Changes in the volume and histology of endometriosis foci in rats treated with copaiba oil (Copaiferalangsdorffii)

Abstract

The aim of this study was to analyze the changes that occur in rats with experimental endometriosis after treatment with copaiba oil.
Experimental endometriosis was induced in rats. The experimental group received copaiba oil (Copaiferalangsdorffii) orally (0.63 mg/day), and the control group received a 0.9% sodium chloride solution orally (1 ml/100 g of body weight/day). Both groups were treated with gavage for 14 days. After this period, the animals were euthanized, and the implant volume was calculated. The autologous transplants were removed, dyed with hematoxylin-eosin, and analyzed by light microscopy.
The average final volumes were significantly different between the groups (p=0.007). There was a significant increase (p=0.012) between the initial and final volumes in the control group, whereas treatment with Copaiferalangsdorffii caused a marked reduction in endometrial growth over time (p=0.016). Histologically, 6/11 (55.00%) rats in the experimental group had a well-preserved epithelial layer, and 3 (45.00%) had mildly preserved epithelium. The control group had seven cases (58.30%) of well-preserved epithelial cells and five cases (41.70%) of mildly preserved epithelial cells (p>0.05).
Copaiba oil (Copaiferalangsdorffii) appears to be a promising alternative treatment for endometriosis.

Full text

20 - Acta Cirúrgica Brasileira - Vol. 26 (Suppl. 2) 2011
5 – ORIGINAL ARTICLE
MODELS, BIOLOGICAL
Changes in the volume and histology of endometriosis foci in rats treated with copaiba
oil (Copaiferalangsdorffii)1
Mudanças no volume e histologia do foco de endometriose em ratas tratadas com óleo de
Copaíba (Copaiferalangsdorffii)
João Nogueira NetoI, Márcio Jorge de Souza LindosoII, Laura Fernandes CoelhoII, Rafael Antonio Freire CarvalhoII,
Taciana Gabrielle Pinheiro de Moura RodriguesII, Ana Gisélia Portela de AraújoIII, Manuel João Batista Castelo GirãoIV,
Eduardo SchorV
IMD, Fellow PhD degree, Federal University of Sao Paulo (UNIFESP), Unit of Endometriosis of HU-UFMA, Brazil. Design the protocol, involved
with technical procedures, responsible for manuscript writing, responsible for intellectual and scientific content of the study, supervised all phases of
the study, and provided guidelines for the surgical interventions.
IIGraduate student, UFMA, Maranhao, Brazil. Involved with technical procedures, responsible for manuscript preparation.
IIIMD, HUUFMA, Maranhao, Brazil. Macroscopic and histopathological examinations.
IVPhD, Department of Ginecology, Paulista School of Medicine, Federal University of Sao Paulo (EPM-UNIFESP), Brazil. Responsible for intellectual
and scientific content of the study, critical revision, provide guidelines for the surgical interventions.
VPhD, Affiliate Professor, UNIFESP, Head of Unit of Pelvic Pain and Endometriosis, EPM-UNIFESP, Sao Paulo, Brazil. Responsible for acquisition
and interpretation of data, statistical analysis, helped with technical procedures, collection and processing of study informations, provide guidelines
for the surgical interventions
ABSTRACT
PURPOSE: The aim of this study was to analyze the changes that occur in rats with experimental endometriosis after treatment
with copaiba oil.
METHODS: Experimental endometriosis was induced in rats. The experimental group received copaiba oil (Copaiferalangsdorffii)
orally (0.63 mg/day), and the control group received a 0.9% sodium chloride solution orally (1 ml/100 g of body weight/day). Both
groups were treated with gavage for 14 days. After this period, the animals were euthanized, and the implant volume was calculated.
The autologous transplants were removed, dyed with hematoxylin-eosin, and analyzed by light microscopy.
RESULTS: The average final volumes were significantly different between the groups (p=0.007). There was a significant increase
(p=0.012) between the initial and final volumes in the control group, whereas treatment with Copaiferalangsdorffii caused a
marked reduction in endometrial growth over time (p=0.016). Histologically, 6/11 (55.00%) rats in the experimental group had a
well-preserved epithelial layer, and 3 (45.00%) had mildly preserved epithelium. The control group had seven cases (58.30%) of
well-preserved epithelial cells and five cases (41.70%) of mildly preserved epithelial cells (p>0.05).
