Copaiba oil effect under different pathways in mice subjected to sepsis 1

PURPOSE: To evaluate the effects of copaiba oil administered by different routes on survival of mices subjected to cecal ligation and puncture. METHODS: Thirty two mice were distributed into four study groups (N=8): Sham group: normal standard animals; Control group: submitted a cecal ligation and puncture (CLP); Gavage group: submitted a CLP, and treat with copaiba oil by gavage; and Subcutaneous group: submitted a CLP, and treat with copaiba oil by subcutaneous injection. After the death of the histological analysis were performed. The Kaplan-Meier curves of surviving time were realized. RESULTS: All animals that received copaiba, regardless of the route used, survived longer when compared to the control group (p<0.0001), whereas the survival time ranged from 20 hours for the control group up to 32 hours for the animals of gavage group and 52 for subcutaneous group. The animals that received gavage copaiba lived about and about 20 hours unless the subcutaneous group (p=0.0042). There was no statistical difference when compared the intensity of inflammatory response (p>0.05) CONCLUSION: Prophylactic subcutaneous administration of copaiba in mice subjected to severe sepsis by cecal ligation and puncture, resulted in a survival time higher than non-use or use of this oil by gavage.


Introduction
Sepsis is the leading cause of death in intensive care units (ICU) with a mortality rateranging from 20 to 80%, being diagnosed 18 million new cases of severe sepsis per year worldwide [1][2][3][4] .
The incidence of severe sepsis increased 91.3% over the past 10 years occurring in 1 to 3 per 1000 individuals in different regions, with annual growth of 1% per year 1,5 , representing still a severe public health problem in the world, both from the point of view of social and economic terms 6 . Brazil is appointed, along with the United Kingdon, as one of the countries with the highest rate of occurrence of sepsis in ICU, responsible for occupying about one quarter of the ICU beds 3 .
The treatment of sepsis represents a challenge for medicine 7 , because it still remains an entity of difficult clinical management.
Possible interventions in the inflammatory response and coagulation with the goal of reducing morbidity and mortality, and improve the prognosis of sepsis, has been extensively investigated 8 .
The use of plants for medicinal purposes, for treatment, prevention and cure of diseases, is one of the oldest forms of medical practice of mankind. In the early 1990s, the World Health Organization reported that 65-80% of the population in developing countries depended on medicinal plants as the only form of access to basic health care 9 , and in relation to world population, about 60% makes use of medicinal plants 10 .
In Brazil, many species are still used empirically, without scientific support for efficacy and safety, which shows that, in a country with enormous biodiversity, there is a huge gap between the supply of plants and little research 11 .
One of the main medicinal plants used in the Amazon region, and the subject of many scientific studies is the copaiba oils, whose effects were tested and found effective in various activities, such as anti-inflammatory and antimicrobial 12 , especially against gram-positive bacteria 13 .
It is, therefore, a potential source of new agents against major infectious diseases 13 having already reports in the literature about its benefits in sepsis induced in mice 14 . Still, the oil released by copaiba tree resin is low cost and easy access 15 present in pharmacies throughout Brazilin various presentations 16 . Thus, the aim of this study is to evaluate the effects of copaiba oil administered by different routes on survival of mices subjected to cecal ligation and puncture. After opening the abdominal cavity, the cecum was located, exposed and isolated, leaving the rest of the small and large intestine into the peritoneal cavity, taking care not to violate or damage the mesenteriais' vessels. The cecal ligation and puncture as realized by a 75% cecum ligated with silk 4-0 immediately after the ileocecal valve, to induce a high-grade sepis. After this, the cecal stump was transfixed by a single through-and-through puncture with a 21 G needle. After the surgical procedure, was administered pre-heated saline (5 ml per100 g) by subcutaneously.

Research approved by the Ethics
The gavage and subcutaneous group was tread by copaiba oil (Copaifera officinalis) 0,63ml/Kg once, for five days before the cecal ligation and puncture, differentiating according to the administration route. Kruskall-Wallis and Fisher's exact test was used to compare the histopathological results. Survival curves of groups were plotted using the Kaplan-Meier method and then compared by the log-rank test. Was adopted a significance level of 5% to reject the null hypothesis.

Results
No animals died in the Sham group, presenting good surgical recovery, and euthanized 30 days after the death of the animals in the other groups.
All animals that received copaiba, regardless of the route used, survived longer when compared to the control group

Discussion
The present study demonstrated that there is significant difference in the organic response to severe sepsis when using different routes for the administration of copaiba oil. This maybe related to the longer time required for drug absorption according to the specific route, thus delaying the on set of it, which is a critical factor to the prognosis and survival of animals with sepsis.
Furthermore, the bioavailability of active molecules with antimicrobial and anti-inflammatory present in copaiba oil may be changed due to the mechanism of the first passage through the liver when administered by gavage. Thus, using the same dose for both routes (subcutaneous and gavage) probably resulted in increased action of the copaiba in subcutaneous group, culminating in the increased animal survival on this group.
Copaiba oil has been used by several authors to test its anti-inflammatory and antimicrobial effects [17][18][19] , and the results have shown that the main active components responsible for these properties are the diterpenes and sesquiterpenes, as bisabolol and beta betacariofileno 9 . Knowing that the copaiba used in this study does not show effects against gram-negative bacteria 13 and that the model of CLP primarily deflagrates gramnegative septicemia, it is believed that the determining factor for increasing animal survival was the anti-inflammatory action of this plant, by modulating the inflammatory response and subsequent issue damage mediated by free radicals and proinflammatory citocinais against sepsis.
When analyzed histologically in the lungs of mice, was observed that the use of copaiba oil was crucial to the progression of pulmonary status, been observed pattern of focal pulmonary necrosis only in animals that don't received the oil. This finding suggests appropriate systemic anti-inflammatory action of copaiba, in agreement with that described by Veiga Junior et al. 20 , which showed a decrease in the number of lung neutrophils and leukocytes, besides inhibiting the production of nitric oxide. As regards the histopathological (Table 1)   Regardless of theroute used, the prophylactic use of copaiba oil for five days was effective to delay and mitigate the evolution of sepsis, and, judging by its wide use in Brazilian culture, especially Amazon, shows itself as an important component to be further studied for evidence of its role in popular usage.

Conclusion
Prophylactic subcutaneous administration of copaiba (copaifera reticulada) in mice subjected to severe sepsis by cecal ligation and puncture, resulted in a survival time of 2.6 and 1.5 times higher compared to non-use or use of this oil by gavage, respectively.