In vitro effect of aegle marmelos fruit extract and imperatorin on cytochrome P450

Abstract

Aegle marmelos or Matoom is a medicinal plant. Unripe fruit had been traditionally used as an antidiarrhoea and antidysentery. Several chemical constituents of A. marmelos fruit have also been reported for its pharmaceutical activities. The fruit of A. marmelos was reported to be the source of imperatorin and other bioactive substances. Imperatorin indicated some pharmacological activities such as antibacterial, anticoagulant, antiplatelet aggregation. It can have an effect on drug or xenobiotic metabolism by being cytochrome P450 or CYP inhibition. In this study, A. marmelos fruits were extracted with hexane, ethanol and water then they were dried and prepared for inhibition test on human CYP isoforms (CYP1A2, CYP2C9, CYP2C19 and CYP3A4). Using the conversion of specific probe substrates, the inhibitory effect could be measured fluorometrically in a 96-well plate format. Moreover, the fruit extracts were analyzed for imperatorin by using RP-HPLC. A. marmelos dried fruit contained 0.56% w/w of n-hexane extract, 7.69% w/w of ethanolic extract and 21.54% w/w of aqueous extract. Hexane extract of A. marmelos showed the highest content of imperatorin. total yields (%) of imperatorin in hexane and ethanolic extracts were 5.78% w/w and 0.12% w/w, respectively, based on weight of dried extracts. For each test substance, the IC₅₀ (the concentration required to inhibit metabolism of CYP activities by 50%) was estimated. Imperatorin showed potent inhibitory effect on human CYP1A2 (IC₅₀ of 0.42±0.02 µM), CYP2C9 (IC₅₀ of 3.59±0.02 µM), CYP2C19 (IC₅₀ of 2.15±0.02 µM) and CYP3A4 (IC₅₀ of 1.63±0.02 µM) at concentrations of less than 10uM. Whereas hexane extracts of A. marmelos did not showed significantly different inhibition when compared with imperatorin. Hexane extract showed the highest inhibitory effect for CYP1A2 (IC₅₀ of 0.73+-0.03 µg/ml), CYP2C9 (IC₅₀ of 3.24±0.03 ug/ml), CYP2C19 (IC₅₀ of 4.04±0.02 µg/ml) and CYP3A4 (IC₅₀ of 1.65±0.05 ug/ml). While ethanolic extract showed lower inhibitory effect for CYP1A2 (IC₅₀ of 39.40±0.02 ug/ml), CYP2C9 (IC₅₀ of 197.60±0.04 µg/ml), CYP2C19 (IC₅₀ of 107.90±0.02 µg/ml) and CYP3A4 (IC₅₀ of 86.59+-0.05 µg/ml) and aqueous extract showed the least inhibitory effect, CYP1A2 (IC₅₀ of 352.30±0.04 µg/ml), CYP2C9 (IC₅₀ of 965.00±0.02 µg/ml), CYP2C19 (IC₅₀ of 414.00±0.20 µg/ml) and CYP3A4 (IC₅₀ of 842.40±0.04 µg/ml). This study provided the information of effects of imperatorin and A. marmelos extracts on phase I drug-metabolizing enzymes. These data suggest that imperatorin and A. marmelos fruit extracted with hexane potentially inhibit the metabolism of co-administered medication whose primary route of elimination is via those CYPs, thus interpretation of these data from in vitro to human should be further studied