Evaluation of grapefruit extractfor the prevention of paracetamol-induced hepatotoxicity after overdose in rats

[摘要] English: Paracetamol is a widely used analgesic and antipyretic agent. While it is generally safe for use at recommended doses, acute overdose of paracetamol can cause potentially fatal liver damage. Despite the understanding that some cytochrome P450 isoforms are responsible for activation of paracetamol to the hepatotoxic metabolite, N-acetyl-p-benzoquinine-imine (NAPQI), the use of enzyme inhibitors for prevention and/or treatment of paracetamol hepatotoxicity is still not well researched. Therefore grapefruit juice was evaluated for prevention of paracetamol-induced hepatotoxicity. A high performance liquid chromatography (HPLC) method for the determination of paracetamol in plasma was developed. It involved protein precipitation of 50 μl of paracetamol spiked plasma with zinc sulphate followed by centrifugation. The supernatant was directly injected into the HPLC. The sample was eluted with a mobile phase of 0.01% trifluoroacetic acid in distilled water: acetonitrile (75: 25, v/v) over a Phenomenex C18 (4.60 x 250 mm) 5 μ analytical column at 1 ml/min. 4'Aminoacetophenone was used as the internal standard. Under these conditions paracetamol and 4'aminoacetophenone eluted at retention times of 4.2 minutes and 6.2 minutes, respectively. The average calibration curve (0 - 20 μg/ml) was linear with a regression equation of y = 0.0603x + 0.089, and regression coefficient of r2 = 0.9957. The method was used to measure paracetamol concentrations in rat plasma. Evaluation of grapefruit juice for the prevention of paracetamol-induced hepatotoxicity was performed with Sprague Dawley rats. The rats were treated with one-off oral dose of saline, paracetamol only, paracetamol + grapefruit juice low dose and paracetamol + grapefruit juice high dose. A commercially available grapefruit derivative, bergamottin, was also evaluated. Thereafter, 5 rats from each group were sacrificed after 24, 48 and 72 hours. After the treatment period the blood samples were collected for liver function tests, full blood count and paracetamol concentration. A piece of liver was sent for histopathology. Administration of a toxic dose of paracetamol (1725 mg/kg) elevated the liver enzymes (ALT and AST) significantly when compared to the control group. Upon physical observation of the liver during surgery, the livers of the rats at 48 hours exhibited moderate to severe hepatic injury. The haematology results, especially platelet count, revealed a very low amount of platelets at 48 hours, which is indicative of thrombocytopenia. Paracetamol plasma concentrations of rats treated with paracetamol only were higher than at 24 hours and there was a slight decrease at 48 and 72 hours due to the drug metabolism. However, the hepatic injuries of the rats treated with paracetamol only were antagonised by co-administration of paracetamol with grapefruit juice and also co-administration with bergamottin. The significant decrease in liver enzymes (ALT and AST) and the slight increase in platelet count at 72 hours revealed that grapefruit juice and bergamottin may have hepatoprotective effects against paracetamol-induced hepatotoxicity. Similarly, the concentration of paracetamol in the plasma of rats treated with paracetamol + grapefruit juice/bergamottin were lowered even further as opposed to when paracetamol was administered alone. The results of this study imply that both grapefruit juice and bergamottin have enzyme inhibition ability and hence were able to play a role in inhibiting the enzymes which are involved in the production of a toxic metabolite known as NAPQI, which is produced in high quantity during overdose of paracetamol and is a major cause of liver injury.

[发布机构] University of the Free State