Effects Of The Extracts And Active Constituents Of Andrographis Paniculata (Hempedu Bumi), Centella Asiatica (Pegaga) And Orthosiphon Stamineus (Misai Kucing) On Four Important Human Hepatic Cytochrome P450 Isoforms

Abstract

Herbal products are widely consumed all around the world. Since they are natural products, they are perceived as safe. However, because they are plant extracts, they contain phytochemicals with capabilities to inhibit or induce drug-metabolising enzymes. Cytochrome P450 (CYP) enzymes are fixed-function monooxygenases, which are expressed abundantly in human liver and play central roles in drug metabolism. Four isoforms, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were selected in this study as they are expressed at high levels in human liver and collectively they metabolise more than 80% of drugs known to be CYP substrates. Andrographis paniculata (hempedu bumi) (AP), Centella asiatica (pegaga) (CA) and Orthosiphon stamineus (misai kucing) (OS) are three popular local herbs traditionally used for various conditions, including hypertension, diabetes, skin disorders, ailments of the kidneys, liver and bladder. Current research on these herbs mostly concentrates on the chemical and botanical analyses, standardisation of extract preparations and pharmacological elucidation of the extracts and bioactive constituents of the plants. To date, limited studies have been carried out to investigate CYP modulating effects of these herbs, hence their potential to cause pharmacokinetic interaction with CYP substrates. Our objective was to develop specific in vitro assays for screening of the modulatory effects of these commonly used Malaysian herbs on activities of major cytochrome P450 enzymes. Four in vitro assays, CYP2C9-mediated tolbutamide methylhydroxylase assay, CYP2C19-mediated S-mephenytoin 4-hydroxylase assay, CYP2D6-mediated dextromethorphan O-demethylase assay and CYP3A4-mediated testosterone 6-hydroxylase assay, were selected and established using published high performance liquid chromatography (HPLC) methods. Herbal extracts together with important known active compounds of the herbs were evaluated by acquiring relevant pharmacokinetic parameters including IC50 and Ki to predict their potential to cause clinically significant interactions. Generally, all the herbal preparations and constituents were found to cause differential modulatory effects on the four CYP isoforms investigated. AP ethanol and methanol extracts inhibited CYP isoforms activities more potently compared with aqueous and hexane extracts. Similarly, CYP2C9 and CYP3A4 were more susceptible to AP inhibitory effects compared to other two isoforms. Potent inhibition observed for CYP2C9 and CYP3A4 (Ki values below 20 μg/ml) implying potential risk of drug-herb interactions for common drug substrates of the isoforms especially when patients are consuming large amount of AP preparations. As for CA, two active constituents, asiatic acid and madecassic acid, were found to selectively inhibited CYP isoforms activities to different extents, with CYP2C9 and CYP2D6 inhibited more than the rest. Of particular interesting is the potent inhibitory effect of asiatic acid on CYP2C9 (Ki below 20 μM). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Only CA ethanol and dichloromethane extracts potently to moderately inhibited CYP2C9 and CYP2C19 activities among all the CA extracts, indicating the involvement of semi-polar constituents including asiatic acid or madecassic acid in the inhibitory effect. While CA showed propensity to inhibit CYP2C9, OS appeared to selectively inhibit CYP3A4 activity. OS dichloromethane extract inhibited CYP3A4 activity with moderate effect whereas petroleum ether extract moderately inhibited both CYP3A4 and CYP2C19. Eupatorin was the most potent inhibitor among the four OS active constituents investigated, showing moderate to strong inhibitory effects on CYP2C19, CYP2D6 and CYP3A4. Of particular clinical relevant was the potent inhibition of eupatorin on CYP2D6 and CYP3A4, with both Ki values fell below 11 µM, implying good potential for eupatorin interaction with substrates of these two isoforms. Caffeic acid, on the other hand, showed significant inductive effect on CYP2C9 and CYP3A4. Hence, the net effect of intake of OS products would depend on the relative concentrations of various constituents in the products, the CYP isoforms involved and the drug substrates taken by the patients. In conclusion, studies carried out in the present project have provided more insight into the guideline of rational administration and precaution to be taken for using A. paniculata, C. asiatica and O. stamineous. The four in vitro enzyme assays developed in this project have served as convenient and robust models for screening and predicting herb–drug interactions in humans. The in vitro inhibition data generated indicate that certain extracts and constituents of the three herbs are sufficiently inhibitory for important CYP isoforms to suggest the need for additional in vitro and in vivo studies to evaluate the possibility of clinically significant drug interactions.