Journal of Ethnopharmacology 155 (2014) 841–846
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
The effect of Cree traditional medicinal teas on the activity of human cytochrome P450-mediated metabolism Teresa W. Tam a,b, Rui Liu a,b, Ammar Saleem b, John T. Arnason b, Anthony Krantis a,b, Pierre S. Haddad c, Brian C. Foster a,b,n a
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa, Ottawa, ON, Canada K1H 8M5 c Department of Pharmacology, Université de Montréal, Montréal, QC, Canada H3C 3J7 b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 April 2014 Received in revised form 16 June 2014 Accepted 17 June 2014 Available online 24 June 2014
Ethnopharmacological relevance: Rhododendron groenlandicum (Bog Labrador tea), Rhododendron tomentosum (Marsh Labrador tea) and Juniperus communis (Juniper) are used in medicinal teas by Canadian aboriginal cultures alone and in combination with conventional drug products. The safety of this combination had not been previously examined and this study was initiated to examine the potential of medicinal teas to inhibit the major human drug metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). Materials and methods: The decoctions of Rhododendron groenlandicum and Rhododendron tomentosum leaves and Juniperus communis berries were examined in a microtiter fluorometric assay to examine their potential to inhibit CYP-mediated metabolism. Results: The decoctions showed progressive inhibition towards CYP3A4 the longer the leaves or berries were brewed. R. Rhododendron groenlandicum and Juniperus communis may have the potential to inhibit CYP3A4-mediated metabolism. Conclusions: The findings of this study with these traditional medicines are significant in that they provide mechanistic support that these products have the potential to affect the safety and efficacy of other health and medicinal products. As this study only examined CYP3A4, it is possible that these medicinals contain substances that could also affect other metabolic enzymes. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytochrome P450 Labrador tea Juniperus communis Rhododendron groenlandicum Traditional medicine Cree
1. Introduction The use of traditional medicinal plants to treat ailments is still commonly practiced among First Nations in Canada, and teas or decoctions are the most common preparation (Ritch-Krc et al., 1996). In a collaborative ethnobotanical study undertaken by the CIHR team in aboriginal antidiabetic medicines with the Cree Healers of Eeyou Istchee (Quebec, Canada) two of the common medicinal teas used were Juniper and Labrador teas (Leduc et al., 2006; Spoor et al., 2006). These species are also widely used by other First Nations (Arnason et al., 1981; Moerman, 1998). There are two Eastern Canadian species of Labrador tea: Rhododendron groenlandicum (Bog Labrador tea, synonym Ledum groenlandicum) and Rhododendron tomentosum (Marsh Labrador tea, synonym
n Corresponding author at: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5. Tel.: þ1 613 224 7244. E-mail address: [email protected] (B.C. Foster).
http://dx.doi.org/10.1016/j.jep.2014.06.045 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Ledum tomentosum) (Black et al., 2011). Labrador tea is an Ericaceae shrub with evergreen leaves and is used traditionally by First Nations of Eastern Canada to treat a variety of ailments such as inflammation, asthma, rheumatism, digestive and skin disorders, or kidney and liver diseases (Arnason et al., 1981; Chartier et al., 2005; Dufour et al., 2007). Rhododendron tomentosum is found in subarctic habitats, and is used by both Inuit and Northern Cree communities while Rhododendron groenlandicum is found in boreal and mixed forest regions and is used by Cree, Algonquin and other First Nations (Arnason et al., 1981). The leaves are the most commonly used part for medicinal teas and decoctions, and are prepared by brewing the leaves in hot or boiling water. Juniper, Juniperus communis is a small shrub or tree belonging in the family Cupressaceae and is found in boreal and deciduous regions of Eastern Canada. The berry-like seed cones, which are green ripening to purple black, are used for cystitis by the Cree (Arnason et al., 1981). Through discussions in our ethnobotanical study with Cree healers and Elders, they became aware of studies that have
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demonstrated that some herbal and traditional medicines can interact with conventional and alternative medicines (Foster et al., 2003; Zhou et al., 2003; Marchetti et al., 2007; Nowack, 2008; Tam et al., 2009, 2011). As the Cree healers are very concerned about conditions for safe use of traditional medicines, this study was undertaken at their request to determine whether the decoctions of these botanicals have the capacity to affect the safety of conventional medicines used in their community. As a first step, we studied the potential of the teas to inhibit cytochrome P450 3A4-mediated (CYP3A4) biotransformation of a marker substance in an in vitro system. This assay was examined to provide mechanistic information that could be related to patient safety (US FDA, 1999). Chromatographic analysis was undertaken to provide a baseline of the substances present in the extracts for any future comparative analysis between our findings and others. CYP3A4 is the major human drug metabolizing enzyme of large, diverse, lipophilic substances (Anzenbacher and Anzenbacherová, 2001).
