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).     

Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevance

Kaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer.

Aim of the study

In order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated.

Materials and methods

The impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively.

Results

In vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of V_max (15.276 ± 0.206 nmol/min) and lowest of K_i value (0.008 ± 0.002 μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low K_m value (1.599 ± 0.147 pmol). For in vivo studies, mice were orally treated with 250 mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity.

Conclusions

Kaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.     

Cocktail探针药物法评价百解胶囊对肝药酶亚型CYP2C19、CYP2E1的影响

目的:研究百解胶囊对大鼠肝药酶CYP2C19、CYP2E1活性的影响,探讨其解药物毒作用的机制。方法:将大鼠随机分为4组,即空白对照组、百解胶囊组(2.43g生药/kg)、百解胶囊组(0.27g生药/kg)、苯巴比妥钠组(10.8mg/kg),按上述剂量灌胃给药。以甲硝唑为内标,建立HPLC方法检测Cocktail探针药物奥美拉唑和氯唑沙宗在大鼠体内的代谢情况,评价百解胶囊对肝药酶CYP450的影响。结果:与空白对照组比较,百解胶囊组(2.43g生药/kg)对奥美拉唑的清除率(CL/F)明显增强,曲线下面积(AUC)明显减少,其半衰期(t1/2)亦有减少趋势;百解胶囊组(2.43g生药/kg)和百解胶囊组(0.27g生药/kg)对氯唑沙宗的清除率(CL/F)明显增强,曲线下面积(AUC)明显减少,半衰期(t1/2)明显缩短。结论:百解胶囊对大鼠肝药酶CYP2C19、CYP2E1具有诱导作用,可能是其解药物毒作用的机制之一。     

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 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, IC₅₀ 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 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 (IC₅₀'s of 123.3 μg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC₅₀'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 IC₅₀ values for asiaticoside were high (1070.2 μg/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.     

天王补心丸中生地黄、玄参、天冬和麦冬水提液对大鼠肝CYP450酶含量及其亚型CYP3A, CYP2E1和CYP1A2活性的影响

目的:研究天王补心丸中滋阴类药味生地黄、玄参、天冬和麦冬水提物对大鼠肝肝药酶(CYP450)含量及对亚型CYP3A,CYP2E1,CYP1A2活性的影响,探讨CYP450在天王补心丸代谢中的作用.方法:大鼠灌胃给予生地黄、玄参、天冬和麦冬水提物7d后取肝脏称重并制备肝微粒体,紫外分光光度法测定肝微粒体细胞色素b5(Cytb5),P450的含量及CYP3A的活性,高效液相法测定CYP2E1和CYP1A2的活性.结果:与空白对照组比较,各给药组大鼠肝指数无显著变化;生地黄组CYP450含量极显著减少(P＜0.01),CYP3A活性极显著增加(P＜0.01),CYP1A2活性显著增加(P＜0.05);玄参组CYP450含量减少(P＜0.05),CYP3A和CYP1A2活性均极显著增加(P＜0.01);天冬组Cytb5含量增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01);麦冬组cytb5,CYP450含量无统计学差异,CYP3A活性增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01).结论:天王补心丸中生地黄、玄参降低大鼠肝脏CYP450酶含量并均诱导CYP3A和CYP1A2活性,天冬诱导CYP2E1和CYP1A2活性,麦冬诱导CYP3A,CYP2E1和CYP1A2活性.由于抑制CYP450活性,减少对其他药代谢,滋阴药组在天王补心丸全方配伍中可增强其他各功能组比如补血养血、宁心安神类药味的作用,为探讨天王补心丸的配伍机制提供了理论基础.     

In vitro Inhibition of Human CYP1A2, CYP2D6, and CYP3A4 by Six Herbs Commonly Used in Pregnancy

Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaf, herbs frequently used in pregnancy, were investigated for their in vitro CYP1A2, 2D6, and 3A4 inhibitory potential. Aqueous or ethanolic extracts were made from commercially available herbal products, and incubations were performed with recombinant cDNA‐expressed human CYP enzymes in the presence of positive inhibitory controls. Metabolite formation was determined by validated LCMS/MS or HPLC methodologies. IC₅₀ inhibition constants were estimated from CYP activity inhibition plots using non‐linear regression. The most potent inhibition was shown for fennel towards CYP2D6 and 3A4 with respective IC₅₀ constants of 23 ± 2 and 40 ± 4 µg/ml, horsetail towards CYP1A2 with an IC₅₀ constant of 27 ± 1 µg/ml, and raspberry leaf towards CYP1A2, 2D6, and 3A4 with IC₅₀ constants of 44 ± 2, 47 ± 8, and 81 ± 11 µg/ml, respectively. Based on the recommended dosing of the different commercial herbal products, clinically relevant systemic CYP inhibitions could be possible for fennel, horsetail, and raspberry leaf. In addition, fennel and raspberry leaf might cause a clinically relevant inhibition of intestinal CYP3A4. The in vivo inhibitory potential of these herbs towards specific CYP enzymes should be further investigated. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytochrome P450 2C19 inhibitory activity of common berry constituents

The cytochrome P450 enzyme CYP2C19 is involved in the metabolism of many commonly prescribed drugs, including proton pump inhibitors, antiepileptics and antidepressants. CYP2C19 inhibitors from food and food supplements may augment the toxicity of these agents and lead to noncompliance with treatment. The present investigation addresses CYP2C19 inhibition by 18 berry constituents using a chemiluminescent assay. Test compounds displayed inhibitory properties in a concentration‐dependent fashion, with IC₅₀ values ranging from 20.2 µM up to >316 µM . In the order of decreasing effect size, anthocyanidins were followed by anthocyanidin‐monoglycosides and procyanidins. Anthocyanidin‐diglucosides exhibited weak and biphasic effects. When compared with the CYP2C19 inhibitor fluvoxamine, the flavonoids under study were 50‐ to 750‐fold less potent. It is concluded that the above natural substances are moderate to poor inhibitors of CYP2C19 in vitro. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of unprocessed versus vinegar-processed Schisandra chinensis on the activity and mRNA expression of CYP1A2, CYP2E1 and CYP3A4 enzymes in rats

Ethnopharmacological relevance

Schisandra chinensis (SC) is a well-known traditional Chinese herbal medicine that has been used in clinical practices for thousands of years. However, the differences between the effects of unprocessed and vinegar-processed Schisandra chinensis (VSC) on cytochrome P450 (CYP450) activities are poorly understood.

Aim of the study

To evaluate the differences between processed and unprocessed SC on the metabolism of CYP1A2, CYP2E1 and CYP3A4 substrates in rats using a cocktail method based on a developed and validated HPLC method. We also investigate the influence of processing on the levels of CYP mRNA.

Materials and methods

Three probe substrates (theophylline, dapsone and chlorzoxazone) were delivered simultaneously into rats treated with single or multiple doses of processed or unprocessed SC extract. The plasma concentrations of the three probes were profiled by HPLC, and their corresponding pharmacokinetic parameters were calculated. Real-time RT-PCR was performed to determine the effects of processed and unprocessed SC on the mRNA expression of CYP1A2, CYP2E1 and CYP3A4 in the liver.

Results

Treatment with single or multiple doses of either extract of SC induced CYP3A4 enzyme activity and inhibited CYP1A2 enzyme activity in rats. Furthermore, the inhibitory effect of SC was more potent after vinegar processing than without vinegar processing. CYP2E1 enzyme activity was induced after treatment with a single dose but was inhibited after multiple doses. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

These results provide useful scientific data for the safe clinical application of either extract of SC in combination with other drugs, which should lack the side effects induced by other herb–drug interactions.     

Effects of Panax notoginseng saponins on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats in vivo

The aim of this study was to assess the influence of the Panax notoginseng saponins (PNS) on the activities of the drug‐metabolizing enzymes cytochrome P450 (CYP450) 1A2, 2 C9, 2D6 and 3A4 in rats. The activities of CYP1A2, 2 C9, 2D6 and 3A4 were measured using specific probe drugs. After pretreatment for 1 week with PNS or physiological saline (control group), probe drugs caffeine (10 mg/kg; CYP1A2 activity), tolbutamide (15 mg/kg; CYP2C9 activity), metoprolol (20 mg/kg; CYP2D6 activity) and dapsone (10 mg/kg; CYP3A4 activity) were administered to rats by intraperitoneal injection. The blood was then collected at different times for ultra performance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) analysis. The data showed that PNS exhibited an induction effect on CYP1A2 by decreasing caffeine C_max (36.3%, p < 0.01) and AUC_0‐∞ (22.77%, p < 0.05) and increasing CL/F (27.03%, p < 0.05) compared with those of the control group. Western blot analysis was used to detect the effect of PNS on the protein level of CYP1A2, and the results showed that PNS could upregulate the protein expression of CYP1A2. However, no significant changes in CYP2C9, 2D6 or 3A4 activities were observed. In conclusion, the results indicate that PNS could induce CYP1A2, which may affect the disposition of medicines primarily dependent on the CYP1A2 pathway. Our work may be the basis of related herb–drug interactions in the clinic. Copyright © 2011 John Wiley & Sons, Ltd.     

