Hui Meng Er

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19).

ETHNO PHARMACOLOGICAL RELEVANCE Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date. AIM OF THE STUDY In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated. MATERIALS AND METHODS S-mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects. RESULTS Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K(i) values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K(i)=39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K(i)=7.1 μg/ml or 20.6 μM). CONCLUSIONS It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).

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

ETHNOPHARMACOLOGICAL RELEVANCE 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. AIM OF THE STUDY 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. MATERIALS AND 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. CONCLUSION 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.

Mutagenic and antimutagenic activities of aqueous and methanol extracts of Euphorbia hirta

Euphorbia hirta (E. hirta) is a weed commonly found in tropical countries and has been used traditionally for asthma, bronchitis and conjunctivitis. However, one of the constituents in this plant, quercetin, was previously reported to be mutagenic. This work aimed to determine the level of quercetin in the aqueous and methanol plant extracts and to investigate the mutagenic effects of quercetin and the extracts in the Ames test utilising the mutant Salmonella typhimurium TA98 and TA100 strains. The antimutagenic activity of Euphorbia hirta aqueous and methanol extracts was also studied in Salmonella typhimurium TA98. HPLC analyses showed that quercetin and rutin, a glycosidic form of quercetin, were present in the acid-hydrolysed methanol extract and non-hydrolysed methanol extract respectively. The quercetin concentration was negligible in both non-hydrolysed and acid-hydrolysed aqueous extracts. The total phenolic contents in Euphorbia hirta were determined to be 268 and 93 mg gallic acid equivalent (GAE) per gram of aqueous and methanol extracts, respectively. Quercetin (25 microg/mL) was found to be strongly mutagenic in Salmonella typhimurium TA98 in the absence and presence of S-9 metabolic activation. However, both the aqueous and methanol extracts did not demonstrate any mutagenic properties when tested with Salmonella typhimurium TA98 and TA100 strains at concentrations up to 100 microg/mL in the absence and presence of S-9 metabolic activation. In the absence of S-9 metabolic activation, both the extracts were unable to inhibit the mutagenicity of the known mutagen, 2-nitrofluorene, in Salmonella typhimurium TA98. On the other hand, the aqueous extracts at 100 microg/mL and methanol extracts at 10 and 100 microg/mL exhibited strong antimutagenic activity against the mutagenicity of 2-aminoanthracene, a known mutagen, in the presence of S-9 metabolic activating enzymes. The results indicated that these extracts could modulate the xenobiotic metabolising enzymes in the liver at the higher concentrations.

In vitro effects of active constituents and extracts of Orthosiphon stamineus on the activities of three major human cDNA-expressed cytochrome P450 enzymes.

Orthosiphon stamineus (OS) has been traditionally used to treat diabetes, kidney and urinary disorders, high blood pressure and bone or muscular pain. To assess the possibility of drug-herb interaction via interference of metabolism, effects of four OS extracts of different polarity and three active constituents (sinensetin, eupatorin and rosmarinic acid) on major human cDNA-expressed cytochrome P450 (CYP) enzymes were investigated. Three substrate-probe based high-performance liquid chromatography (HPLC) assays were established to serve as activity markers for CYP2C9, CYP2D6 and CYP3A4. Our results indicate that OS extracts and constituents exhibited differential modulatory effects on different CYPs. While none of the OS components showed significant inhibition on CYP2C9, eupatorin strongly and uncompetitively inhibited CYP2D6 activity with a K(i) value of 10.2μM. CYP3A4 appeared to be the most susceptible enzyme to OS inhibitory effects. It was moderately inhibited by OS dichloromethane and petroleum ether extract with mixed-type and noncompetitive inhibitions (K(i)=93.7 and 44.9μg/mL), respectively. Correlation study indicated that the inhibition was accounted for by the presence of eupatorin in the extracts. When IC(50) values of these extracts were expressed in volume per dose unit to reflect inhibitory effect at recommended human doses from commercially available products, moderate inhibition was also observed. In addition, CYP3A4 was strongly and noncompetitively inhibited by eupatorin alone, with a K(i) value of 9.3μM. These findings suggest that co-administration of OS products, especially those with high eupatorin content, with conventional drugs may have the potential to cause drug-herb interactions involving inhibition of major CYP enzymes.

Development of an institutional assessment framework

the mean, the correlation with item difficulty improved significantly (r = 0.402, Z = 3.33). Correlations became even higher if authors had attended a workshop for MCQ construction (r = 0.490), received feedback on the difficulty of earlier questions (r = 0.426), or had experience as reviewers (r = 0.537). (A level of significance of p < 0.05 was used in all analyses.) What lessons were learned? If a regular, a priori standard-setting procedure is not feasible because resources are limited, a rough estimation of item difficulty by both authors and reviewers can represent a way to resolve the issue. The accuracy of item authors’ predictions improves after participation in MCQ workshops and after the receipt of feedback on the difficulty of items authored for earlier tests. The best estimate of item difficulty can be obtained by combining the estimates of reviewers and authors who have experience as reviewers.