were photographed with a SONY NEX 5 N digital device
coupled to a SONY E lens 18−200 mm mount, f3.5–6.3, right after
surgery for up to day 10, using a millimeter ruler as a scale bar posi-
tioned on the same plane as the scars. After calibrating the software
based on the scale bar present in each figure, the area was measured
blindly using the polygon tool in image J freeware, with which the
wound was contoured.
Results for the Partial Area Reduction (PAR) were expressed con-
cerning the initial moment day 0, according to the formula:
PAR = [1 -(Area of injury on study day/Area of injury on day 0)]
× 100%
Thus, the PAR on day 0 is 0%, while the area healed at 100 % means
total scar closure.
2.7. Histological and immunohistochemical analysis
On 3rd and 10th days, the rats were euthanized with sodium thio-
pental (120 mg/kg) preceded by intraperitoneal lidocaine (10 mg/kg).
After death, each circular incision (cranial and caudal) was fixed in
methacarn (60 % methanol, 30 % chloroform, and 10 % glacial acetic
acid) for 24 h, subsequently processed and included in Paraplast®. The
5 μm cross-sections were Hematoxylin-Eosin (HE) stained for evaluation
of the overall wound structure. And to highlight the collagen fibers,
picrosirius staining was performed [26].
Photomicrographs were obtained on the same microscope set
(Nikon Eclipse 80i coupled with Nikon Ri1 camera), at the same light
intensity and for the same exposure time. Five photomicrographs of the
papillary layer of the dermis were captured in the 40x objective in the
animals on day 10, in the cranial lesions. Such requirements are ne-
cessary because the percentage of fiber area per field and intensity of
fiber staining were measured by freeware Image J 1.47c [27].
The counts were made with freeware Image J 1.47c [28–31].

2
L.P. Azevedo, et al.
Fig. 1. Assembly with scars in the kinetics of 0 h to 10 days for Saline Solution (SS), Essential Fatty Acids (EFA), and Caiman oil treatments.
Fig. 2. Averages and standard deviations of Partial Area Reduction of cranial
and caudal wounds of the different groups on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10. The negative control group (SS), reference substance (EFA), and Caiman oil.
* p < 0.05; ** p < 0.01.
Histological analysis was performed by the same professional,
without prior knowledge of the identification of treatment groups.
At the 3rd day, the area of edema was observed in the scar tissue,
classified according to the intensity and transformed into semi-
quantitative variables, grading the edema as discrete (+), moderate (+
+) or accentuated (+++).
At the 10th day, the number of papillae was quantified by 10x ob-
jective active search treatment by counting the number of epidermal
papillae in the region of scar tissue.
Counting of infiltrating cells in the regenerating dermis was done on
HE-stained histological sections, where five fields of the dermis were
photomicrographed at the locations with the highest infiltration in a
3
Wound Medicine 31 (2020) 100195
100x objective. Polymorphonuclear cells (PMNs) and monomorpho-
nuclear cells (MNs) were counted in the 3-day scars and, at the 10th
day, PMNs, MNs, and fibroblasts were counted.
Immunohistochemical labels were given by antigen recovery in
10 mM citrate buffer solution (pH 6,0) for 15 min under pressure. We
performed the washes of this protocol with TBS-Tween Buffer solution
(pH 7,4). After that, the peroxidase blocking steps with 6% hydrogen
peroxide in distilled water was performed for 30 min. Immunostainings
were performed with smooth muscle anti-actin antibody (monoclonal,
mouse, Sigma-Aldrich, 1A4, anti-α-SMA), diluted 1: 10,000, and in-
cubated for 1 h. Finally, SuperPicture™ 3rd Gen IHC (Life Technologies,
Carlsbad, CA, United States) polymer binding was performed for 30 min
and DAB precipitation for 5 min during the revelation. Negative control
was obtained by suppression of the primary antibody.
Immunostaining of the smooth muscle of the middle blood vessel
tunic with anti-α-SMA allowed the quantification of the sectioned area
of the blood vessels with the aid of freeware Image J 1.47c. Five fields
per scar on the papillary layer of the dermis were photomicrographed,
with a 40x objective, and each photo was marked by 2500 crosspieces,
where all the crosspieces arranged over the blood vessels were counted.
This procedure was performed exclusively on cranial injuries at 10th
day. Results were expressed as the sum of crosspieces on blood vessels
counted in the five photomicrographs of each rat, and the five images
contain a total of 12,500 crosspieces.
2.8. Caiman oil cytotoxicity assay
For the cytotoxicity assay, the non-tumoral retinal pigment epithe-
lium cell line RPE-1 was accessed. This cell line was maintained in
RPMI 1640 medium supplemented with 10 % fetal bovine serum (v/v),
2 mmol/L glutamine, 100 U/mL penicillin and 100 μg/mL strepto-
mycin. Cells were cultured at 37 °C under a 5% CO2 atmosphere.
To evaluate the cytotoxicity of the oil, RPE-1 cells were plated per
well in 96 well plates (5 × 104 cells/mL in 200 μL medium). A stock
solution of CyO diluted in dimethylsulfoxide (DMSO) at a concentration
of 10 mg/mL was previously prepared. After 24 h, the CyO/DMSO stock
solution was added to the medium containing cells at 5-fold dilution
from 0.005 to 0.50 μL/mL (equivalent to 5–500 μg/mL), each well
maintained 0.5 % DMSO. Each concentration was tested in duplicate
and incubated for 72 h. Negative control with DMSO and positive
control with doxorubicin (0.001–5.43 μg/mL) were performed. After
72 h of incubation, the supernatant was replaced by culture medium
L.P. Azevedo, et al. Wound Medicine 31 (2020) 100195