2. Materials and methods 2.1. Chemicals and reagents All solvents for HPLC analysis were optima grade including methanol (MeOH) and acetonitrile (ACN) (Fisher Scientific, Ottawa, ON, Canada). Dibenzylfluorescein (DBF) and microsomes derived from Baculovirus infected insect cells expressing CYP3A4 and CYPreductase 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). Samples of the selected plant parts of the Cree Nation of Eeyou Istchee (CEI) medicines were harvested in Mistissini, Quebec, Canada following the instructions of the Elders and healers of this community (Leduc et al., 2006). These plants were identified by Dr. A. Cuerrier and voucher specimens were deposited at the Marie-Victorin herbarium at the Montreal Botanic Garden, Montreal, Quebec, Canada. Additional Canadian specimens were obtained from commercial sources. A list of the teas examined is provided in Table 1. 2.2. Aqueous sample preparation A representative sample of each botanical was ground to a fine consistency using a mortar and pestle. Water or ethanol (EtOH) extracts were prepared by vigorously vortexing 5 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
1000g at room temperature. Decoctions were prepared from Rhododendron groenlandicum leaves, Rhododendron tomentosa leaves, or Juniperus communis berries by adding 1 g of the dried or fresh material to 250 ml of boiling water. The water was maintained at a boiling temperature and aliquots of the tea were taken after at various boiling times ranging from 0 to 180 min. 2.3. 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 (Foster et al., 2001; Tam et al., 2009). 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 sample, 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 7.4) at a final volume of 200 μl for 20 min. The initial and final fluorescence was read at 485 nm excitation and 530 nm emissions with a gain of 50. For alcohol extracts, the extract was diluted tenfold prior to testing. The positive inhibitor used was 1.9 μM ketoconazole (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 water vehicle control. 2.3.1. CYP3A4 mechanism-based inactivation assay The assays were performed in similar conditions as stated above. The procedure was adapted from Tam et al. (2009). Briefly, two types of solutions were prepared for each experiment: a preincubation solution where conditions are set to screen for mechanism-based inactivation (MBI), containing 0.25 μM EDTA, the tea sample or water (vehicle control) and 100 nM CYP3A4 in 0.14 M buffer; and a reaction solution which was used to determine the remaining activity of the CYP3A4, containing 1.1 mM NADPH and 1 μM DBF in 0.13 M buffer. For each experiment, tea samples were compared to a water control to determine percent inhibition. To test for NADPH-dependence, pre-incubation solutions were prepared as stated above as well as identical preincubation solutions containing 1.1 mM NADPH, for both the tea sample and vehicle control. CYP3A4 was added last to the preincubation solutions and incubated for 15 min at 37 1C. Aliquots of the pre-incubation solutions were added to the incubation solution and diluted ten-fold. After a 20 min incubation period at 37 1C, fluorescence released from metabolized DBF (fluorescein) was measured to determine the activity of the remaining active enzymes.
Table 1 List of the medicinal teas tested. Reference
Species [voucher number]
Medicinal use
CEI-3 NRP 216
Juniperus communis, Northern Quebec, Canada. [Whap04-6] Rhododendron groenlandicum, Saskatchewan, Canada
NRP 217
Rhododendron groenlandicum, Ontario, Canada
NRP 218
Rhododendron groenlandicum, Manitoba, Canada
CEI-1
Rhododendron groenlandicum (Oeder) Kron Judd, Northern Quebec, Canada. [Mis03-2] Rhododendron tomentosum Harmaja, Northern Quebec, Canada. [Whap04-33]
Urinary tract infections Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Diuretic, analgesic; rheumatism, skin disorders, and arthritisa
CEI-2 a b
Chartier et al., 2005. Dufour et al., 2007.
Diuretic, analgesic, rheumatism, skin disorders, and arthritisa
T.W. Tam et al. / Journal of Ethnopharmacology 155 (2014) 841–846
2.4.1. HPLC-PAD-APCI/MS analyses Water and EtOH extracts of dried and fresh berries were re-dissolved at 40 mg/ml in HPLC grade methanol, sonicated for 5 min, filtered through 0.2 μm syringe filters (Chromspec Inc.) and 20 μl was injected on an HPLC DAD APCI MSD system (1100 series, 1946C VL), Agilent Technologies Inc. (Palo Alto, CA, USA). The system consisted of an autosampler with 100 μl built in loop, a quaternary pump (maximum pressure limit 400 bars), a photodiode array, a column thermostat, and an online APCI/MSD with mass range of 50–15,000 amu. The separations were performed on a Synergi Fusion – RP 150 mm 3.00 mm, 4 μm particle size and pore an average diameter of 80 Å (Catalog number PN 00F-4424YO, SN 355361-1) at an oven temperature of 50 1C with a flow rate of 0.4 ml/min using a linear gradient of a binary solvent system comprising A; H2O was 10–100% B (acetonitrile) in 30 min followed by the column wash with 100% B for 2 min. The solvent composition was returned to the initial conditions in 0.1 min, and re-equilibrated for 5 min before next injection (total run time 35 min). The elutions were monitored by total-ion chromatography (TIC).
