Inhibition of Caffeine Metabolism by Apiaceous and Rutaceae Families of Plant Products in Humans: In Vivo and In Vitro Studies.

Daily consumption of caffeinated beverages is considered safe but serious health consequences do happen in some individuals. The Apiaceous and Rutaceae families of plant (ARFP) products are popular foods and medicines in the world. We previously reported significant amounts of furanocoumarin bioactive such as 8-methoxypsoralen, 5-methoxypsoralen, and isopimpinellin in ARFP products. As both caffeine and furanocoumarin bioactive are metabolized by the same hepatic CYP1A1/2 isozyme in humans, caffeine/ARFP product interactions may occur after co-administration. The objectives of the present study were to study in vivo loss of caffeine metabolizing activity by comparing the pharmacokinetics of caffeine in volunteers before and after pre-treatment with an ARFP extract, study the correlation between the decrease in hepatic CYP1A2 activity and the content of furanocoumarin bioactive in ARFP extracts, characterize CYP1A2 inactivation using in vitro incubations containing 14C-caffeine, a furanocoumarin bioactive, and human liver microsomes (HLMs), and provide a mechanistic explanation for both in vivo and in vitro data using the irreversible inhibition mechanism. The study results showed pre-treatment of volunteers with four ARFP extracts increased the area-under-the-concentration-time-curve (AUC0-inf) ratio of caffeine in the plasma ranging from 1.3 to 4.3-fold compared to the untreated volunteers indicating significant caffeine metabolism inhibition. The increases in AUC0-inf ratio also were linearly related to the effect-based doses of the furanocoumarins in the ARFP extracts, a finding which indicated caffeine metabolism inhibition was related to the content of furanocoumarin bioactive in an ARFP product. In vitro incubation studies also showed individual furanocoumarin bioactive were potent inhibitors of caffeine-N-demethylation; the IC50 for 8-methoxypsoralen 5-methoxypsoralen, and isopimpinellin were 0.09, 0.13, and 0.29 µM, respectively. In addition, CYP1A2 inactivation by individual furanocoumarin bioactive was concentration- and time-dependent involving the irreversible inhibition mechanism. The proposed irreversible inhibition mechanism was investigated further using 14C-labeled 8-methoxypsoralen and HLMs. The formation of 14C-adducts due to 14C-8-MOP-derived radioactivity bound to HLMs confirmed the irreversible inhibition of CYP1A2 activity. Thus, furanocoumarin bioactive metabolism in humans would result in reactive metabolite(s) formation inactivating CYP1A2 isozyme and inhibiting caffeine metabolism. Once the CYP1A2 isozyme was deactivated, the enzymic activity could only be regained by isozyme re-synthesis which took a long time. As a result, a single oral dose of ARFP extract administered to the human volunteers 3.0 h before still was able to inhibit caffeine metabolism.

Caffeine/Angelica dahurica and caffeine/Salvia miltiorrhiza metabolic inhibition in humans: In vitro and in vivo studies.

BACKGROUND: caffeine is a major constituent in numerous foods, beverages, dietary supplements and medications.Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav, and Salvia miltiorrhiza Bunge are traditional medicines commonly used in Asia. OBJECTIVES: to compare the pharmacokinetics of caffeine in humans before and after consuming an aqueous extract of A. dahurica or S. miltiorrhiza, and to propose a mechanistic explanation for in vivo caffeine metabolism inhibition based on in vitro data obtained with human liver microsomes. METHODS: Each of the four human volunteers was given a single oral dose of caffeine before and after consuming an A. dahurica or S. miltiorrhiza extract. Saliva samples were collected from the volunteers at pre-determined time points after receiving caffeine. The saliva samples were analyzed for unchanged caffeine using liquid chromatography. RESULTS: A. dahurica and S. miltiorrhiza extracts were capable of inhibiting caffeine metabolism in the human volunteers. In a separate study, cytochrome (CYP) 1A2-mediated caffeine demethylase activity was studied in incubation containing human liver microsomes, ß-nicotinamide adenine dinucleotide phosphate, and an herbal extract (or a pure bioactive chemical from the herbs). In all cases, CYP1A2 activity was decreased with an increasing inhibitor concentration, confirming the inhibition of caffeine metabolism in vivo. Caffeine metabolism inhibition most likely involved the competitive and/or non-competitive mechanism. CONCLUSION: Because a high level of caffeine in the plasma may result in adverse health effects in humans, care must be exercised when caffeine is consumed together with A. dahurica or S. miltiorrhiza.

Predicting the content of anthraquinone bioactive in Rhei rhizome (Rheum officinale Baill.) with the concentration addition model.