Fig. 3. An assembly containing three photomicrographs of the lesion site at 3 days of the SS (A), EFA (B), and Caiman oil (C) treatments of the cranial scars. HE 40x,
thickness: 5 μm.

Table 2
Number of cells at 3 and 10 days and semiquantitative classification of edema at 10 days of healing for cranial scars. Values are represented as Mean ± S.D.
Significant differences (p < 0.05) compared to other treatments were marked with * , and # indicates a significant difference in relation to EFA. MN:
Monomorphonuclear cells; PMN: Polymorphonuclear cells; SS: Saline Solution; EFA: essential fatty acids.
MN PMN Fibroblasts Edema

3 days SS 231.25 ± 51.87 14.38 ± 6.72 – +

EFA 221.63 ± 40.83 22.38* ± 9.38 – ++
Caiman oil 216.63 ± 43.73 16.38 ± 12.93 – +++
10 days SS 14.63 ± 9.30 6.63 ± 5.93 194.75 ± 44.48 –
EFA 12.33 ± 4.93 8.17 ± 8.93 190.33 ± 80.24 –
Caiman oil 21.38* ± 12.25 5.13# ± 5.54 226.13* ± 42.98 –

Fig. 4. An assembly containing three photomicrographs of the injury site at 10 days of the SS (A), EFA (B), and Caiman oil (C) treatments of the cranial scars, along
with the graph with the counts and significant differences between the treatments. *p < 0.05 and **p < 0.01.