丹参酚酸A对大鼠肝微粒体细胞色素P450酶系的影响

目的:研究丹参酚酸A对大鼠肝微粒体细胞色素P450和细胞色素b_5含量以及CYP1A2和CYP2E1活性的影响.方法:将大鼠分成溶剂对照组和丹参酚酸A给药组,每组10只,雌雄各半,丹参酚酸A给药组尾静脉注射给予丹参酚酸A 20 mg·kg~(-1)·d~(-1),连续给药5 d;溶剂对照组给予相同剂量的溶剂,紫外分光光度法测定大鼠肝微粒体细胞色素P450和细胞色素b_5含量;探针底物法评价CYP1A2和CYP2E1的活性.结果:丹参酚酸A尾静脉注射连续给药5 d后,大鼠细胞色素P450和细胞色素b_5含量与对照组比较均无显著性差异;CYP1A2和CYP2E1的活性与对照组比较也无显著性差异.结论:丹参酚酸A对CYP1A2和CYP2E1没有诱导或抑制作用,与经过CYP1A2和CYP2E1代谢的药物发生相互作用的可能性较小.     

一步法分析细胞色素P450 2D6酶活性缺陷等位基因CYP2D6A和CYP2D6B

 目的：CYP2D6A和CYP2D6B是引起细胞色素P4502D6(CYP2D6)酶活性缺陷的最主要的等位基因 ，对CYP2D6A和CYP2D6B的检测可准确(>92%)预测CYP2D6慢代谢者。本研究利用等位基因特异扩增法，建立了一步PCR法测定CYP2D6A和CYP2D6B等位基因。方法：等位基因特异扩增法分析CYP2D6A和CYP2D6B等位基因；右美沙芬作为探针药物测定表型。结果：经130例测定，说明本法更为快捷、更少污染。结论：本法的建立为该项测定应用于临床、指导临床合理用药奠定基础。     

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevance

The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes.

Material and methods

A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression.

Results

The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Cl_int) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment.

Conclusion

We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.     

Identification of cytochrome P450 (CYP) isoforms involved in the metabolism of corynoline,and assessment of its herb–drug interactions

Corynoline, an isoquinoline alkaloid isolated from the genus Corydalis, has been demonstrated to show multiple pharmacological effects including inhibition of acetylcholinesterase, inhibition of cell adhesion, fungitoxic and cytotoxic activity. The present study focused on its metabolism and metabolism‐based herb–drug interactions. After corynoline was incubated with human liver microsomes (HLMs) in the presence of NADPH, two metabolites (M‐1 and M‐2) were formed. Chemical inhibition experiments and assays with recombinant CYP isoforms showed that CYP2C9 was mainly involved in the formation of M‐1 and CYP3A4 mainly catalysed the production of M‐2. Among seven major CYP isoforms tested, corynoline showed strong inhibitory effects on the activities of CYP3A4 and CYP2C9, with an IC₅₀ of 3.3 ± 0.9 µm and 31.5 ± 0.5 µm , respectively. Kinetic analysis showed that inhibition of CYP3A4 by corynoline was best fit to a noncompetitive manner with K_i of 3.2 µm , while inhibition of CYP2C9 by corynoline was best fit to a competitive manner with K_i of 6.3 µm . Additionally, corynoline exhibited time‐dependent inhibition (TDI) toward CYP3A4. The inactivation kinetic parameters (K_I and k_inact) were calculated to be 6.8 µm and 0.07 min^‐1, respectively. These data are of significance for the application of corynoline and corynoline‐containing herbs. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves

Ethnopharmacological relevance

The herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a “tea drink” or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro.

Material and methods

Five probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC–MS/MS.