3. Results The assay system used a series of internal controls to determine which samples, if any, had constituents capable of quenching the fluorescence monitored in the CYP 3A4 assay. 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 products was then undertaken at a single lower concentration where most if not all of the products had an inhibitory effect of 95% or less. 3.1. Rhododendron sp. medicinal teas Four different samples of Rhododendron groenlandicum and one sample of Rhododendron tomentosum collected at different locations in Canada were examined (Table 1). Ethanolic extracts from ground leaves of the two Rhododendron sp. samples from different regions in Northern Quebec (CEI 1 and 2) have been examined previously for their ability to inhibit CYP3A4-mediated metabolism (Tam et al., 2009). As the leaves are generally boiled as a decoction for their medicinal use, whole leaves were examined at several boiling times up to 30 min to determine if the strength of the teas affected the capacity to inhibit CYP3A4 activity. Common to all the different samples examined was a progressive increasing inhibition of CYP3A4 activity with time of boiling (Fig. 1). The results for the two species (CEI 1 and 2) were similar up to the
120
CYP3A4 activity ( % water control)
A 1 ml aliquot of the boiled tea was added to an equal volume of 99% EtOH and placed in a 10 1C freezer for 30 min to precipitate out the polysaccharides. The mixture was then centrifuged and the supernatant was filtered through a 0.2 mm PTTE filter before a 50 μl aliquot of reaction mixture was injected into a Phenomenex C18 column (3 μm practical size, 150 mM 2 mm; Phenomenex, Torrance, CA, US) in an Agilent 1100 Series HPLC system with a diode array detector (DAD). A gradient eluted method initially with a ratio of MeOH:H2O, 0.1% formic acid (5:95, v/v) and gradient change to a ratio of MeOH:ACN:H2O, 0.1% formic acid (10:50:40, v/v) in 25 min, the column was then washed with ACN for 5 min before the next injection. Flow rate was set at 0.3 ml/min, column temperature was 55 1C, and wavelengths 280 nm and 350 nm.
10 min time point but diverged by 20 min. Rhododendron tomentosum (CEI-2) tea was more inhibitory than the other teas. After 5 to 10 min of boiling, it had twice the inhibition of the other teas and inhibited at least 75.3 7 5.5% of CYP3A4 activity. The progressive increase in inhibition with CEI-2 started to slow after 20 min; whereas for the other collections, the progressive increase was steady and linear. After 30 min, CYP3A4 inhibition ranged from 66.2 72.3% to 95.27 2.7% relative to the water vehicle control. According to our phytochemistry study of Rhododendron sp. samples (Black et al., 2011), phenolic substances are major secondary substances in this botanical. Five major compounds were selected for further study in the assay to determine if they were responsible for the inhibition of CYP3A4 (Fig. 2). At a concentration of 10 mM, chlorogenic acid, quercetin-3-rhamnoside, catechin hydrate, and p-coumaric acid had no or a minor effect on CYP3A4mediated metabolism. Quercetin (10 mM ) inhibited 48.6 70.2% of CYP3A4 activity relative to a vehicle control.
NRP216 NRP217 NRP218 CEI-1 CEI-2
100 80 60 40 20 0
0
5
10
20
30
Boiling Time (min) Fig. 1. The progressive inhibition in human cytochrome P450 3A4-mediated metabolism by 10 ml aliquots of a decoction prepared by the extended boiling of whole Rhododendron groenlandicum (Bog Labrador tea) and Rhododendron tomentosum (Marsh Labrador tea) leaves (n¼ 2–3; mean 7 SEM). CEI, Cree Nation of Eeyou Istchee; NRP, Nutraceutical Research Programme number.
120
CYP3A4 Activity (% Vehicle Control)
2.4. HPLC-DAD analyses
843
100
80
60
40
20
0
ke
o toc
na
zo
ch
le
lor
o
n ge
qu
ic a
e erc
tin
cid
-3-
m rha
no
sid
te
e
dra
ca
tec
hin
hy
p-c
ou
ri ma
ca
cid qu
erc
eti
n
Fig. 2. The effect of various authentic reference compounds (10 mM) present in the Labrador tea samples on human cytochrome P450 3A4 activity (n¼ 2; mean 7 SEM). Ketoconazole (1.9 mM) was used as a positive control.
T.W. Tam et al. / Journal of Ethnopharmacology 155 (2014) 841–846
4. Discussion
Undiluted Reconstituted
80
60
40
20
0 NRP 216 NRP 217 NRP 218
100 Without NADPH With NADPH
80
60
40
20
0
a
Az
to Ke
I-1
CE
I-2
CE
Sample Fig. 3. The NADPH-dependent human cytochrome P450 3A4 inhibition effect of decoction prepared by boiling whole Labrador Tea (CEI-1, Rhododendron groenlandicum and CEI-2, Rhododendron tomentosum) leaves for 30 min (n¼ 2; mean 7 SEM). Solid bar, with NADPH; hatching, without NADPH. CEI, Cree Nation of Eeyou Istchee. Azamulin (0.1 mM, Aza) and ketoconazole (0.9 mM, Keto) were used as a positive and negative controls, respectively.
CEI-1
CEI-2
Sample Fig. 4. The effect of boiling on Rhododendron groenlandicum (Bog Labrador tea) activity towards human cytochrome P450 3A4 activity. The decoction was tested with and without reconstitution to the initial volume of 250 ml at 30 min (n¼ 2 and 3; mean 7SEM). CEI, Cree Nation of Eeyou Istchee; NRP, Nutraceutical Research Programme number.
