Fractionation using supercritical CO₂ influences the antioxidant and hepatoprotective activity of propolis against liver damage induced by tert-butyl hydroperoxide

The ethanolic extract (E) of propolis was further fractionated with supercritical CO₂ into four fractions (R, F1, F2 and F3). The extracts and the four fractions were characterised in terms of antioxidant and hepatoprotective activity against tert-butyl hydroperoxide (t-BHP)-induced damage in vitro and in a rat model. The in vitro study revealed that pre-treatment with propolis extract or its fractions significantly protected the primary hepatocytes against damage by t-BHP (P < 0.05). The hepatoprotective capacity increased with the dose of propolis. The R and F1 fractions had the highest flavinoid contents and most effectively protected the liver from damage by t-BHP. This study also demonstrated that the oral pretreatment with propolis (50 and 100 mg kg^-1) 5 days before a single dose of t-BHP (1.5 mM kg^-1, s.c. injection) was administered significantly kept the serum levels of hepatic enzyme markers (aspirate aminotransferase and alanine aminotransferase) low, even after treatment with t-BHP (P < 0.05). A pathological examination showed that lesions of liver were partially protected by treatment with propolis extract and fractions. Oxidative stress induced by t-BHP led to lipid peroxidation (malondialdehyde) and changes in the levels of the antioxidant enzymes. However, all the fractions, except F3 at low concentration (50 mg kg^-1), markedly suppressed lipid peroxidation and any increase in the activity of antioxidant enzymes.