4
L.P. Azevedo, et al.
Fig. 5. (A) Averages and standard deviations of relative areas covered by col-
lagen fibers (%), (B) intensity of red (0-255) in cranial scars and (C) the number
of blood vessel crossheads from cranial scars. Significant differences are in-
dicated by *p < 0,05 and **p < 0,01.
containing MTT (0.5 mg/mL). Three hours later, the supernatant was
removed and, after drying the plate, the MTT formazan blue-containing
precipitate was dissolved in 150 μL DMSO, and the absorbance was
measured at 570 nm [32]. Two independent experiments were per-
formed in duplicate.
2.9. Statistical analysis
To determine whether there was a significant difference in PAR
among treatments, we excluded the outlier values and compared the
averages by the One-Way ANOVA, and post hoc Fisher LSD test to
identify significant differences. Regarding the number of different cell
types in the papillary dermis, the number of crosses in the blood vessels,
and the number of epidermal papillae, the Poisson distribution
Generalized Linear Model (GLzM) test was used.
Regarding the area of collagen fibers and the intensity of staining of
collagen fibers, the ANOVA Repeated Measures and post hoc Tukey test
were used.
For the cytotoxicity assay, the values were calculated together with
the respective 95 % confidence intervals by nonlinear regression using
GraphPad Prism 8.0.
3. Results
3.1. Chromatographic analysis of C. yacare oil
Among the fatty acids sought by GC from C. yacare oil, nine fatty
acids were identified and quantified. Saturated fatty acids correspond to
42.95 ± 1.03 % of oil, and unsaturated fatty acids correspond to
5
Wound Medicine 31 (2020) 100195
43.74 ± 0.74 % of oil, of which 34.65 ± 0.39 % monounsaturated
and 9.09 ± 0.42 % polyunsaturated (Table 1).
3.2. Macroscopic analysis
The cranial and caudal scars were distinguished as to their shape
from 7th day, the cranial scars close centrally, while the caudal scars
closed predominantly lateral-laterally, slit-like (Fig. 1).
In cranial scars, we found that the PAR in EFA was significantly
smaller than in SS at 1st, 2nd, 3rd, 4th, 5th, and 6th days, while the
scarred area was significantly larger than in SS on day 10. In Caiman
oil, the PAR was significantly smaller than in SS on 2nd, 3rd, 4th, 5th,
and 6th days, while the PAR was significantly larger than in SS at 10th
day. There were no significant differences found between EFA and
Caiman oil (Fig. 2A).
There was no significant difference between EFA and SS in caudal
scars. Caiman oil PAR were significantly smaller than SS at 5th and 6th
days. No significant differences were found between EFA and Caiman
oil (Fig. 2B).
3.3. Histological/structural analysis
In cranial scars, at 3rd day, its most superficial portion consists of
erythrocytic clot and leukocytes, underlying the dermis containing
granulation tissue, characterized by the presence of vessels with active
endothelium, and inflammatory cells near the clot in EFA and SS
treatments. In Caiman oil treatment, often the inflammatory infiltrate is
distanced from the clot by the presence of edema and hemorrhagic foci,
with sparse leukocytes in these regions (Fig. 3). The edema area was
characterized by the accumulation of interstitial fluid permeated by
amorphous forms, being larger in Caiman oil and EFA compared to SS
(Table 2). We identified adipocytes immersed in the stroma with leu-
kocyte infiltration in the hypodermis region.
In cranial scars, at the 10th day, in lesions with complete closure,
the basal, spiny, granular, and corneal layers of the epidermis can be
identified. The dermis has fibroblasts, inflammatory infiltrate, and
blood vessels. EFA and Caiman oil were found to have significantly
more epidermal papillae than SS, and EFA had significantly more pa-
pillae than Caiman oil (Fig. 4). Incomplete closing scars showed clot on
the dermis.
3.4. Cell count
In cranial scars, at 3rd days, the PMNs count was significantly
higher in EFA compared to SS and Caiman oil. At 10th day, the MNs and
Fibroblasts count was significantly higher in Caiman oil compared to SS
and EFA. PMN count was significantly higher in EFA compared to
Caiman oil (Table 2).
4. Collagen fibers
In cranial scars, at 10th day, a relative area of collagen fibers stained
in red by picrosirius was significantly higher in Caiman oil than in SS
and EFA. The red intensity of picrosirius staining was significantly
lower (darker reds) in Caiman oil compared to EFA and SS (Fig. 5A).
In the cranial scars, it was verified that the number of crosses on the
blood vessels marked by anti-α-SMA differ significantly in the three
treatments tested, being EFA the lowest value and Caiman oil the
highest (Fig. 5C).
5. Cytotoxicity test
C. yacare visceral corpus adiposum oil presented no significant cy-
totoxicity against RPE-1 cells, with growth inhibitory values below 50
% even at the highest tested concentration (500 μg/mL).
L.P. Azevedo, et al. Wound Medicine 31 (2020) 100195