100 Dry Fresh
80
60
40
20
0
0
As a previous study in this laboratory had demonstrated the potential of the Cree botanical extracts to mediate CYP metabolism
CYP3A4 Activity (% Vehicle Control)
100
CYP3A4 Activity (% Water Control)
Teas boiled for five and 30 mins were examined to identify compounds present in the tea, and to determine if changes in the concentrations of the compounds occurred with different boiling times. A representative HPLC phytochemical profile of CE1 and CE2 are given in Supplementary information, Figs. S1 and S2. After 30 min of boiling, the relative peak profiles were similar but in higher concentrations than the 5 min samples. Chlorogenic acid, catechin, epi-catechin, quercetin-3-galactoside and rutin (quercetin 3-O-rutinoside) were commonly identified in the different Labrador teas. Strong CYP3A4 inhibition was observed with whole CEI-1 and CEI-2 leaves after 30 min of boiling. To determine if the inhibition was mechanism-based, the inhibition was tested for NADPH-dependence, a requirement for MBI (Fig. 3). Only CEI-1 and CEI-2 were examined as representative samples for these studies. Azamulin (0.1 μM) and ketoconazole (0.9 μM) were used a positive and negative control of mechanism-based inactivation, respectively. The results indicate that the inhibition was not mechanism-based as the presence of NADPH during the pre-incubation period did not affect the relative degree of inhibition. It was observed that the volume of the water decreased during the boiling process. Approximately half the water had evaporated after 30 min of boiling, consequently concentrating and increasing the strength of the tea. After the 30 min of boiling, the tea was reconstituted with water to a final volume of 250 ml to match the initial water volume. Undiluted and reconstituted samples were examined for inhibition of CYP3A4-mediated metabolism (Fig. 4). As expected, the reconstituted sampled caused less inhibition of CYP3A4 activity. The CYP3A4 present with the reconstituted tea samples had approximately 1.4 to 1.9 times greater activity than CYP3A4 present with the undiluted tea samples. A pilot study with Juniperus communis found similar CYP3A4 inhibitory results that increased with time of preparation (Fig. 5). Information on relative amounts of substances detected is presented in Supplementary information, Fig. S3.
CYP3A4 Inhibition (% Water Control)
844
5
10
20
30
60
120
180
Boiling Time (min) Fig. 5. The effect of increased boiling time on human cytochrome P450 3A4mediated metabolism by Juniperus communis dry or fresh berry tea (n¼ 3; mean7 SEM). An aliquot of 10 mL of the decoction was sampled at various time points.
(Tam et al., 2009), this study examined the effect of a decoction on the inhibitory potential. Specific details on how traditional aboriginal medicines are prepared are considered a sacred ceremony and consequently could not be shared freely by the Elders and healers. However, the mechanical processing is generally known to follow the basic boiling method of preparing traditional medicine decoctions. As expected, the inhibitory potential increased markedly after an initial lag with time. Chromatographic analysis of the Cree samples confirmed that the relative amounts of the nonvolatile soluble constituents increased with time, a finding consistent from discussions with the Elders and healers that the medicinal use will change as the length of preparation. 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 (US FDA, 1999). The findings here demonstrate the need to examine both aqueous and ethanolic extracts for their potential to affect a biotransformation enzyme. There can be substantial variation in the composition of botanical products due to several factors
T.W. Tam et al. / Journal of Ethnopharmacology 155 (2014) 841–846
including environmental conditions, time and year of harvest, and the manufacturing and storage processes (Ho et al., 2009; Erturk et al., 2010). With the broad range of substances present in many botanicals, it is not unrealistic that other solvents could also be required to give a fuller understanding of any potential interactions. The complex nature of botanicals can effectively preclude unequivocal association of a pharmacological effect to a single substance as there may be multiple pharmacologically active substances affecting one or more pathways that can affect bioavailability and hence, pharmacodynamic activity. Thus, it is important to note that some substances may act through synergism or antagonism depending of their concentrations which could have a profound effect on the clinical efficacy and safety. Quercetin is a flavonol present as an aglycone or conjugate in many fruits, grains, vegetables and botanicals used as traditional medicines. The finding of its moderate potential to affect CYP3A4mediated metabolism is significant and could underlie some fooddrug or herb-drug interactions. However, the dose of 10 mM used in our studies lies in the higher range of concentrations to be expected in biological fluids and it is not clear if such concentrations will be reached in the intracellular milieu where the CYP3A4 isozyme normally exists. The finding of its' potential to affect CYP3A4-mediated metabolism underlies the need for further study of the interaction of foods and medicines, especially in light of popular trends to consume more phytochemically rich plants for their health benefits where an additive or synergistic effect with other substances may occur. Further studies will be required to fully address the relevance of this finding. Teas and decoctions have long been 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 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 interactions with these botanicals and other health products metabolized by CYP3A4.
5. Conclusion The findings of this study with these two traditional medicines are significant in that they provide mechanistic support that 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 CYP3A4, it is possible that these teas contain additional substances that could also affect other metabolic enzymes. This uncertainty warrants further investigation.
Source of funding This project was funded by a strategic grant from the Canadian Institutes of Health Research.