Rhei rhizome (Rheum officinale Baill.) (RR) contains a large number of anthraquinone bioactive, yet little is known of the combined effect of these anthraquinones in a mixture. The goals of this study were: to determine the inhibitory potencies of individual anthraquinones and whole RR extract against human liver microsomal CYP1A2/3A4 activity, to predict the content of anthraquinones in RR using the concentration addition (CA) model, and to compare predicted and empirical contents in the same RR sample. Anthraquinone concentrations in the RR extract were determined using HPLC. The inhibitory potencies of individual anthraquinones were determined in incubations containing human liver microsomes. The study results were used to predict an effect-based dose measure of the anthraquinones in RR using the CA model. An empirical dose measure also was determined in the whole RR extract using the CYP1A2/3A4-based bioassay. For the CYP1A2-based studies, the predicted and empirical dose measures of anthraquinones were identical; they were 12.0 ±â€¯1.80 and 12.20 ±â€¯0.81 mg aloe-emodin equivalents/g RR, respectively. For the CYP3A4-based studies, the predicted and empirical dose measures were different; they were 2.80 ±â€¯0.10 and 19.04 ±â€¯0.41 mg aloe-emodin equivalents/g RR, respectively. Only the CYP1A2-based CA model which assumed additive effects of RR anthraquinones predicted an effect-based dose measure that was verifiable by empirical data. The CA model provides an alternative approach to the CYP1A2/3A4-based bioassay or empirical method to screen for the anthraquinones in RR. The CA model as described in this study is applicable to other botanical drugs, plant-based foods and dietary supplements.

Physiologically based pharmacokinetic modeling of tea catechin mixture in rats and humans.

Although green tea (Camellia sinensis) (GT) contains a large number of polyphenolic compounds with anti-oxidative and anti-proliferative activities, little is known of the pharmacokinetics and tissue dose of tea catechins (TCs) as a chemical mixture in humans. The objectives of this study were to develop and validate a physiologically based pharmacokinetic (PBPK) model of tea catechin mixture (TCM) in rats and humans, and to predict an integrated or total concentration of TCM in the plasma of humans after consuming GT or Polyphenon E (PE). To this end, a PBPK model of epigallocatechin gallate (EGCg) consisting of 13 first-order, blood flow-limited tissue compartments was first developed in rats. The rat model was scaled up to humans by replacing its physiological parameters, pharmacokinetic parameters and tissue/blood partition coefficients (PCs) with human-specific values. Both rat and human EGCg models were then extrapolated to other TCs by substituting its physicochemical parameters, pharmacokinetic parameters, and PCs with catechin-specific values. Finally, a PBPK model of TCM was constructed by linking three rat (or human) tea catechin models together without including a description for pharmacokinetic interaction between the TCs. The mixture PBPK model accurately predicted the pharmacokinetic behaviors of three individual TCs in the plasma of rats and humans after GT or PE consumption. Model-predicted total TCM concentration in the plasma was linearly related to the dose consumed by humans. The mixture PBPK model is able to translate an external dose of TCM into internal target tissue doses for future safety assessment and dose-response analysis studies in humans. The modeling framework as described in this paper is also applicable to the bioactive chemical in other plant-based health products.

Factors Affecting Distribution of Estrogenicity in the Influents, Effluents, and Biosolids of Canadian Wastewater Treatment Plants.

Canadian wastewater treatment plants (WWTPs) release significant amounts of estrogenic chemicals to nearby surface waters. Environmental estrogens have been implicated as the causative agents of many developmental and reproductive problems in animals, including fish. The goals of this study were to assess the estrogenic activity in the influents, effluents, and biosolids of thirteen Canadian WWTPs using the yeast estrogen screen (YES) bioassay and to investigate whether factors, such as wastewater treatment method, sample storage, extraction efficiency, population, and summer/winter temperature had any effects on the distribution of estrogenicity in the WWTPs. Results of the study showed that estrogenicity from the influent to the effluent decreased in seven WWTPs, increased in two WWTPs, and did not change in four WWTPs during the winter. Estrogenic concentrations generally decreased in the order of biosolids > influents > effluents and ranged from 1.57 to 24.6, 1.25E-02 to 3.84E-01, and 9.46E-03 to 3.90E-01 ng estradiol equivalents/g or ml, respectively. The estrogenicity in the final effluents, but not those in the influents and biosolids, was significantly higher in the summer than the winter. Among the WWTP treatment methods, advanced, biological nutrient removal appeared to be the most effective method to remove estrogenic chemicals from wastewaters in Canada. Our studies help to identify factors or mechanisms that affect the distribution of estrogenicity in WWTPs, providing a better understanding on the discharges of estrogenic chemicals from WWTPs.

Assessing estrogenic chemicals in anchovy and mussel samples from Karachi, Pakistan with the yeast estrogen screen bioassay.

