Lotus Metabolic Drug-drug Interactions And In Vivo Constituents

Cytochrome P450(CYP) is a major metabolizing enzyme system in the body and plays an important role in the metabolism of many endogenous and exogenous substances. Five P450isoenzymes, namely, CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9are particularly important because they are responsible for the metabolism of approximately90%of the currently known therapeutic drugs. Drug-drug interactions are frequently occurrence when drugs are co-administered and one drug modifies the metabolic clearance of the other drugs by inhibition or induction of some P450isoenzymes. In general, inhibition is much more harmful than induction in clinic settings. The inhibition of P450activity may cause an increase in the blood concentrations of the drug that may lead to toxic symptoms and sometimes even fatal interactions. Many drugs have been withdrawn from the market due to their significant inhibition of some isoenzymes that resulted in serious clinical damages.Traditional Chinese medicines (TCMs) are often considered as safe and harmless natural products and are conventionally used with prescription drugs. However, many different side effects have been reported for some herbs and/or their formulations recently. Many TCMs have been reported to exhibit inhibitory or induction effects on the P450enzymes, this cause to herb-drug interactions (HDI) and have attracted much attention. Thus, it is extremely important to evaluate the potential HDI of the commonly used herbs for their safe and effective use.Nelumbinis Folium, the dried leaves of lotus (Nelumbo nucifera Gaertn), is utilized not only as a dietary staple but also as a TCM to treat sunstroke, assuage thirst, and cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the flavonoids and/or alkaloids of the herb exhibit various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-diabetic, anti-microbial, and anti-HIV 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, information on the leaf extract and its active compounds that cause HDI by inhibition of P450is limited. In the present study, we investigated the potential HDI of lotus leaves that mediated by P450through in vitro or in vivo models. Firsty, the inhibitory effects on the CYP activities in human liver microsomes were evaluated by in vitro model, and later were verified by in vivo model. Finally, the analysis of chemical constituents of lotus leaves in rats and their tissue distribution were investigated to interpret the mechanism of lotus leaves leading to the potential HDI. In addition, the P-gp function was preliminary investgated by Caco-2cell model. The main contents of this paper are as follows:1. By using LC-MS/MS techniques, an in vitro cocktail method was established for simultaneous determination of five major cytochrome P450isoenzymes activities by monitoring the metabolites of probe substrates, including omeprazole (CYP2C19), dextromethorphan (CYP2D6), testosterone (CYP3A4), chlorzoxazone (CYP2E1), tolbutamide (CYP2C9), in incubation human liver microsomes with mixed the five probe substrates. Inhibition activities of12TCMs including Nelumbinis Folium, Ziziphi Spinosae Semen, Cassiae Semen, Schisandrae Chinensis Fructus, Crataegi Fructus, Lycii Fructus, Nelumbinis Plumula, Ginseng Radix Et Rhizoma, Chrysanthemi Flos, Polygonati Rhizoma, Poria and Lonicerae Japonicae Flos on the five isoenzymes were evaluated using established method. The results indicated that the method possessed a good linearity and sensitivity, as well as the excellent precession and accuracy, and can be used for the high throughput screening of P450enzyme activities. In addition, aqueous extracts of12TCMs showed a weak or no inhibitory effects on P450enzymes, however, their ethanolic extracts possessed more potent and concentration-dependent inhibition effects (except for the Lycii Fructus). Among them, the lotus leaf ethanolic extract strongly inhibited the CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9activities with IC50values of77.38,12.05,98.01,61.43and83.46μg/mL, respectively; chrysanthemum Flos ethanolic extract inhibited the CYP2C9activity with IC50value of45.25μg/mL; Schisandrae Chinensis Fructus ethanolic extract inhibited CYP3A4and CYP2C19activities with IC50values of47.24and64.42μg/mL, respectively. These results suggest that the possible HDI between the herb ethanolic extracts and their preparations with conventional medicines should thus be taken into account.2. The inhibitory effects of the total flavonoids and total alkaloids of lotus leaves, as well as its main alkaloid compounds on the activities of human liver microsomal P450enzymes were investigated by the established cocktail probe substrate method. The results showed that the total flavonoids weakly inhibited P450enzyme activities, with IC50values around100μg/mL; while, the total alkaloids strongly inhibited the activities of CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9with IC50value of40.79,0.96, 44.87,63.84and52.58μg/mL, respectively. The individual alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and2-hydroxy-l-methoxyaporphine (HMA) competitively inhibited CYP2D6activity with Ki values of1.88,2.34, and1.56μM, respectively. And the three alkaloids are not mechanism-based inhibitors of CYP2D6.The effects of lotus leaf ethanolic extract, total flavonoids and total alkaloids on the function of P-glycoprotein (P-gp) were investigated by using Caco-2cell model and a P-gp substrate Rhodamine-123(Rho-123). The result showed that the ethanolic extract and total alkaloids could significantly increase the uptake of Rho-123by Caco-2cells at the concentration of100μg/mL (P<0.05), this indicates that they can inhibit the function of P-gp. The total flavonoids showed a weak inhibition effect on P-gp, but no significant difference comparing with negative control (P>0.05).All these results suggest that lotus leaves have inhibitory effects on P450enzyme activities and P-gp function and the effects may probable related with its alkaloid components. There are potential drug interactions between total alkaloids and other therapeutic drugs.3. The in vivo inhibition effects of lotus leaf total alkaloids on CYP2D6activity was investigated in rats using dextromethorphan and metoprolol as probe substrates. After being pretreated with total alkaloids (50mg/kg) for continuous7days, the rats was intravenously administrated with dextromethorphan (5mg/kg) or metoprolol (10mg/kg) at2or12h time point of the last alkaloid dosing. The plasma concentrations of the substrate metabolites, dextrorphan and a-hydroxymetoprolol were determined by LC-MS/MS method, for comparing their pharmacokinetic properties between the treated and untreated rats, and protein expression of CYP2D6in rat liver was also determined by western blot. The results showed total alkaloids significantly decreased the plasma concentrations of the two probe metabolites, AUCall of dextrorphan was significantly decreased from167.27to43.13h-ng/mL (P<0.01)(given at2h after the last dose of alkaloids) and62.25h-ng/mL (P<0.05)(given at12h after the last dose of alkaloids), respectively; and AUCall of a-hydroxy-metoprolol were also significantly decreased from347.68to223.24h-ng/mL (P<0.05)(given at2h after the last dose of alkaloids). The total alkaloids had no effect on the protein expression of CYP2D6in rat liver. These results suggest that the total alkaloids have significant inhibition on rat CYP2D6enzyme, which was consistent with the in vitro results.4. Based on developed LC-MS/MS methods, the in vivo components of lotus leaf extracts were identified after oral administration of total flavonoids and total alkaloids to rats. In addition, the tissue distribution of alkaloids was also studied after intravenous administration to rats. A total of37flavonoid compounds, including parent flavonoids and their glucuronidation and sulfation metabolites, were identified in rat plasma and urine. This indicates that flavonoids are mainly biotransformed by phase II metabolism pathway. A total of18alkaloids were identified, including parent alkaloids and their oxidation, methylation, glucuronidation and sulfation metabolites, were also identified in rat plasma and urine. This indicates that the alkaloids were biotransformed by both phase I and phase II metabolism pathways. The studies on tissue distribution of alkaloids showed that NF, N-NF and HMA could quickly (within5min) distribute to various tissues, especially in the liver, lungs, spleens and kidneys with a relative high concentrations, and could remained in the body for a relative long period. It is speculated that the inhibitory effects of lotus leaves on P450enzyme activities are likely to relate to these in vivo components.In conclusion, the present study provides systematic investigations on the in vitro inhibitory effects of lotus leaf ethanolic extracts, and its main two fractions total flavonoids and total alkaloids on P450enzyme activities, particularly the CYP2D6isoenzyme, which was further confirmed by an in vivo study. Meanwhile, the effect of lotus leaf ethanolic extract and the two fractions on the function of P-gp was preliminary evaluated by using a Caco-2cell model. In addition, the in vivo components of lotus leave flavonoids and alkaloids, as well as the tissue distribution of alkaloids in rats were also investigated. The results reveals that the lotus leaves can inhibit the activities of P450enzymes, especially for CYP2D6, and inhibit P-gp function. These effects are mainly related to the alkaloids existed in lotus leaves, this might cause the potential HDI and should pay much attentions when used in clinic. The present studies provide new information for safe and appropriate use of lotus leaves, and also provide ideas for further investigation of pharmacology and toxicology, and further development and utilization of the herb.