CONCLUSION: Copaiba oil (Copaiferalangsdorffii) appears to be a promising alternative treatment for endometriosis.
Key words: Phytotherapy. Endometriosis. Drug Therapy. Rats.
RESUMO
OBJETIVO: O objetivo deste estudo foi analisar as mudanças que ocorreram em ratas com endometrioses experimental tratadas
com óleo de copaíba.
MÉTODOS: Foi induzida a endometriose experimental nas ratas. O grupo experimental recebeu óleo de copaíba
(Copaiferalangsdorffii) oralmente (0,63mg/dia) e o grupo controle recebeu oralmente solução salina 0,9% (1mL/100g/dia). Ambos
grupos foram tratados por gavagem por 14 dias. Depois desse período, foi realizada a eutanásia dos animais e calculado o volume
do implante. Os transplantes autólogos foram removidos, corados com Hematoxilina-eosina e realizada a microscopia óptica.
RESULTADOS: A média final dos volumes foi significativamente diferente entre os grupos (p=0,007). Houve um aumente
significante (p=0,12) entre o volume inicial e final do grupo controle, enquanto no grupo tratado com Copaiferalangsdorffii causou
uma redução acentuada no crescimento endometrial ao longo durante o período (p=0,016). Histologicamente,6 das 11 (55%) ratas
do grupo experimental tinha uma camada epitelial bem preservada e 3 (45%) apresentaram epitélio levemente preservado. O grupo
controle apresentou sete casos (58,3%) de células epiteliais bem preservadas e cinco casos (41,7%) de células epiteliais levemente
preservadas (p>0,05).
CONCLUSÃO: O óleo de copaíba (Copaiferalangsdorffii) parece ser um tratamento alternativo promissor para endometriose.
Descritores: Fitoterapia. Endometriose. Quimioterapia. Ratos.

Changes in the volume and histology of endometriosis foci in rats treated with copaiba oil (Copaiferalangsdorffii)
Acta Cirúrgica Brasileira - Vol. 26 (Suppl. 2) 2011 - 21
Introduction
Endometriosis is a condition that affects 10% of women,
and often causing pelvic pain and infertility. Diagnosis is
sometimes difficult and the optimal treatment is yet to come. Some
events of the pathophysiology of endometriosis are well known,
such as inflammation, immunomodulatory disorders, increased
local and systemic oxidative stress, among others. The search for
new treatments for endometriosis is justified when interfering in
the events mentioned above1.
The copaibas are trees in the Leguminosae-
Caesalpinoideae family, reaching 25-40 feet tall and can live upto
400 years. Copaiba oil, in biological terms, is a product of excretion
or detoxification of the plant organism, and acts as a plant defence
against animals, fungi and bacteria2. The attributed effects to
Copaiba oils in folk medicine are anti-inflammatory, anti-tumour,
anti-tetanus, anti-blenorrhagea, as a urinary anti-septic, to treat
bronchitis, syphilis, skin diseases, ulcers, as well as for healing
wounds3.
Some properties of Copaiba oil are well documented in
the scientific world, which could justify its use in the treatment of
endometriosis. Its anti-inflammatory effect had been observed in
the research, which used kaurenoico acid, extracted from
Coapiferalangsdorffii, and used in Wistar rats with induced colitis.
Furthermore, light microscopy revealed the marked reduction of
inflammatory cell infiltration and submucosal oedema formation
in the colon segments of rats treated with the test compound4. The
anti-inflammatory activity was confirmed in a comparative study
between oils from Copaiferacearensis, Copaifera reticulate and
Copaiferamultijuga in vitro and in vivo5. The study indicated that
Copaiba oils demonstrate peripheral and central antinociceptive
effect and may be useful in the treatment of algesic disorders6.
Another study examined the effects of
Copaiferalangsdorffii resin on intestinal damage associated with
mesenteric ischemia/reperfusion (I/R) in rats. Caused significant
attenuations in I/R – associated increases of myeloperoxidase,
malondialdehyde, catalytic activities and nitrite level.
