title: In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6beta-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.
  
  
  RESULTS
  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 microg/ml) and dichloromethane (K(i)=26.6 microg/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 microg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC(50)'s of 117.9 microg/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 microg/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 microg/ml). On the other hand, the IC(50) values for asiaticoside were high (1070.2 microg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity.
  
  
  CONCLUSIONS
  Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug-herb interactions through CYP2C9 inhibition.
authors.affiliation: School of Pharmacy and Health Sciences, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.
authors.forename: Yan
authors.initials: Y
authors.lastname: Pan
authors.forename: Badrul Amini
authors.initials: BA
authors.lastname: Abd-Rashid
authors.forename: Zakiah
authors.initials: Z
authors.lastname: Ismail
authors.forename: Rusli
authors.initials: R
authors.lastname: Ismail
authors.forename: Joon Wah
authors.initials: JW
authors.lastname: Mak
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Pook
authors.forename: Hui Meng
authors.initials: HM
authors.lastname: Er
authors.forename: Chin Eng
authors.initials: CE
authors.lastname: Ong
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D028041:Centella; D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors; D003640:Dealkylation; D003915:Dextromethorphan / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D041743:Herb-Drug Interactions; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008745:Methylation; D053978:Pentacyclic Triterpenes / Q000302:isolation & purification / Q000494:pharmacology; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism; D014315:Triterpenes / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D053978:Pentacyclic Triterpenes; D010936:Plant Extracts; D011994:Recombinant Proteins; D014315:Triterpenes; D013739:Testosterone; D003915:Dextromethorphan; D014044:Tolbutamide; C017032:asiatic acid; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C001669:madecassic acid; C004446:asiaticoside