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Acknowledgments Special thanks to Elizabeth Coon Come, Harriet Matoush, Sandy Matoush, Charlotte Petawabano, Laurie Petawabano, Pat Petawabano, Sam Petawabano, Simeon Petawabano, and Smally Petawabano from the Cree Nation of Mistissini, and Agnes Kawapit, Eliza Kawapit, Abraham Mamianskum, and Juliet Mamianskum from Whapmagoostui First Nation as well as to Cree Elders of both nations of the Eeyou Istchee who kindly agreed to be interviewed. They made this article possible by allowing us to use, for the purposes of this research, their knowledge relating to medicinal plants, transmitted to them by their Elders. Their trust has also enabled a useful exchange between Indigenous knowledge and Western science. We also acknowledge the technical expertise of Jingqin Mao, and the support and input from Dr. William Staines.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.06.045.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
The effect of Cree traditional medicinal teas on the activity of human cytochrome P450-mediated metabolism Teresa W. Tam a,b, Rui Liu a,b, Ammar Saleem b, John T. Arnason b, Anthony Krantis a,b, Pierre S. Haddad c, Brian C. Foster a,b,n a
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa, Ottawa, ON, Canada K1H 8M5 c Department of Pharmacology, Université de Montréal, Montréal, QC, Canada H3C 3J7 b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 April 2014 Received in revised form 16 June 2014 Accepted 17 June 2014 Available online 24 June 2014
Ethnopharmacological relevance: Rhododendron groenlandicum (Bog Labrador tea), Rhododendron tomentosum (Marsh Labrador tea) and Juniperus communis (Juniper) are used in medicinal teas by Canadian aboriginal cultures alone and in combination with conventional drug products. The safety of this combination had not been previously examined and this study was initiated to examine the potential of medicinal teas to inhibit the major human drug metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). Materials and methods: The decoctions of Rhododendron groenlandicum and Rhododendron tomentosum leaves and Juniperus communis berries were examined in a microtiter fluorometric assay to examine their potential to inhibit CYP-mediated metabolism. Results: The decoctions showed progressive inhibition towards CYP3A4 the longer the leaves or berries were brewed. R. Rhododendron groenlandicum and Juniperus communis may have the potential to inhibit CYP3A4-mediated metabolism. Conclusions: The findings of this study with these traditional medicines are significant in that they provide mechanistic support that these products have the potential to affect the safety and efficacy of other health and medicinal products. As this study only examined CYP3A4, it is possible that these medicinals contain substances that could also affect other metabolic enzymes. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytochrome P450 Labrador tea Juniperus communis Rhododendron groenlandicum Traditional medicine Cree
1. Introduction The use of traditional medicinal plants to treat ailments is still commonly practiced among First Nations in Canada, and teas or decoctions are the most common preparation (Ritch-Krc et al., 1996). In a collaborative ethnobotanical study undertaken by the CIHR team in aboriginal antidiabetic medicines with the Cree Healers of Eeyou Istchee (Quebec, Canada) two of the common medicinal teas used were Juniper and Labrador teas (Leduc et al., 2006; Spoor et al., 2006). These species are also widely used by other First Nations (Arnason et al., 1981; Moerman, 1998). There are two Eastern Canadian species of Labrador tea: Rhododendron groenlandicum (Bog Labrador tea, synonym Ledum groenlandicum) and Rhododendron tomentosum (Marsh Labrador tea, synonym
n Corresponding author at: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5. Tel.: þ1 613 224 7244. E-mail address: [email protected] (B.C. Foster).
http://dx.doi.org/10.1016/j.jep.2014.06.045 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Ledum tomentosum) (Black et al., 2011). Labrador tea is an Ericaceae shrub with evergreen leaves and is used traditionally by First Nations of Eastern Canada to treat a variety of ailments such as inflammation, asthma, rheumatism, digestive and skin disorders, or kidney and liver diseases (Arnason et al., 1981; Chartier et al., 2005; Dufour et al., 2007). Rhododendron tomentosum is found in subarctic habitats, and is used by both Inuit and Northern Cree communities while Rhododendron groenlandicum is found in boreal and mixed forest regions and is used by Cree, Algonquin and other First Nations (Arnason et al., 1981). The leaves are the most commonly used part for medicinal teas and decoctions, and are prepared by brewing the leaves in hot or boiling water. Juniper, Juniperus communis is a small shrub or tree belonging in the family Cupressaceae and is found in boreal and deciduous regions of Eastern Canada. The berry-like seed cones, which are green ripening to purple black, are used for cystitis by the Cree (Arnason et al., 1981). Through discussions in our ethnobotanical study with Cree healers and Elders, they became aware of studies that have
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demonstrated that some herbal and traditional medicines can interact with conventional and alternative medicines (Foster et al., 2003; Zhou et al., 2003; Marchetti et al., 2007; Nowack, 2008; Tam et al., 2009, 2011). As the Cree healers are very concerned about conditions for safe use of traditional medicines, this study was undertaken at their request to determine whether the decoctions of these botanicals have the capacity to affect the safety of conventional medicines used in their community. As a first step, we studied the potential of the teas to inhibit cytochrome P450 3A4-mediated (CYP3A4) biotransformation of a marker substance in an in vitro system. This assay was examined to provide mechanistic information that could be related to patient safety (US FDA, 1999). Chromatographic analysis was undertaken to provide a baseline of the substances present in the extracts for any future comparative analysis between our findings and others. CYP3A4 is the major human drug metabolizing enzyme of large, diverse, lipophilic substances (Anzenbacher and Anzenbacherová, 2001).