Copaiferalangsdorffii could effectively prevent the I/R-associated
depletion of glutathione. The data indicate that the copaiba
provided protection against I/R-induced intestinal tissue damage,
which appeared to be, at least in part, due to an antioxidant and
anti-lipid peroxidation mechanism7.
Antimicrobial activity was revealed through disruption
and damage to the cell wall, resulting in the release of cytoplasmic
compounds, alterations in morphology, and a decrease in cell
volume, indicating that copaiba oil may affect the cell wall. Another
effect that suggests anti-cell-proliferation was shown in a study
that showed significant activity against the parasite Leishmania
amazonensis8.
The development of experimental endometriosis in female
rats through auto-transplantation techniques prompted research on
additional medical treatments for endometriosis. These treatments
were analyzed for their efficacy via macroscopic and histological
parameters, among others9,10.
Because of the known properties of Copaiferalangsdorffii
and its relationship to mechanisms involved in the etiopathogeny
of endometriosis, the present study aimed to analyze its effects on
experimental endometriosis implants in rats.
Methods
The study was carried out between March and Julyof
2010, using 40 60-day-old (adult) female Wistar rats (Rattus
norvegicus albinus), weighing 180-250g. The rats were obtained
from the bioterium of the Federal University of Maranhao
(UFMA).
The study was developed in the Experimental Surgery
Laboratory of the University Hospital of UFMA, Brazil. Study
procedures followed the regulations of the Brazilian Legislation
for the use of experimental animals (Arouca Act n° 11.794 /2008)
and of the Brazilian Animal Experimentation College (COBEA),
an institution affiliated with the International Council for
Laboratory Animal Science. The study was approved by the Animal
Experimentation Ethics Committee (CEEA-UEMA) under
protocol number 035/2010.
The animals were grouped four per polypropylene cage
(46 cm x 31 cm x 16 cm) with a stainless steel grid lid and a paper
covered floor that was replaced every 48 hours. The animals were
divided into four groups and maintained under constant
environmental conditions. These including rat rations (PURINA®,
São Paulo, Brazil) and water ad libitum for seven days for
adaptation; noise control; 22°C ± 2°C temperature; 40% to 60%
relative humidity; and 12/12 hour light/dark cycles.
The autotransplantation technique was performed
according to the methodology proposed by Nogueira et al.1. Shortly
after the midway incision, the uterine horns were identified;
fragments of the medium third were resected, immersed in saline
solution and cut into 4mm by 4mm fragments. The fragments were
sutured to the mesentery adjacent to the artery that irrigates the
cecum, with the serosa faces turned to the peritoneum and the
endometrial layer turned to the cavity (Figure 1).
After the first surgery, the animals were kept in the lab
for a period of 21 days. Following this period, the rats underwent
an additional operation. An inventory of the peritoneal cavity was
taken using a digital pachymeter to identify the success of the
autotransplantation, followed by a volume calculation using the
following formula: [4
ʌ
(lenght/2)x(width/2)x(hight/2)/3]11.
Classification of the experimental endometriosis implant growth
was performed9, and only those animals that progressed to a growth
score of III remained in the study (Figure 2).
FIGURE 1 - Photomicrography showing construction of
autotransplant according to Nogueira et al.1

22 - Acta Cirúrgica Brasileira - Vol. 26 (Suppl. 2) 2011
Nogueira Neto J et al.
FIGURE 2 - Photomicrography showing an autotransplant
with a diameter longer than 4.5 mm, classified as grade III
according to Quereda et al.9
After the surgical approach, the rats were identified and
randomly divided into the Copaiferalangsdorffii (COP group) and
the control group (C group), both containing 12 rats.
The COP group gavaged oil (Óleo de Copaiba® All
Chemistry, lot 34883, São Paulo, Brazil) at 0.63mg/day for 14
days was carried out for Brito et al. in 200012. The C group received
1 ml daily gavage of 0.9% saline solution for 14 days.
After the end of the medical treatments, the rats were
anesthetised using ketamine, and a third laparotomy was
performed.
After opening the abdominal wall, an inventory of the
peritoneal cavity and measurements of autotransplant volumes
were performed; the transplant and the middle third of the uterine
horn remaining were then removed. The salvaged tissue was rinsed
with 0.9% saline solution and stored in 10% formaldehyde buffer
for later anatomopathological analysis.