Table 3
Lipid profile of oils extracted from crocodilian fat.
Authors Li et al., 2012 Buthelezi et al., 2012 Ayalasomayajula, 2012 Vicente-Neto et al., 2010
Fatty acids Caiman yacare Crocodylus siamensis Crocodylus niloticus Alligator mississipiensis Caiman yacare

Total Fat 86.95 – – –

Saturated Fat 42.95 38.89 – – 35.12
Monounsaturated Fat 34.65 36.86 – – 37.57
Polyunsaturated Fat 9.09 12.81 – – 27.33
Trans Fat 0.26 – – – –
Unsaturated Fat 43.74 49.67 – – 64.09
Palmitic Acid C16:0 21.61 29.22 15.43 23.3 22.00
Heptadecanoic Acid C17:0 0.95 – 0.47 – –
Stearic Acid C18:0 18.50 1.84 1.35 5.3 11.62
Palmitoleic Acid C16:1 2.33 5.67 3.13 12.11 4.22
Oleic Acid C18:1n9c 32.31 30.47 19.59 55.54 25.86
Linoleic Acid C18:2n6c 5.94 11.74 4.03 – 12.74
Arachidonic Acid C20:4n6 3.15 – – 0.64 8.76
Elaidic Acid C18:1n9t 0.26 – 0.2 – –
Myristic Acid C14:0 2.29 3.23 1.15 12.7 1.50
Lauric Acid C12:0 – 4.6 – – –
Linolenic Acid C18:3 – 1.07 – – 0.75
Erucic Acid C22:1 – 0.42 < 0.001 – –
Nervic Acid C24:1 – 0.31 – – –
Undecanoic Acid C11:0 – – – – –
Tridecanoic Acid C13:0 – – 0.01 – –
Pentadecanoic Acid C15:0 – – 0.25 – –
Linolelaidic Acid C18:2 – – 0.16 – –
Eicosanoid Acid C20:1 – – 0.05 1.56 2.95
Araquic Acid C20:0 – – 0.002 – –
Lignoceric Acid C24:0 – – 1.33 – –
Eicotrienoic acid C20:3 – – – 0.64 –
Eicosanoid acid C18:1n11c – – – 4.55 –
Adrenic Acid C22:4n6 – – – – 1.80
α-linolenic acid C18:3n-3 – – – – 1.80
Eicosapentaenoic acid C20:5n-3 – – – – 0.78
Docosahexaenoic acid C22:6n-3 – – – – 0.72