2. Materials and methods 2.1. Chemicals and reagents All solvents for HPLC analysis were optima grade including methanol (MeOH) and acetonitrile (ACN) (Fisher Scientific, Ottawa, ON, Canada). Dibenzylfluorescein (DBF) and microsomes derived from Baculovirus infected insect cells expressing CYP3A4 and CYPreductase 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). Samples of the selected plant parts of the Cree Nation of Eeyou Istchee (CEI) medicines were harvested in Mistissini, Quebec, Canada following the instructions of the Elders and healers of this community (Leduc et al., 2006). These plants were identified by Dr. A. Cuerrier and voucher specimens were deposited at the Marie-Victorin herbarium at the Montreal Botanic Garden, Montreal, Quebec, Canada. Additional Canadian specimens were obtained from commercial sources. A list of the teas examined is provided in Table 1. 2.2. Aqueous sample preparation A representative sample of each botanical was ground to a fine consistency using a mortar and pestle. Water or ethanol (EtOH) extracts were prepared by vigorously vortexing 5 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
1000g at room temperature. Decoctions were prepared from Rhododendron groenlandicum leaves, Rhododendron tomentosa leaves, or Juniperus communis berries by adding 1 g of the dried or fresh material to 250 ml of boiling water. The water was maintained at a boiling temperature and aliquots of the tea were taken after at various boiling times ranging from 0 to 180 min. 2.3. 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 (Foster et al., 2001; Tam et al., 2009). 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 sample, 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 7.4) at a final volume of 200 μl for 20 min. The initial and final fluorescence was read at 485 nm excitation and 530 nm emissions with a gain of 50. For alcohol extracts, the extract was diluted tenfold prior to testing. The positive inhibitor used was 1.9 μM ketoconazole (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 water vehicle control. 2.3.1. CYP3A4 mechanism-based inactivation assay The assays were performed in similar conditions as stated above. The procedure was adapted from Tam et al. (2009). Briefly, two types of solutions were prepared for each experiment: a preincubation solution where conditions are set to screen for mechanism-based inactivation (MBI), containing 0.25 μM EDTA, the tea sample or water (vehicle control) and 100 nM CYP3A4 in 0.14 M buffer; and a reaction solution which was used to determine the remaining activity of the CYP3A4, containing 1.1 mM NADPH and 1 μM DBF in 0.13 M buffer. For each experiment, tea samples were compared to a water control to determine percent inhibition. To test for NADPH-dependence, pre-incubation solutions were prepared as stated above as well as identical preincubation solutions containing 1.1 mM NADPH, for both the tea sample and vehicle control. CYP3A4 was added last to the preincubation solutions and incubated for 15 min at 37 1C. Aliquots of the pre-incubation solutions were added to the incubation solution and diluted ten-fold. After a 20 min incubation period at 37 1C, fluorescence released from metabolized DBF (fluorescein) was measured to determine the activity of the remaining active enzymes.
Table 1 List of the medicinal teas tested. Reference
Species [voucher number]
Medicinal use
CEI-3 NRP 216
Juniperus communis, Northern Quebec, Canada. [Whap04-6] Rhododendron groenlandicum, Saskatchewan, Canada
NRP 217
Rhododendron groenlandicum, Ontario, Canada
NRP 218
Rhododendron groenlandicum, Manitoba, Canada
CEI-1
Rhododendron groenlandicum (Oeder) Kron Judd, Northern Quebec, Canada. [Mis03-2] Rhododendron tomentosum Harmaja, Northern Quebec, Canada. [Whap04-33]
Urinary tract infections Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Anti-inflammatory, kidney and liver diseases; asthma; rheumatism; digestive disturbancesa,b Diuretic, analgesic; rheumatism, skin disorders, and arthritisa
CEI-2 a b
Chartier et al., 2005. Dufour et al., 2007.
Diuretic, analgesic, rheumatism, skin disorders, and arthritisa
T.W. Tam et al. / Journal of Ethnopharmacology 155 (2014) 841–846
2.4.1. HPLC-PAD-APCI/MS analyses Water and EtOH extracts of dried and fresh berries were re-dissolved at 40 mg/ml in HPLC grade methanol, sonicated for 5 min, filtered through 0.2 μm syringe filters (Chromspec Inc.) and 20 μl was injected on an HPLC DAD APCI MSD system (1100 series, 1946C VL), Agilent Technologies Inc. (Palo Alto, CA, USA). The system consisted of an autosampler with 100 μl built in loop, a quaternary pump (maximum pressure limit 400 bars), a photodiode array, a column thermostat, and an online APCI/MSD with mass range of 50–15,000 amu. The separations were performed on a Synergi Fusion – RP 150 mm 3.00 mm, 4 μm particle size and pore an average diameter of 80 Å (Catalog number PN 00F-4424YO, SN 355361-1) at an oven temperature of 50 1C with a flow rate of 0.4 ml/min using a linear gradient of a binary solvent system comprising A; H2O was 10–100% B (acetonitrile) in 30 min followed by the column wash with 100% B for 2 min. The solvent composition was returned to the initial conditions in 0.1 min, and re-equilibrated for 5 min before next injection (total run time 35 min). The elutions were monitored by total-ion chromatography (TIC).
