Cytochrome P450 3a4 and 2d6-mediated Metabolism of Leisure and Medicinal Teas

PURPOSE: Thirty-five commercially available Camellia sinensis (black and green) and herbal leisure teas and an assortment of Traditional Chinese medicinal teas were randomly selected and examined for their potential to inhibit the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). The study was then extended to examine CYP2D6*1 and CYP2D6*10. METHODS: Microtiter fluorometric assays were utilized to examine the potential for the teas to inhibit CYP-mediated metabolism. Aqueous or alcoholic extracts of the dried tea plant material were examined. METHODS: Most of the black and green leisure teas generally inhibited CYP3A4 more than the Chinese medicinal teas. The medicinal Chinese teas were generally more inhibitory towards CYP3A4 compared to the CYP2D6 isozymes, and the aqueous extracts displayed more potency than the alcoholic extracts. CONCLUSIONS: Tea whether used for leisure or medicinal purposes has the potential to inhibit CYP3A4-mediated drug metabolism particularly black tea.


INTRODUCTION
Tea next to water is the second most consumed beverage in the world and has been consumed for thousands of years for their alluring flavors and health benefits; however their potential affect on the safety and efficacy of medicinal compounds has not been adequately established (1,2).Peng et al, (3) found that Camellia sinensis extract weakened the effect of β-lactam antibiotics.In vitro studies showed that the minimal inhibitory concentration of ampicillin, cefazolin, amoxicillin, and oxacillin were greatly decreased by 0.25% tea extract.In vivo findings showed that 5% tea extract with amoxicillin conferred a higher median efficacy dose (ED 50 ) than with antibiotic alone.Several studies have demonstrated either antagonistic or synergistic interactions between tea substances and drugs (4)(5)(6)(7).Numerous cultures across the world drink a leisure or medicinal tea as an infusion or tisane or as a decoction prepared by boiling in water.With the broader understanding that botanicals contain a wide range of substances including the anti-oxidant flavonoids the demarcation between what is a leisure and medicinal product has softened and many teas are now being used as both.
Most teas are concoctions of various herbal materials but single-entity products are available.All 6 varieties of teas (white, yellow, green, oolong, black, and pu-erh) are derived from C. sinensis (L) Kuntze but differ in their processing which include steps such as drying steaming or fermentation (2).Processing of green tea includes steaming or frying which inactivates the oxidase and peroxidase and prevents fermentation from occurring.The antioxidant and anticarcinogenic properties of green teas has been attributed to its higher levels of catechins particularly (-)-epigallocatechin gallate (EGCG) (2).The main difference between black and green teas is that black teas are fermented.Oolong oxidation ranges from 10% to 70% oxidation somewhere between green and black.The fermentation process utilizes endogenous polyphenol oxidase and peroxidase to catalyze the transformation of catechins to theaflavins, thearubigins, and other compounds, which are responsible for the colour and flavour of black tea (2).
___________________________________________________ Corresponding Author at: Brian C Foster, Ph.D. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; Email: bfoste2@uottawa.ca The total catechin content in green tea is 35% to 50% whereas in black tea it ranges at approximately 10% to 12% (2).In addition to polyphenols, green tea contains carotenoids; tocopherols; ascorbic acid (vitamin C); minerals such as chromium manganese selenium or zinc; and other phytochemical compounds.Green teas also contain 2 caffeine metabolites: theophylline which is a stronger stimulant than caffeine, and theobromine which is slightly weaker than caffeine.Black tea is generally stronger in flavor and contains more caffeine than the less oxidized teas.Green tea is considered to be a more potent antioxidant than black tea although black tea has substances which green tea does not such as theaflavin.The amount of caffeine and other phytochemical compounds in any single serving of tea varies significantly depending on variety and how the infusion is prepared.
Herbal teas are tisanes and are typically are composed of dried plant material such as flowers, leaves, roots, or rinds.Many Traditional Chinese Medicines (TCMs) are prepared as aqueous decoctions with some containing up to 30 herbs (8).
A wide range of constituents including polyphenols such as catechins and flavonoids which are also present in teas, have been shown to have anti-oxidant, anti-cancer, anti-diabetes, antiinflammatory, anti-hypertensive, or anticarcinogenic properties in vitro and in vivo (9)(10)(11)(12)(13)(14)(15).Recent studies have demonstrated that some of these compounds can interact with conventional and alternative medicines (16)(17)(18)(19)(20)(21)(22).The potential effect of some teas to mediate serious adverse events may be under estimated as they also contain furanocoumarin derivatives such as bergapten which can elicit mechanism-based inactivation of the major human drug metabolizing enzyme as seen with grapefruit juice (23).
This study was undertaken to determine whether different types of teas have the capacity to affect cytochrome P450 3A4-mediated biotransformation of a marker substance in an in vitro system which would provide mechanistic information that could be related to a patient (24).CYP3A4 is the major human drug metabolizing enzyme of large diverse lipophilic substances (25)(26)(27)(28).Studies were extended to examine the potential of the TCM products to affect CYP2D6*1-and *10-mediated metabolism of a marker substance as the 2D6*10 polymorphism is common within Chinese populations (23,29).CYP2D6 is selective for basic nitrogen ionized hydrophobic substances.