Endocrine disrupting chemicals (EDCs) are introduced into the aquatic environment through industrial and municipal effluents along with urban and agricultural runoffs. Exposure of aquatic organisms to EDCs may lead to hormonal disruption and adverse health effects. The goals of our study were: to collect anchovy and mussel samples from the coastal region of Karachi, to use the yeast estrogen screen (YES) bioassay in estimating xeno-estrogen content in these samples, and to investigate if the bioassay could be used to quantify known amounts of 17ß-estradiol (E2) injected into cod and salmon fillets. Results of the studies showed that mussel estrogenic activity in Karachi decreased in the order of Buleji point 1 (8.91 ± 4.77, mean ± SD) > Paradise point 1 (1.72 ± 0.81) > Paradise point 2 (0.61 ± 0.84) ng E2 equivalents/g wet wt (p < 0.05). By comparison, anchovy estrogenic activity at Korangi/Phitti Creek was much higher than at Manora. Together, these results confirmed previous reports that both Buleji point 1 and Korangi/Phitti Creek were the most contaminated areas of Karachi. The YES bioassay was only a semi-quantitative method in determining the contents of xeno-estrogens in aquatic organisms; it consistently overestimated the amounts of E2 injected into cod and salmon fillets due to additive and/or non-additive interactions between E2 and endogenous estrogens. Nevertheless, the YES bioassay was able to identify the contaminated sites in the coastal region of Karachi.

Physiologically based pharmacokinetics of matrine in the rat after oral administration of pure chemical and ACAPHA.

ACAPHA, a botanical drug for the treatment of human esophageal cancer in China, is under investigation as a lung cancer chemoprevention agent at the BC Cancer Agency (Vancouver, BC, Canada). Little or no information is available on the pharmacokinetics of ACAPHA in animals. The objectives of this study were as follows: to examine the disposition kinetics of matrine, a bioactive marker of ACAPHA in the rat; to develop a physiologically based pharmacokinetic (PBPK) model for pure matrine; and to characterize the absorption and clearance of crude matrine in ACAPHA-treated rats using the PBPK model. Pure matrine (15 mg/kg) or crude matrine in the form of ACAPHA (0.38 or 3.8 g/kg) was administered to the rat by gavages. The rats were sacrificed at different time points postdosing. Blood and major organs were removed from the rat, extracted with toluene/butanol, and quantified for matrine using gas chromatography-mass spectrometry. An 11-compartment, flow-limited PBPK model of matrine was developed. The PBPK model was able to simulate closely the empirical data of rats treated with pure matrine. Because the absorption and clearance of crude matrine in ACAPHA-treated rats could not be parameterized a priori, they were estimated by fitting the experimental data to the PBPK model. Results of the study show that pure matrine is absorbed and eliminated by the rat at faster rates than crude matrine. Moreover, the ACAPHA matrix may change the pharmacokinetics of matrine in the rat significantly. The PBPK model is a valuable tool to gain insights into the disposition kinetics of a botanical drug.

Study of tanshinone IIA tissue distribution in rat by liquid chromatography-tandem mass spectrometry method.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for determining tanshinone IIA in rat tissues. After a single step liquid-liquid extraction with diethyl ether, tanshinone IIA and loratadine (internal standard) was subjected to LC/MS/MS analysis using positive electro-spray ionization under selected reaction monitoring mode. Chromatographic separation of tanshinone IIA and loratadine was achieved on a Hypersil BDS C(18) column (i.d. 2.1 x 50 mm, 5 microm) with a mobile phase consisting of methanol-1% formic acid (90:10, v/v) at a flow rate of 300 microL/min. The intra-day and inter-day precision of the method were less than 10.2 and 12.4%, respectively. The intra-day and inter-day accuracies ranged from 99.7 to 109.7%. The lowest limit of quantification for tanshinone IIA was 1 ng/mL. The method was applied to a tanshinone IIA tissue distribution study after an oral dose of 60 mg/kg to rats. Tanshinone IIA tissue concentrations decreased in the order of stomach > small intestine > lung > liver > fat > muscle > kidneys > spleen > heart > plasma > brain > testes. Tanshinone IIA still could be detected in most of the tissues at 20 h post-dosing. These results indicate that the LC/MS/MS method was rapid and sensitive to quantify tanshinone IIA in different rat tissues.

The use of in vitro bioassays to quantify endocrine disrupting chemicals in municipal wastewater treatment plant effluents.