Paraffin tissues were sectioned in 5µm widths and placed
in a warm bath; the slides were incubated with Meyer albumin
and dried afterwards. Tissue sections were stained with
hematoxylin-eosin (HE), and a histological analysis was performed
by a pathologist. The oestrous cycles were analyzed in the middle
third of the uterine horn remaining and drug efficacy was
evaluated10. The persistence of epithelial cells in uterine autografts
was evaluated as follows: a well-preserved epithelial layer = score
3, a moderately-preserved epithelium with leukocyte infiltrate = score
2, a poorly-preserved epithelium (occasional epithelial cell
only) = score 1, and no epithelium = score 0 (Figure 3).
FIGURE 3 - Photomicrography of moderately-preserved
epithelium, with mildly-preserved (40X) cytoplasm from the group
of rats treated with 0,63 mg/day Copaiferalangsdorffii, administered
for a 14-day period.
Biostat 3.0 Windows XP was used for statistical analysis,
where the significance level (
Į
) used to reject the null hypothesis
was 5% (p<0.05). The Mann-Whitney test was used for
independent samplings, and the Wicoxon test was performed for
related samples. Fisher’s exact test was used to analyze the
histopathologic score.
Results
Initially there were 12 samples in each group. Due to
technical problems, one sample of the experimental group was
lost. This left 11 samples in the experimental group. All of the
middle third of the uterine horn remaining was in either the
proestrus or oestrus phase of oestrous cycle.
There was no significant difference between the mean
volume (38.09 mm3) in the C group and (47.00mm3) that in the
COP group at 21 days following the induction of endometriosis
(p=0.470). Two weeks after administering copaiba oil and saline
solution, the final average volumes were 65.40mm3 for the C group
and 23.38mm3 for the COP group (p=0.007).
Average volumes for the initial control group and the
saline-treated group two weeks later were 38.09mm3 and
65.70mm3, respectively. This increase in average volume was
significantly different (p=0.012). For the COP group, the initial
average volume was 47.00mm3, in contrast to the 23.38mm3
final average, and these measurements were also significant
(p=0.016) (Table 1).

Changes in the volume and histology of endometriosis foci in rats treated with copaiba oil (Copaiferalangsdorffii)
Acta Cirúrgica Brasileira - Vol. 26 (Suppl. 2) 2011 - 23
TABLE 1- Characteristics and results for a group of rats treated orally for 14 days with
Copaiba oil (0,63 mg/day) or saline solution (1 ml/100 g body weight).
Regarding preservation of the epithelial layer, upon
histological evaluation of the autotransplant focus the experimental
group (n=11) contained six cases (55.00%) with a well-preserved
epithelial layers (Figure 2), and five cases (45.00%) with
mildly-preserved epitheliums. The control group (n=12) included
seven cases (58.30%) with well-preserved epithelial cells and five
cases (41.70%) of mildly-preserved epithelial cells. No significant
differences were presented (p>0.05).
Discussion
The treatment of endometriosis is either clinical or surgical
or both. The drugs commonly used interfere with the menstrual
cycle, and after the drug taking is suspended, there is a
reoccurrence of the pathology. Although the surgeries can be
simple, they are often mutilating. It is important to do the
experimental tests with therapeutic options that go against what
has happened in pathogenesis of endometriosis, mainly in the
interference of the inflammatory process and oxidative stress1.
The copaiba oil is extracted from trees of the species
Copaifera, popularly known as “copaiba”, “copaíva” or “pau-de-
óleo”. It is known to have the following medicinal qualities: anti-
inflammatory; anti-tumoral against Walker sarcoma and melanoma
cell line; anti-ulcerogenic, antioxidant and anti-lipoperoxidative;
cercaricide and anti- helmintic; insect repellent and antimicrobial.
The cosmetic industry uses copaiba oil in shampoos, capillary
lotion and bathing foams6.
The copaiba oil used in this research was extracted from
the species Copaiferalangsdorffii, which has scientifically
confirmed anti-inflammatory and antioxidant effects4,7, justifying
its experimental use in endometriosis.
