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Acta Cirurgica Brasileira

On-line version ISSN 1678-2674

Acta Cir. Bras. vol.29 no.12 São Paulo Dec. 2014

http://dx.doi.org/10.1590/S0102-86502014001900002 

Original Articles

Attenuation of copaiba oil in hepatic damage in rats1

Marcus Vinicius Henriques Brito I  

Felipe D'Almeida Costa II  

Diego Matos de Vasconcelos III  

Lauro Augusto Veloso Costa IV  

Edson Yuzur Yasojima V  

Renan Kleber Costa Teixeira VI  

Vitor Nagai Yamaki VII  

IPhD, Full Professor, Head, Department of Integrate Health, Medical School, Para State University (UEPA), Belem-PA, Brazil. Scientific and intellectual content of the study and critical and final revision

IIGraduate student, School of Medicine, UEPA, Brazil. Conception and design of the study, technical procedures

IIIGraduate student, School of Medicine, UEPA, Belem-PA, Brazil. Technical procedures, interpretation of data, biochemistry analysis

VIGraduate student, School of Medicine, UEPA, Belem-PA, Brazil. Technical procedures, Interpretation of data, critical revision

VPhD, Associate Professor, Medical School, UEPA, Belem-PA, Brazil. Conception and design of the study, manuscript writing, critical revision

VIGraduate student, School of Medicine, UEPA, Belem-PA, Brazil. Grant from Institutional Program for Scientific Initiation (PIBIC) of the National Council of Scientific and Technological Development (CNPq), Ministry of Science, Technology and Inovation, Brazil. Technical procedures, care of animals

VIIGraduate student, School of Medicine, UEPA, Belem-PA, Brazil. Grant from Institutional Program for Scientific Initiation (PIBIC) of the National Council of Scientific and Technological Development (CNPq), Ministry of Science, Technology and Inovation, Brazil. Biostatistics analysis, care of animals, manuscript preparation

ABSTRACT

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.

Key words: Plant Oils; Hepatitis; Liver; Acetaminophen; Rats

Introduction

Acute liver failure (ALF) is characterized as a syndrome with sudden progression and severe hepatocellular dysfunction1. 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 drugs2 , 3.

This syndrome has multiple etiologies, such as viral hepatitis, poisoning, ischemic events and induced by drugs4. 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 children5 - 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 disorder8 - 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, anti-inflammatory and hepato protective effects13 - 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 liver16. 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.

Methods

Approved by the Ethics 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.

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. The acetaminophen and N-acetyl-cysteine were administered by gavage at a dose of 2 g/kg and 1.2 g/kg, respectively9 , 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.

Table 1 Mean and standard deviation of serum AST and ALT according to the groups. 

Group AST ALT
CG 131.66 ±17.71 74.16 ±15.18
AG* 373.00 ±103.91 311.00 ±137.77
PCG1* 303.00 ±214.85 226.83 ±159.90
PCG7# 225.00 ±95.29 164.33 ±62.05
TCG# 198.33 ±91.99 168.16 ±41.84
PCOG1* 333.50 ±79.97 322.00 ±164.29
PCOG7* 263.66 ±100.73 253.00 ±127.19
TCOG* 424.33 ±103.50 403.50 ±102.99
NG# 175.33 ±97.08 104.50 ±73.90

Source: Protocol search

*p<0.01 relative to CG (ANOVA)

#p<0.01 relative to AG (ANOVA)

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.

Table 2 Mean and standard deviation of serum alkaline phosphatase and ? GT according to the groups 

Group alkalinephosphatase ɤ GT
CG 150.33 ±88.14 6.83 ±1.83
AG 195.16 ±58.43 7.16 ±1.60
PCG1 145.66 ±30.91 7.83 ±2.40
PCG7 181.50 ±29.04 6.00 ±1.26
TCG 112.50 ±65.49 5.66 ±1.03
PCOG1 210.16 ±27.66 6.66 ±1.86
PCOG7 182.16 ±34.10 5.50 ±1.22
TCOG 262.50 ±16.22 5.66 ±1.36
NG 145.00 ±52.82 7.50 ±1.87

Source: Protocol search p>0.05 (ANOVA)

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

Group Total bilirubin* Direct bilirubin Indirect bilirubin*
CG 0.0783 ±0.04 0.0283 ±0.01 0.0500 ±0.02
AG 0.2133 ±0.11 0.0433 ±0.02 0.1700 ±0.11
PCG1 0.2567 ±0.09 0.0617 ±0.02 0.1950 ±0.09
PCG7 0.1633 ±0.08 0.0450 ±0.02 0.1183 ±0.06
TCG 0.5100 ±0.46 0.1033 ±0.12 0.4067 ±0.35
PCOG1 0.2417 ±0.13 0.0617 ±0.03 0.1867 ±0.12
PCOG7 0.2033 ±0.06 0.0633 ±0.03 0.1400 ±0.04
TCOG 0.2217 ±0.13 0.0750 ±0.05 0.1467 ±0.08
NG 0.1167 ±0.04 0.0385 ±0.03 0.0733 ±0.03

Source: Protocol search

*p<0.01 TCG relative to outhers groups (ANOVA)

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 hepatocytes17 , 18.

In our previous study16 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 oil19. 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 regeneration20. 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 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 injury attenuation by copaiba oil was not due to stimulation of liver regeneration9 , 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.

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 status20. 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 step21 , 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 function8 - 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.

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Financial source: Institutional Program for Scientific Initiation Scholarship (PIBIC), National Council for Scientific and Technological Development (CNPq)

1 Research performed at Experimental Surgery Laboratory, School of Medicine, State University of Para (UEPA), Belem-PA, Brazil.

Received: August 11, 2014; Revised: September 12, 2014; Accepted: October 13, 2014

Correspondence: Marcus Vinicius Henriques Brito Rua Apinagés, 630/202 66.033-170 Belém - PA Brasil Tel.: (55 91)3222-9752/8854-8895 marcusvhbrito@gmail.com

Conflict of interest: none

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