3. Results The assay system used a series of internal controls to determine which samples, if any, had constituents capable of quenching the fluorescence monitored in the CYP 3A4 assay. 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 products was then undertaken at a single lower concentration where most if not all of the products had an inhibitory effect of 95% or less. 3.1. Rhododendron sp. medicinal teas Four different samples of Rhododendron groenlandicum and one sample of Rhododendron tomentosum collected at different locations in Canada were examined (Table 1). Ethanolic extracts from ground leaves of the two Rhododendron sp. samples from different regions in Northern Quebec (CEI 1 and 2) have been examined previously for their ability to inhibit CYP3A4-mediated metabolism (Tam et al., 2009). As the leaves are generally boiled as a decoction for their medicinal use, whole leaves were examined at several boiling times up to 30 min to determine if the strength of the teas affected the capacity to inhibit CYP3A4 activity. Common to all the different samples examined was a progressive increasing inhibition of CYP3A4 activity with time of boiling (Fig. 1). The results for the two species (CEI 1 and 2) were similar up to the
120
CYP3A4 activity ( % water control)
A 1 ml aliquot of the boiled tea was added to an equal volume of 99% EtOH and placed in a 10 1C freezer for 30 min to precipitate out the polysaccharides. The mixture was then centrifuged and the supernatant was filtered through a 0.2 mm PTTE filter before a 50 μl aliquot of reaction mixture was injected into a Phenomenex C18 column (3 μm practical size, 150 mM 2 mm; Phenomenex, Torrance, CA, US) in an Agilent 1100 Series HPLC system with a diode array detector (DAD). A gradient eluted method initially with a ratio of MeOH:H2O, 0.1% formic acid (5:95, v/v) and gradient change to a ratio of MeOH:ACN:H2O, 0.1% formic acid (10:50:40, v/v) in 25 min, the column was then washed with ACN for 5 min before the next injection. Flow rate was set at 0.3 ml/min, column temperature was 55 1C, and wavelengths 280 nm and 350 nm.
10 min time point but diverged by 20 min. Rhododendron tomentosum (CEI-2) tea was more inhibitory than the other teas. After 5 to 10 min of boiling, it had twice the inhibition of the other teas and inhibited at least 75.3 7 5.5% of CYP3A4 activity. The progressive increase in inhibition with CEI-2 started to slow after 20 min; whereas for the other collections, the progressive increase was steady and linear. After 30 min, CYP3A4 inhibition ranged from 66.2 72.3% to 95.27 2.7% relative to the water vehicle control. According to our phytochemistry study of Rhododendron sp. samples (Black et al., 2011), phenolic substances are major secondary substances in this botanical. Five major compounds were selected for further study in the assay to determine if they were responsible for the inhibition of CYP3A4 (Fig. 2). At a concentration of 10 mM, chlorogenic acid, quercetin-3-rhamnoside, catechin hydrate, and p-coumaric acid had no or a minor effect on CYP3A4mediated metabolism. Quercetin (10 mM ) inhibited 48.6 70.2% of CYP3A4 activity relative to a vehicle control.
NRP216 NRP217 NRP218 CEI-1 CEI-2
100 80 60 40 20 0
0
5
10
20
30
Boiling Time (min) Fig. 1. The progressive inhibition in human cytochrome P450 3A4-mediated metabolism by 10 ml aliquots of a decoction prepared by the extended boiling of whole Rhododendron groenlandicum (Bog Labrador tea) and Rhododendron tomentosum (Marsh Labrador tea) leaves (n¼ 2–3; mean 7 SEM). CEI, Cree Nation of Eeyou Istchee; NRP, Nutraceutical Research Programme number.
120
CYP3A4 Activity (% Vehicle Control)
2.4. HPLC-DAD analyses
843
100
80
60
40
20
0
ke
o toc
na
zo
ch
le
lor
o
n ge
qu
ic a
e erc
tin
cid
-3-
m rha
no
sid
te
e
dra
ca
tec
hin
hy
p-c
ou
ri ma
ca
cid qu
erc
eti
n
Fig. 2. The effect of various authentic reference compounds (10 mM) present in the Labrador tea samples on human cytochrome P450 3A4 activity (n¼ 2; mean 7 SEM). Ketoconazole (1.9 mM) was used as a positive control.
T.W. Tam et al. / Journal of Ethnopharmacology 155 (2014) 841–846
4. Discussion
Undiluted Reconstituted
80
60
40
20
0 NRP 216 NRP 217 NRP 218
100 Without NADPH With NADPH
80
60
40
20
0
a
Az
to Ke
I-1
CE
I-2
CE
Sample Fig. 3. The NADPH-dependent human cytochrome P450 3A4 inhibition effect of decoction prepared by boiling whole Labrador Tea (CEI-1, Rhododendron groenlandicum and CEI-2, Rhododendron tomentosum) leaves for 30 min (n¼ 2; mean 7 SEM). Solid bar, with NADPH; hatching, without NADPH. CEI, Cree Nation of Eeyou Istchee. Azamulin (0.1 mM, Aza) and ketoconazole (0.9 mM, Keto) were used as a positive and negative controls, respectively.
CEI-1
CEI-2
Sample Fig. 4. The effect of boiling on Rhododendron groenlandicum (Bog Labrador tea) activity towards human cytochrome P450 3A4 activity. The decoction was tested with and without reconstitution to the initial volume of 250 ml at 30 min (n¼ 2 and 3; mean 7SEM). CEI, Cree Nation of Eeyou Istchee; NRP, Nutraceutical Research Programme number.