6. Discussion
Reptiles exhibit varying lipid storage patterns, with the main form
of storage being visceral fat bodies [33], whose triglyceride composi-
tion can be directly influenced by diet [34–37]. C. yacare is tolerant to
the addition of vegetable oils in the diet [38] and may interfere with the
fatty acid profile of oil sources, suggesting possible modulation of the
lipid profile for compounds of higher interest.
Oils extracted from crocodilian fat are often cited by popular
knowledge [9,39–44] for the treatment of skin diseases whose main-
tenance over generations signals their effectiveness and may lower the
cost of drug development as well as increase the chances of success
[45,46].
C. yacare (oleic, palmitic, and stearic acid) resembles the oils de-
scribed for other different species of crocodilians. However, there is an
intraspecific variation in the proportion of these oils (Table 3).
Regarding healing, there was a different behavior between the
cranial and caudal scars, the latter being less representative in the
healing process concerning the cranial scars. The different behavior of
cranial and caudal healing can be interpreted considering the diver-
gence of physiological characteristics of the skin in different anatomical
regions and may differ in the vascularization, skin thickness, and bio-
mechanics of the adjacent musculature [47,48]. This argument corro-
borates with the circular shape of the cranial scar closure while the
caudal scar closes in the slit. In this sense, we decided to analyze mi-
croscopically only the head injuries, enabling the proper comparison
between the treatments.
In cranial scars, the smaller area of scar coverage in the EFA and
Caiman oil treatments, with subsequent inversion to a larger area at 10
days, is in agreement with the findings for the effects of topical use of
oleic acid on scars in Wistar rat [49].
Among the possibilities that explain the largest edema observed in
Caiman oil, at 3rd day, it is worth mentioning the composition with
42.95 % of saturated fatty acids in Caiman oil, which solidifies at room
temperature, forming a hydrophobic protective film over the scar,
which makes water evaporation difficult, a function similar to hydra-
tion by decreasing dermal evaporation through the trans-epidermal
barrier of an integral skin [50]. A similar result was found in scars
treated with animal oil, which presented higher edema compared to
those treated with honey [51], and moist crust in animals treated with
capybara oil [31].
Another assumption possible is that the oils have a higher viscosity
than saline [52,53] so that the oils tend to stay over the scar longer
radiating more heat to the skin, thus contributing to local vasodilation
and, possibly interfering with the inflammatory phase of wound healing
differently to saline [54]. These effects can be related to the perpe-
tuation of the edema. These explanations are not self-exclusive and may
have interfered concurrently. The prolongation of the inflammatory
phase was observed in rats treated with Emu oil, characterized by the
perpetuation of edema and delayed scar closure [55], similar to our
results. Both edema and perpetuation of inflammation contribute to the
maintenance of inflammatory mediators in the focus of injury [1,56].
Capybara, C. siamensis and Emu oils [31,55,57] obtained a higher
density of collagen fibers in the dermis when compared to their con-
trols, in agreement with our results for Caiman oil. The higher fiber
density may be related to the higher frequency of fibroblasts in Caiman
oil, a protagonist cell type in the deposition of collagen fibers [58].
Fibroblasts predominate in Caiman oil treatment. However, EFA is
predominantly PMNs. Probably, different mechanisms of action were
triggered by the use of these oils, since the compositions of EFA and
Caiman oil are different [59,60]. The prolongation of the inflammatory
phase and concomitant maintenance of cytokines for a longer time may
have contributed to increased fibroblast recruitment and vasculariza-
tion in Caiman oil [61,62]. However, the prolongation of the in-
flammatory phase in EFA had a higher number of PMNs, less vascu-
larization, and more discrete edema, so further studies should focus on

6
L.P. Azevedo, et al.
understanding the mechanisms of action of these two oils.
The larger number of epidermal papillae observed in the EFA and
Caiman oil treatments suggests an advanced stage of tissue reconstitu-
tion due to the higher dermal-epidermal junction, providing higher
adhesion and dynamic interaction for skin integrity [63].
Cytotoxic products are those products with IC50 less than 30 μg/mL
for plant derivatives and IC50 less than or equal to 4 μg/mL for pure
substances [64]. Our result showed IC50 higher than 500 μg/mL, even
increasing the permeability through surfactation of the oil in 5% DMSO.
Thus, we can infer that there is evidence of pharmacological safety
regarding the use of this compound topically.
7. Limitations
We studied the therapeutic effect of caiman oil under a morpholo-
gical analysis of wound closure. The molecular mechanisms of tissue
repair remain uncertain and need further investigation. Our study was
conducted in an in vivo animal model, requiring clinical tests to assess
the healing potential in humans. Finally, additional studies using
chronic wound models treated with semi-thin creams or soluble nano-
particles-based formulations should be performed in order to exclude
the adverse effects of oil temperature during the experimental proce-
dure.
8. Conclusions
Caiman oil is extracted from a by-product of the C. yacare zoo-
technical disposal and has demonstrated aptitude as a healer, as pre-
dicted by specific ethnoknowledge. Lesions treated with Caiman oil
have similar progression to EFA. It should be noted that EFA is a shelf
product widely used in healthcare routines [65]. Given the findings, we
conclude that Caiman oil stimulated skin healing and that there is a
need to continue studies to verify the mechanisms of action that sti-
mulated healing.
Funding sources
FAPEMATnº 0229029/2017 e 160048/2014.
Declaration of Competing Interest
The authors declares that there is no conflict of interest.
Acknowledgments
Lucas Azevedo was a FAPEMAT fellowship grant.
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