Chemicals and reagents
Aminoethyl-7-methoxy-4-methyl coumarin (AMMC), dibenzylfluorescein (DBF) and microsomes derived from Baculovirus infected insect cells expressing CYP3A4 and CYP-reductase were purchased from BD Biosciences (Mississauga ON Canada).Nicotinamide adenine dinucleotide phosphate reduced form (NADPH), quinidine and azamulin were purchased from Sigma-Aldrich (Oakville ON Canada).Ketoconazole was purchased from Calbiochem (Gibbstown NJ USA).Black, green and herbal teas were randomly selected from different commercial outlets.Medicinal TCM tea sachets were randomly selected by a local Chinese herbalist.Voucher specimens were deposited at the University of Ottawa herbarium.A list of the teas examined is provided in Table 1.

Aqueous sample preparation
A representative sample of each tea was ground to a fine consistency using a mortar and pestle.The TCM material did not require any processing as the material was provided in the sachets in a usable ground form.Water and alcohol (methanol or ethanol) extracts were prepared by vigorously vortexing 25 mg of ground material in 1 ml of the solvent for 1 min.The extract was separated from the undissolved material by centrifugation for 15 min at 1000 x g at room temperature.

CYP inhibition assay
The assays were performed in triplicate in 96-well plates with white walls and clear flat bottoms under red-colored light to minimize the exposure of fluorescent light to photosensitive material using a previously described method (20,30).In brief, the fluorescence was measured using a Cytofluor 4000 Fluorescence Measurement System (Applied Biosystems Foster City CA) using active and inactivated enzyme.For CYP3A4, a volume of 10 μl of the tea extract, 10 nM CYP3A4, 1 μM DBF (dissolved in acetonitrile), and 0.6 mM NADPH, were incubated in 0.19 M phosphate buffer solution (buffer pH 74) at a final volume of 200 μl for 20 min.The initial and final fluorescence was read at 485 nm excitation and 530 nm emission with a gain of 50.For ethanol extracts the extract was diluted ten-fold prior to testing.The positive inhibitor used was 19 μM ketoconazole (dissolved in MeOH).A similar method to the CYP3A4 inhibition assay was used for CYP2D6*1 and *10 except 10 nM CYP2D6*1 or *10, 0.12 μM AMMC (dissolved in acetonitrile), and 0.2 mM NADPH was used.The initial and final fluorescence was read at 409 nm excitation and 460 nm emission with a gain of 85 and an incubation time of 40 min.The positive inhibitor used was 2 μM quinidine (dissolved in MeOH).Initial fluorescence was subtracted from respective final fluorescence for the calculations.The percent inhibition of each extract was calculated relative to the CYP activity with the vehicle control.

RESULTS
The assay system used a series of internal controls to determine which samples if any had constituents capable of quenching the fluorescence.Quenching was not detected in any of the samples under these test conditions.Initial screening determined that all products completely inhibited CYP3A4-mediated metabolism of DBF.Subsequent testing of aqueous black, green, and herbal tea extracts were then undertaken at a single concentration of 25 mg/ml where most if not all of these products had an inhibitory effect of 95% or less.
In total, aqueous extracts from 19 different varieties of black tea, 5 selections of green teas, 11 varieties of herbal teas, and aqueous and alcohol extracts of 7 TCMs were tested for their potential to inhibit CYP3A4-mediated metabolism of DBF.These teas were randomly selected from a wider range of teas from several commercial outlets.Several of the teas contained a mixture of herbal and green teas and were grouped with both categories.
The black teas were generally more inhibitory than the green and herbal teas with 12 of the 19 black teas inhibiting more than 75% of the CYP3A4 activity relative to a vehicle control (Figure 1).Only 2 black tea extracts, NRP427 and 433, had weak inhibition towards CYP3A4 inhibiting less than 10% of CYP3A4 activity.Herbal tea extracts were generally the least inhibitory towards CYP3A4 activity which ranged from -4.3 ± 16% to 60.8 ± 30% inhibition.Four of the herbal teas NRP391, 435, 437, and 438 had weak inhibition towards CYP3A4 activity.Many of the herbal teas which had a weak or moderate CYP3A4 inhibition had a common main ingredient of chamomile.Green tea extracts were generally less inhibitory than those from black teas but more inhibitory than the herbal teas.All of the green teas examined effected greater than 45% inhibition of CYP3A4 activity and 3 of the 5 green teas (NRP389, 430, and 434) had greater than 75% inhibition of CYP3A4 activity.
Seven different TCMs (NRP 265 to 271) were examined for their capacity to inhibit CYP3A4mediated metabolism (Table 2).TCM material was randomly selected by a Chinese herbalist who made an a priori decision to provide products that were both readily available and in sachets in a usable extract or ground form.At a concentration of 25 mg/ml the water extracts were more potent CYP3A4 inhibitors than the alcohol extracts except for NRP270 and 271 (Table 3).Methanolic extracts from these 2 TCMs inhibited approximately 71% of CYP3A4 activity.Four of the water extracts from NRP265, 266, 268, and 269 inhibited at least 75% of CYP3A4 activity.The Chinese medicinal tea extracts uniformly had a high inhibitory effect on 3A4-mediated metabolism.
Testing of NRP 265 to 271 was then extended to examined their capacity to inhibit CYP2D6*1and 2D6*10-mediated metabolism.Less inhibition was observed against CYP2D6*1 and *10 activity for both the TCM water and alcohol extracts at 25 mg/ml.For most of the TCMs similar inhibition against CYP2D6*1 activity was observed for both aqueous and ethanolic extracts.NRP265 was the most inhibitory TCM inhibiting 64.3% ± 4.4% and 62.3% ± 7.3% of CYP2D6*1 activity for the aqueous and ethanolic extracts respectively.All of the other TCM extracts inhibited CYP2D6*1 activity with less than 40% relative to the vehicle control and several of these TCMs (NRP267, 268, and 271) inhibited less than 10% of CYP2D6*1 activity.
Similar results were obtained with CYP2D6*10.Aqueous and ethanolic extracts of the TCMs had similar inhibition towards CYP2D6*10 and NRP265 was the most inhibitory TCM inhibiting 57.8% ± 6.2% and 71.3% ± 3.7% of CYP2D6*10 activity for the aqueous and ethanolic extracts, respectively.All of the other TCM extracts inhibited CYP2D6*10 activity with less than 40% relative to the vehicle control.