The doses used in this research were based on studies
that used the copaiba oil in different doses (0.63 ml/kg and 0.06
ml/kg during 14 days), followed by microscopic hepatic evaluation
of the rats, in which no hepatocelular alteration was found. The
volume used in the pilot plan was 1.26 ml/kg during 14 days, where
on the 5th day 90 % of the samples died. The volume was fractioned
to 0.98 ml/kg ((3/4 de 1.26 ml/kg), 0.63 ml/kg (1/2 of 1.26 ml/kg)
and 0.31 ml//kg ¼ de 1, 26 ml/kg). This way, the doses of 0.63
ml/kg was standardized, representing the highest volume from
which there were no mortalities in the period of 14 days, and
considered sufficient to realize the major part of the experiment
that used copaiba oil12. Our research reassured the security of the
Group Average Initial Volume Average Final Volume p*
Oil copaiba 47.00 mm3 23.38 mm3 0.016
Control
p**
38.09 mm³
0.470
65.40 mm³
0.007
0.012
p* The Wilcoxon Test for samples (with p<0.05 for rejecting the null hypothesis) comparing
averages of the initial and final volumes between the groups.
p** Mann-Whitney test for independent samples (with p<0.05 for rejecting the null
hypothesis) comparing averages of the initial and final volumes between the oil copaiba
and the control treated groups.
mentioned doses as there were no mortalities in our study.
Statins represent a class of drugs that can effectively
decrease the serum level of cholesterol and is largely used in the
treatment of hypercholesterolemia. Experimental studies show
that these drugs effectively decreased the inflammatory processes
and are anti-oxidant, and so were used in endometriosis
experiments with similar methodology employed in this research.
The sinvastatin used in the dose of 20 mg/kg/day during 14 days
showed a significant regression of the focus of autotransplants
when comparing the final volumes between the experimental and
the control group1. Studies that used atorvastatin in high doses
showed similar results13; results that are compatible with our
research on copaiba oil.
Melatonin is the product of the secretion of the pineal
gland whose main function is regulating sleep, but also has
anti-inflammatory, antioxidant and immunomodulator properties.
It was used in an experimental endometriosis rat model treated
with melatonin (10 mg/kg) intraperitoneally, with the evaluation
of the morphology of the autotransplants, the expression of
COX-2 and antioxidants enzymes. After four weeks of treatment
it showed significant reduction of the autotransplant volumes,
COX-2 and antioxidant activities in the experimental group14. The
morphological evaluation with the significant reduction in volume
agreed with our results.
Strong evidence of the anti-inflammatory effect in the
reduction of the volume of the focus of the autotransplants in rat
model endometriosis is proven in studies that use dexametason
(0.8 mg/kg/day) for 13 days in Wistar rats. The dexametason
significantly reduced inflammatory processes, the amount of
collagen in the stroma, and the area occupied by the glands of the
autotransplants15.
The histological evaluation didn’t show compatible
alterations with what was seen macroscopically without significant
alterations. The use of melatonin showed significant histological
changes in the experimental group when compared to the control
group, but the author himself criticizes this type of evaluation
because it is semi quantitative14.
The similar volume reduction of the copaiba oil when
compared to anti-inflammatory, anti-oxidants and even
imunommodulators confirmed the theoretical suspicion of the
possible action against the development of focus of experimental
endometriosis and opens one more possible line of research for
the treatment of endometriosis.

24 - Acta Cirúrgica Brasileira - Vol. 26 (Suppl. 2) 2011
Nogueira Neto J et al.
Conclusion
The copaiba oil, at least of the species
Copaiferalangsdorfii, seems to be a promising therapeutic option
in the clinical treatment of endometriosis.
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Correspondence:
João Nogueira Neto
Av. dos Holandeses S/N Qd 24 Lote 05/901
65071-380 São Luís – MA Brasil
j.nogueira.n@uol.com.br
Conflict of interest: none
Financial source: none
1Research performed at Experimental Surgery Laboratory, University Hospital, Federal University of Maranhao (LACEMA-HUUFMA), Brazil.
Presented at the XII National Congress on Experimental Surgery of the Brazilian Society for Development of Research in Surgery-SOBRADPEC,
2011 October 26-29 Ribeirao Preto-SP, Brazil.