Results

The results showed that the LAE strongly inhibited CYP2D6 with an IC₅₀ value of 12.05 µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC₅₀ value of 0.96 µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC₅₀ values of 3.78, 3.76, and 3.15 µM, respectively. Their Lineweaver–Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with K_i values of 1.88, 2.34, and 1.56 µM, respectively.

Conclusion

The study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.     

Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes

Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki—64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki—36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC₅₀ values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co‐administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb‐drug interactions in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

Andrographolide inhibits the expression and metabolic activity of cytochrome P450 3A4 in the modified Caco-2 cells

Aim of the study

The aim of this study is to examine the effects of andrographolide on intestinal enzyme cytochrome P450 3A4 (CYP3A4) and predict whether oral administration of andrographolide-containing remedy leads to herb–drug interaction.

Materials and methods

Caco-2 cells are treated with 1α, 25-dihydroxyvitamin D3 for 3 wks to induce the expression of CYP3A4, and then andrographolide (1, 10, 100 μM) is added and treated for 72 h. Upon the further 4-h testosterone (250 μM) or nifedipine (200 μM) treatment, the basolateral medium samples and the Caco-2 monolayers are collected for analyses.

Results

Andrographolide (1, 10, 100 μM) significantly down-regulates the mRNA level and protein level of CYP3A4, and inhibits nifedipine oxidation and testosterone 6β-hydroxylation.

Conclusion

Oral administration of andrographolide likely leads to reduction of the metabolic activity of intestinal CYP3A4, therefore herb preparations containing andrographolide may result to herb–drug interactions in combination therapy.     

Cytochrome P450 inhibitory potential and RP-HPLC standardization of trikatu—A Rasayana from Indian Ayurveda

Ethnopharmacological relevance

Trikatu is a very well known ‘Rasayana’ in Ayurveda and widely used as a polyherbal ayurvedic formulation in India. It consists of three well known plants, viz., Piper longum (PL), Piper nigrum (PN) and Zingiber officinale (ZO) in equal ratio. Trikatu has been prescribed for cough, cold, fever, asthma, respiratory problems and improvement of digestive disorders. The aim of the present study was to investigate the effect of individual ingredients of trikatu namely PL, PN, and ZO and formulations [Marketed formulation (MF) and laboratory formulation (LF)] on drug metabolizing enzymes (CYP3A4 and CYP2D6), to assess its herb–drug interaction potential through cytochrome P450 inhibition assays. Further this work was aimed to develop an RP-HPLC method for the identification and quantification of piperine and 6-gingerol in the crude drug trikatu.

Materials and methods

Enzyme inhibition effect of LF, MF, PL, PN and ZO was explored through CYP450–CO complex assay using rat liver microsomes (RLM) and a fluorescence screening method using individual isoenzymes (CYP3A4 and CYP2D6). The RP-HPLC method was developed for the identification and quantification of piperine and 6-gingerol in LF, MF and individual plant materials at the concentration of 1 mg/mL.

Results

RP-HPLC analysis confirmed the presence of piperine and 6-gingerol in LF and MF [Piperine: 7.89±2.12% (w/w) (MF), 6.70±2.13% (w/w) (LF)]; [6-gingerol: 5.3±1.21% (w/w) (MF), 4.95±2.34% (w/w) (LF)]. Inhibitory potential of MF and LF in CYP450–CO complex assay was found to be 37.54±3.12% (MF) and 35.12±2.31% (LF) and against CYP2D6 and CYP3A4 was estimated to be IC₅₀ 251.30±3.98 and 245.23±1.92 μg/mL and IC₅₀ 225.50±1.02 and 223.254±0.92 μg/mL respectively.

Conclusions

Different concentrations of the trikatu formulation and its individual components showed significantly (p<0.001) less inhibitory activity on individual isoenzymes as compared to the positive control. The crude drug exhibited inhibitory potential against the CYP450 enzymes in a concentration dependent manner. Outcome of the present study demonstrated that trikatu has less interaction potential with drug metabolizing enzymes.     

Metabolism of the major Echinacea alkylamide N‐isobutyldodeca‐2E,4E,8Z,10Z‐tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes

Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N‐isobutyldodeca‐2E,4E,8Z,10Z‐tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N‐dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N‐dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic‐metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified. Copyright © 2010 John Wiley & Sons, Ltd.