In vitro bioassays are widely used to detect and quantify endocrine disrupting chemicals (EDCs) in the influents and effluents of municipal wastewater treatment plants (WWTP). These assays have sometimes led to false positive or negative results, partly due to the low EDC concentrations in the samples. The objectives of the present study were: (a) to compare the estrogen screen (E-Screen) and the yeast estrogen screen (YES) bioassays using the 17beta-estradiol (E2) or its equivalence and (b) to investigate if a combination of the E-Screen and YES assays can be used to improve the accuracy of EDC detection and quantification. The E-Screen bioassay was conducted with the MCF-7 (BOS) human breast cancer cell line while the YES bioassay employed two different types of recombinant yeast. The influent and effluent samples collected from the five WWTPs operated by the Greater Vancouver Regional District (GVRD) were analyzed by both the E-Screen and the YES bioassays. Since the results of the E-Screen and YES bioassays varied by up to 4-fold on the same split sample of a nominal E2 concentration, the mean value of the E-screen and YES bioassays was used to represent the EDC activity of a given WWTP sample. Results of these studies showed that the E2 equivalent concentration in each WWTP sample was consistently higher than 1 ng/L, a concentration that may potentially cause endocrine disruption in different aquatic species. The composition of selected EDCs in a subset of effluent samples was examined using a gas chromatograph-high resolution mass spectrometer (GC-HRMS). EDC composition in 10 WWTP samples correlated with the mean endocrine disrupting activities of the E-Screen and YES bioassays. Results also indicated that secondary treatment plants are comparable to the primary treatment plants in removing EDCs from the final effluents.

Gas chromatography-mass spectrometry determination of matrine in human plasma.

A method was developed for the quantification of matrine in human plasma using a liquid-liquid extraction procedure followed by gas-chromatography-mass spectrometry (GC/MS) analysis. Deuterated matrine, an internal standard of the analysis, was spiked into the plasma samples before extraction. Linear detection responses were obtained for matrine concentrations ranging from 10 to 500 ng/ml. The intra-day and inter-day precision ranged from 0.4 to 4.0% and 1.0-3.5%, respectively. The intra-day accuracy was between -7.3 and 4.5%. The limit of quantification for matrine was 23 ng/ml. The extraction efficiency averaged about 38%. The validated GC/MS method will be used to quantify matrine in human plasma samples collected in a clinical trial study.

Determination of polycyclic aromatic hydrocarbons in dungeness crabs (Cancer magister) near an aluminum smelter in Kitimat Arm, British Columbia, Canada.

An aluminum smelter situated at the head of Kitimat Arm (BC, Canada) has discharged polycyclic aromatic hydrocarbons (PAHs) into the receiving waters since 1954. The purpose of the present study was to examine the distribution of PAHs contaminants in dungeness crabs (Cancer magister) collected in Kitimat Arm and Douglas Channel (BC, Canada) by determining the concentrations of PAHs in the hepatopancreas and muscle tissues of crabs by using gas chromatography-mass spectrometry. Crabs were collected at specific sites down the Arm from the smelter on four separate occasions over a three-year period. Hepatopancreas and muscle tissues of the crabs were analyzed for 10 of the 16 PAH priority pollutants recommended by the U.S. Environmental Protection Agency. Results of the studies showed that the crabs had detectable levels of PAHs in hepatopancreas and muscle tissues. The highest concentrations of PAHs in the tissues were found at a site near the aluminum smelter, the alleged point source of PAH discharge. The concentrations of PAH analytes were high in crabs collected close to the smelter and at lower levels in crabs collected throughout Douglas Channel. These results show that PAHs discharged by the smelter were bioavailable to the crabs. The concentration of each PAH analyte in the hepatopancreas was found to be strongly related to its water solubility. However, the PAH analyte concentrations in the hepatopancreas and muscle did not appear to correlate highly with each other.

Screening pyrene metabolites in the hemolymph of dungeness crabs (Cancer magister) with synchronous fluorescence spectrometry: method development and application.

The present study examined the metabolic pathways of pyrene in dungeness crabs (Cancer magister) in the laboratory and the potential of using synchronous fluorescence spectrometry (SFS) to determine pyrene metabolite concentrations in the hemolymph of crabs exposed to polycyclic aromatic hydrocarbons (PAHs) in the field. Pyrene was metabolized by crabs mainly to 1-hydroxypyrene and pyrene-1-glucoside. Both pyrene metabolites could be detected by SFS in the hemolymph of crabs. A nondestructive hemolymph collection procedure was developed and used in conjunction with the SFS assay to assess the exposure of crabs to PAHs in Kitimat Arm (British Columbia, Canada). Our results showed that crabs obtained near the source of PAH contamination had the highest level of pyrene-related fluorescence in the hemolymph, whereas concentrations were lower at other sites downstream from the pollution source. In a separate study, the hepatopancreases of crabs were analyzed for parent PAHs by using gas chromatography-mass spectrometry. Pyrene-associated fluorescent responses of the hemolymph were found to correlate positively with the concentration of total PAHs in the hepatopancreas (r = 0.39, p < 0.05).