Attenuation of copaiba oil in hepatic damage in rats 1

PURPOSE: To investigate the copaiba oil on the hepatic damage induced by acetaminophen, comparing against corn oil. METHODS: Fifty four rats were distributed into nine study groups (N=6): control group, that didn’t receive the acetaminophen; Acetaminophen Group, that only received the acetaminophen; Prophylactic Copaiba Group 1, that received copaiba oil two hours before the acetaminophen; Prophylactic Copaiba Group 7, that received copaiba oil seven days, once by day, before the acetaminophen; Therapy Copaiba Group, that received the copaiba oil two hours after the acetaminophen, the corn’s groups were similar than copaiba oil groups; and N-Acetyl-Cysteine Group, that received the N-Acetyl-Cysteine two hours after the acetaminophen. Euthanasia was performed after 24 hours. The serum levels transaminases, bilirubin and canalicular enzymes were analyzed. RESULTS: The prophylactic copaiba group 7, therapy copaiba group and N-Acetyl-Cysteine Group showed amounts of AST and ALT similar to the control group; and the prophylactic copaiba group 1 and corn’s groups showed similar levels to the acetaminophen group. There was no significant difference between the groups regarding the amount of alkaline phosphatase and ɤ GT (p>0.05). The therapy copaiba group showed the highest levels of total bilirubin and was statistically different from the other groups (p<0.01). CONCLUSIONS: Copaiba oil administered prophylactically for seven days and therapeutically 2 hours after the acetaminophen acute intoxication offered a potential hepato protection against paracetamol-induced hepatic damage, normalizing the biochemical parameters similarly to N-Acetyl-Cysteine, and the treatment with corn oil shows no effect on the liver damage.


Introduction
Acute liver failure (ALF) is characterized as a syndrome with sudden progression and severe hepatocellular dysfunction 1 .
The commitment of liver function leads to failure in physiological processes during homeostasis regulation, such as synthesis of plasma proteins, metabolism of lipids, carbohydrates and proteins, as well as performing inactivation of medications and toxic drugs 2,3 .This syndrome has multiple etiologies, such as viral hepatitis, poisoning, ischemic events and induced by drugs 4 .
However, the most important cause is related to overdose of acetaminophen, analgesic and antipyretic widely and indiscriminately used in the word.In the United States nearly 100.000 cases of suicide attempt occur per year by ingestion of acetaminophen, and the unintentional acetaminophen overdose is increasing every year as well, mainly in people with previous liver disease, in use of anticonvulsants, chronic alcoholics and in children [5][6][7] .
There are several studies that search for treatments and antidotes for acute liver toxicity.The use of herbal has shown promising results, when compared with N-acetyl-cysteine (gold standard treatment for acetaminophen's hepatotoxicity), suggesting an alternative handling for the toxic liver disorder [8][9][10][11][12] .
There are considerable amount of herbal among several species in amazon forest, amongst which copaiba oil stands out by scientifically comproved effects in wound healing, antiinflammatory and hepato protective effects [13][14][15] .In our previous study, copaiba oil showed a protective effect against liver injury; however it was not possible to identify the factors that led to decreased damage and a more detailed understand about the histopathological changes in damaged liver 16 .Thus, it was necessary to expand the study to elucidate some questions about the mechanism of this hepatoprotective effect.Therefore, the objective of this study is to investigate the copaiba oil in the acetaminophen hepatic liver failure induced, comparing against corn oil.Copaiba oil was administered by gavage at a dose of 0.63 mL/kg in group PCG7 and 3.8 mL/kg in groups PCG1 and TCG.The acetaminophen and N-acetyl-cysteine were obtained with a concentration of 400mg/mL and 300mg/mL, respectively.

Approved by the Ethics
The acetaminophen and N-acetyl-cysteine were administered by gavage at a dose of 2 g/kg and 1.2 g/kg, respectively 9,11 .
After 24 hours from the acetaminophen administration, a xyphopubic laparotomy was performed to collect 5 ml from inferior vena cava for biochemical measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, gamma glutamyl traspeptidase (ɤGT), total bilirubin (TB) and its fractions, Direct (DB) and indirect (IB).
ANOVA test was used to compare the biochemical results, adopting a 5% significance level to reject the nullity hypothesis.

Results
The mean serum levels of AST and ALT in each group are shown in Table1.On these significant differences between the CG and the groups AG and PCG1 and all groups treated with corn oil, (p<0.01) also was no significant difference between the AG and the groups PCG7, GCT and NG.