DISCUSSION
As may be expected for complex products, there was no clear demarcation between the leisure teas examined.This may relate to the limited number of products tested, minor differences in particle size, and other harvesting and manufacturing processes.The findings revealed that for the leisure teas there was a grouping with high (> 75%), moderate (25 to 75%), and low (<25%) inhibitory potential to affect CYP-mediated metabolism.Within these products the green teas tended to be slightly less inhibitory than black teas towards CYP3A4-mediated metabolism.Overall the herbal teas tended to have a similar or lower inhibitory potential than the green teas.As many Asians have the 2D6*10 polymorphism which reduces the activity of this enzyme it was of interest to determine if these teas may affect this isoform.It was noteworthy that there were some differences in response with the 2D6*1 and *10 isoforms; the differences would not be expected to have a clinically important effect.
Extrapolation of in vitro findings to a clinical situation can be confounded by many intrinsic and extrinsic factors; however an in vitro determination of mechanism for an interaction is valuable in demonstrating the relative importance of a pathway (24).As there can be substantial variation in the composition of botanical products due to several factors including environmental conditions, time and year of harvest, and the manufacturing and storage processes, there may be multiple pharmacologically active substances affecting one or more pathways that can affect bioavailability and hence, pharmacodynamic activity (2,31,32).The complex nature of botanicals can effectively preclude unequivocal association of a pharmacological effect to a single substance.
Teas have long been safely consumed by many cultures for both leisure and their medicinal benefits.As accessibility and understanding of the mechanistic basis for improved health benefits of these products grow, new formulations and uses are being reported.However, there still remains insufficient information on whether the traditional or newer products may present risk when used concomitantly with other health products.This study has provided mechanistic information to assess any potential risk of these products to interact with other products metabolized by these isozymes.Although actual risk of a serious adverse event resulting from an interaction will depend on intrinsic and extrinsic factors including dose and rate of ingestion, these findings demonstrate that there is a mechanistic-basis for tea-mediated interactions with other health products metabolized by these isozymes.
The findings of this study with a wide random selection of leisure medicinal and traditional teas are significant in that they provide mechanistic support that some of these products have the potential to affect the safety and efficacy of other health and medicinal products.Variation in risk between similar products is expected and the findings with these samples cannot be directly extrapolated to other lots or similar products.As this study only examined two of the important human metabolic enzymes (CYP2D6 and CYP3A4) it is possible that teas contain additional substances that could also be affect other metabolic enzymes.This uncertainty warrants further investigation.In particular, white, yellow, oolong and pu-erh teas should be examined for their potential to affect drug metabolism.

Figure 1 .
Figure 1.Inhibition of human cytochrome P450 3A4 mediated-metabolism by aliquots of various black herbal and green teas (each replicate of 3 was repeated 2-3 times; mean ± SEM).A volume of 10 µl of 5 mg/ml aqueous extracts was examined.Ketoconazole (Keto 19 µM) was used as a positive control.NRP, Nutraceutical Research Programme number.

Table 1 .
List of leisure teas tested.The label contents are reported as listed on their package.

Table 2 .
List of Traditional Chinese Medicinal Teas Tested.The label contents are reported as listed on their package.