In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica

Asiatic acid, (1S,2R,4aS,6aR,6aS,6bR,8aR,9S,10S,11R,12aS,14bR)-10,11-dihydroxy-9-(hydro-xymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid, a triterpenoid isolated from Centella asiatica, showed potent inhibitory effect on catalytic activity of human CYP2C9. Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure. The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms. High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6β-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC 50 and K i, to characterize modulatory effects. CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol ( K i = 39.1 μg/ml) and dichloromethane ( K i = 26.6 μg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC 50's of 123.3 μg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC 50's of 117.9 μg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 ( K i = 9.1 μg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate ( K i's ranged from 17.2 to 84.4 μg/ml). On the other hand, the IC 50 values for asiaticoside wer