Discussion
The mechanism of hepatotoxicity caused by acetaminophen is unknown, however, there is a massive production of oxygen free radicals and a direct cytotoxic effect involved in the pathogenesis of this lesion, with a consequent necrosis and apoptosis of the hepatocytes 17,18 .
In our previous study 16 it was proposed that copaiba oil decreased levels of liver injury induced by acetaminophen, reinforcing the herbal anti-inflammatory effect.Nevertheless, to ensure the safety and the effectiveness of this oil its necessary to know detailed mechanism of this herbal, before applying it in large animal models.
Corn oil is a substance containing similar features of viscosity and density compared to copaiba oil and recently has been considered as a control substance in researches involving copaiba oil 19 .Furthermore, it is known that corn oil has significant levels of omega-6 and some studies suggest that diets rich in omega-6 provided improvement in liver regeneration 20 .Thus, we included this oil to evaluate if this substance shows improvement liver regeneration or if it is a standard of control substance to experiments with copaiba.
The use of the copaiba oil's dose of 3.8 ml/Kg, used in PCG1 and TCG, was based on the total amount of oil administered in PCG7 and toxicity studies on copaiba oil.It was an attempt to match the dose offered in all groups that received copaiba.
In addition, the same dose of corn oil was given to simulate the amount of lipids administered to the animals.
According Regarding the measurement of alkaline phosphatase and ɤ GT (Table 2), no significant difference between groups was showed.Regarding total bilirubin and fractions (Table 3) there was a statistical difference (p<0.01) between TCG and all other groups in the dosage of total bilirubin and indirect, but there was no statistical difference between the groups regarding the estimation of direct bilirubin.injury attenuation by copaiba oil was not due to stimulation of liver regeneration 9,11,20 .This evidence suggest the hypothesis that Copaiba`s mechanism takes place reducing free radicals in tissue by herbal anti-inflammatory properties, previously described by Noguchi et al. 15 , suggesting an actual hepato protective effect of this oil.

Group
Another hypothesis that suggests the attenuation in liver tissue damage could be justified by a predominant lipid diet in both prophylactic groups.This is supported due to lipid diet induces a rising in free radicals synthesis, leading the organism to be prepared with antioxidant defenses, such as a preconditioning status 20 .However, lipids concentrations are similar in groups treated seven days before with both oils, although decreases in aminotransferases levels was not observed in groups treated prophylactically with corn oil.
The alkaline phosphatase and ɤGT serum levels usually raise when colestatic liver events occurs.In toxic hepatitis induced by drugs these biochemical markers generally show values within the normal range, whereas the acetaminophen's cytotoxic effect ultimately generates a cytological damage in a physiological phase before the bilirubin excretion step 21,22 .
Studies using fat-rich substances, at high doses, showed that these substances raise the serum levels of those enzymes, whereas the high-fat ends up slowing down the bile excretion.This effect probably did not occur in this experiment due to the short period that the animals were exposed to the substances and the low doses administered as well.
Therapy copaiba group expressed a higher bilirubin level when compared to other experimental groups, though this increase due to unconjugated bilirubin, showing that copaiba could preserve a higher amount of hepatocytes, however with reduction in cells function [8][9][10][11] .Furthermore, rising in bilirubin levels cannot be influenced by increased in lipids consumption, as TCOG group did not show statistical difference.
The copaiba oil attenuated the liver damage caused by acetaminophen, but this protective effect needs further investigation to comprehend the mechanism and main active principle related to this functions.Therefore, additional experiments using oxidative stress analysis and attempts to isolate active principles are extremely important to implementation of this herbal.

Conclusions
Copaiba oil administered prophylactically for seven days and therapeutically 2 hours after the acetaminophen acute intoxication offered a potencial hepatoprotection against paracetamol-induced hepatic damage, normalizing the biochemical parameters similarly to N-Acetyl-Cysteine, and the treatment with corn oil shows no effect on the liver damage.However, the treated with copaiba therapeutically showed increases in bilirubin, at the cost of its indirect fraction.
Committee in the Use of Animals of the State University of Para (UEPA), protocol 08/07.This study used the copaiba oil species Copaiffera officinalis, supplied by Brazilian Agricultural Research Corporation (EMBRAPA) as crude oil, previously submitted to a physicochemical analyze to define its composition.The corn oil was obtained from a conventional commercial source.Fifty four male Wistar rats (Rattus norvegicus) were used, weighing between 200 -250 grams, provided from the animal colony of the Instituto Evandro Chagas and transferred to the animal colony of the Experimental Surgery Laboratory of UEPA, kept in a controlled environment, with food and water ad libitum, where they stayed for 15 days before the beginning of the experiment for adaptation.The animals were randomized distributed into six study groups, with six animals each: -Control Group (CG): The animals were used as normal standard for biochemical and histological analysis; -Acetaminophen Group (AG): The animals received a single dose of acetaminophen; -Prophylactic Copaiba Group 1 (PCG1): The animals received copaiba oil, once, two hours before receiving the acetaminophen dose; -Prophylactic Copaiba Group 7 (PCG7): The animals received copaiba oil, once, seven days before receiving the acetaminophen dose; -Therapy Copaiba Group (TCG): The animals received copaiba oil two hours after receiving the acetaminophen dose; -Prophylactic Corn Group 1 (PCOG1): The animals received corn oil, once, two hours before receiving the acetaminophen dose; -Prophylactic Corn Group 7 (PCOG7): The animals received corn oil, once, seven days before receiving the acetaminophen dose; -Therapy Corn Group (TCOG): The animals received corn oil two hours after receiving the acetaminophen dose; -N-Acetyl-Cysteine Group (NG): The animals received N-Acetyl-Cysteine, two hours after receiving the acetaminophen dose.

TABLE 1 -
Mean and standard deviation of serum AST and ALT according to the groups.
to hepatic enzymes, only PCG7 and TCG groups presented reduced levels in ALF.The absence of a positive outcome in groups treated with corn oil reveals that

TABLE 2 -
Mean and standard deviation of serum alkaline phosphatase and ɤ GT according to the groups.

TABLE 3 -
Mean and standard deviation of serum total bilirubin and fractions according to the groups.