100 Dry Fresh
80
60
40
20
0
0
As a previous study in this laboratory had demonstrated the potential of the Cree botanical extracts to mediate CYP metabolism
CYP3A4 Activity (% Vehicle Control)
100
CYP3A4 Activity (% Water Control)
Teas boiled for five and 30 mins were examined to identify compounds present in the tea, and to determine if changes in the concentrations of the compounds occurred with different boiling times. A representative HPLC phytochemical profile of CE1 and CE2 are given in Supplementary information, Figs. S1 and S2. After 30 min of boiling, the relative peak profiles were similar but in higher concentrations than the 5 min samples. Chlorogenic acid, catechin, epi-catechin, quercetin-3-galactoside and rutin (quercetin 3-O-rutinoside) were commonly identified in the different Labrador teas. Strong CYP3A4 inhibition was observed with whole CEI-1 and CEI-2 leaves after 30 min of boiling. To determine if the inhibition was mechanism-based, the inhibition was tested for NADPH-dependence, a requirement for MBI (Fig. 3). Only CEI-1 and CEI-2 were examined as representative samples for these studies. Azamulin (0.1 μM) and ketoconazole (0.9 μM) were used a positive and negative control of mechanism-based inactivation, respectively. The results indicate that the inhibition was not mechanism-based as the presence of NADPH during the pre-incubation period did not affect the relative degree of inhibition. It was observed that the volume of the water decreased during the boiling process. Approximately half the water had evaporated after 30 min of boiling, consequently concentrating and increasing the strength of the tea. After the 30 min of boiling, the tea was reconstituted with water to a final volume of 250 ml to match the initial water volume. Undiluted and reconstituted samples were examined for inhibition of CYP3A4-mediated metabolism (Fig. 4). As expected, the reconstituted sampled caused less inhibition of CYP3A4 activity. The CYP3A4 present with the reconstituted tea samples had approximately 1.4 to 1.9 times greater activity than CYP3A4 present with the undiluted tea samples. A pilot study with Juniperus communis found similar CYP3A4 inhibitory results that increased with time of preparation (Fig. 5). Information on relative amounts of substances detected is presented in Supplementary information, Fig. S3.
CYP3A4 Inhibition (% Water Control)
844
5
10
20
30
60
120
180
Boiling Time (min) Fig. 5. The effect of increased boiling time on human cytochrome P450 3A4mediated metabolism by Juniperus communis dry or fresh berry tea (n¼ 3; mean7 SEM). An aliquot of 10 mL of the decoction was sampled at various time points.
(Tam et al., 2009), this study examined the effect of a decoction on the inhibitory potential. Specific details on how traditional aboriginal medicines are prepared are considered a sacred ceremony and consequently could not be shared freely by the Elders and healers. However, the mechanical processing is generally known to follow the basic boiling method of preparing traditional medicine decoctions. As expected, the inhibitory potential increased markedly after an initial lag with time. Chromatographic analysis of the Cree samples confirmed that the relative amounts of the nonvolatile soluble constituents increased with time, a finding consistent from discussions with the Elders and healers that the medicinal use will change as the length of preparation. 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 (US FDA, 1999). The findings here demonstrate the need to examine both aqueous and ethanolic extracts for their potential to affect a biotransformation enzyme. There can be substantial variation in the composition of botanical products due to several factors
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including environmental conditions, time and year of harvest, and the manufacturing and storage processes (Ho et al., 2009; Erturk et al., 2010). With the broad range of substances present in many botanicals, it is not unrealistic that other solvents could also be required to give a fuller understanding of any potential interactions. The complex nature of botanicals can effectively preclude unequivocal association of a pharmacological effect to a single substance as there may be multiple pharmacologically active substances affecting one or more pathways that can affect bioavailability and hence, pharmacodynamic activity. Thus, it is important to note that some substances may act through synergism or antagonism depending of their concentrations which could have a profound effect on the clinical efficacy and safety. Quercetin is a flavonol present as an aglycone or conjugate in many fruits, grains, vegetables and botanicals used as traditional medicines. The finding of its moderate potential to affect CYP3A4mediated metabolism is significant and could underlie some fooddrug or herb-drug interactions. However, the dose of 10 mM used in our studies lies in the higher range of concentrations to be expected in biological fluids and it is not clear if such concentrations will be reached in the intracellular milieu where the CYP3A4 isozyme normally exists. The finding of its' potential to affect CYP3A4-mediated metabolism underlies the need for further study of the interaction of foods and medicines, especially in light of popular trends to consume more phytochemically rich plants for their health benefits where an additive or synergistic effect with other substances may occur. Further studies will be required to fully address the relevance of this finding. Teas and decoctions have long been 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 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 interactions with these botanicals and other health products metabolized by CYP3A4.
5. Conclusion The findings of this study with these two traditional medicines are significant in that they provide mechanistic support that 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 CYP3A4, it is possible that these teas contain additional substances that could also affect other metabolic enzymes. This uncertainty warrants further investigation.
Source of funding This project was funded by a strategic grant from the Canadian Institutes of Health Research.
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Acknowledgments Special thanks to Elizabeth Coon Come, Harriet Matoush, Sandy Matoush, Charlotte Petawabano, Laurie Petawabano, Pat Petawabano, Sam Petawabano, Simeon Petawabano, and Smally Petawabano from the Cree Nation of Mistissini, and Agnes Kawapit, Eliza Kawapit, Abraham Mamianskum, and Juliet Mamianskum from Whapmagoostui First Nation as well as to Cree Elders of both nations of the Eeyou Istchee who kindly agreed to be interviewed. They made this article possible by allowing us to use, for the purposes of this research, their knowledge relating to medicinal plants, transmitted to them by their Elders. Their trust has also enabled a useful exchange between Indigenous knowledge and Western science. We also acknowledge the technical expertise of Jingqin Mao, and the support and input from Dr. William Staines.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.06.045.
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