Effects of Standardized Medicinal Plant Extracts on Drug Metabolism Mediated by CYP3A4 and CYP2D6 Enzymes

Article abstract of DOI:10.1021/acs.chemrestox.0c00182

The use of medicinal plants concomitantly with conventional drugs can result in herb-drug interactions that cause fluctuations in drug bioavailability and consequent therapeutic failure and/or toxic effects. The CYP superfamily of enzymes plays an importa

Full text of DOI:10.1021/acs.chemrestox.0c00182

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Article
Effects of standardized medicinal plant extracts on drug
metabolism mediated by CYP3A4 and CYP2D6 enzymes
Clarissa Feltrin, Ingrid Vicente Farias, Louis P Sandjo, Flavio
Henrique Reginatto, and Claudia Maria Oliveira Simoes
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.0c00182 • Publication Date (Web): 29 Jul 2020
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Effects of standardized medicinal plant extracts on drug
metabolism mediated by CYP3A4 and CYP2D6 enzymes
†
†
Clarissa Feltrin ; Ingrid Vicente Farias ; Louis Pergaud Sandjo ‡; Flávio Henrique
†
†
Reginatto ; Cláudia Maria Oliveira Simões *
†
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Programa de Pós-Graduação em Farmácia, Centro de Ciências da Saúde, Universidade Federal de
Santa Catarina, Florianópolis, Santa Catarina, Brazil.
‡ Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina,
Brazil.
*Corresponding author: Cláudia Maria Oliveira Simões, claudia.simoes@ufsc.br. Phone: +55-48 3721
3
499. Address: Programa de Pós-Graduação em Farmácia, Centro de Ciências da Saúde, Universidade
Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, Rua Delfino Conti, s/n, Trindade,
Florianópolis, Santa Catarina, Brazil, CEP: 88040-900.
KEYWORDS. Cecropia glaziovii; CYP2D6; CYP3A4; herb-drug interactions; Ilex paraguariensis; UPLC-
MS/MS.
ABSTRACT: The use of medicinal plants concomitantly with conventional drugs can result in herb-drug
interactions that cause fluctuations in drug bioavailability, and consequent therapeutic failure and/or
toxic effects. The CYP superfamily of enzymes plays an important role in herb-drug interactions. Among
CYP enzymes, CYP3A4 and CYP2D6 are the most relevant since they metabolize about 50% and 30% of
the drugs on the market, respectively. Thus, the main goal of this study was to evaluate the occurrence of
in vitro interactions between medicinal plant extracts and drug substrates of CYP3A4 and CYP2D6
enzymes. Standardized extracts from nine medicinal plants (Bauhinia forficata, Cecropia glaziovii,
Cimicifuga racemosa, Cynara scolymus, Echinacea sp, Ginkgo biloba, Glycine max, Ilex paraguariensis, and
Matricaria recutita) were evaluated for their potential interactions mediated by CYP3A4 and CYP2D6
enzymes. Among the extracts tested, C. glaziovii (red embaúba) showed the most relevant inhibitory
effects of CYP3A4 and CYP2D6 activity, while I. paraguariensis (yerba mate) inhibited CYP3A4 activity.
Both extracts were chemically analyzed by UPLC-MS/MS, and these inhibitory effects could lead to
clinically potential and relevant interactions with the drug substrates of these isoenzymes.
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. INTRODUCTION
interactions through the rapid increase of drug
plasma levels leading to drug-induced toxicity .
Several studies report significant modulation of CYP
3
4
Complementary and alternative medicine (CAM) is
a worldwide culturally-based age-old medicinal
1
, 2
practice . CAM is commonly used by individuals
suffering from chronic pain or chronic diseases ,
such as cancer
obesity, diabetes , arthritis , neurocognitive
disorders and HIV+/AIDS
conventional therapies or for the relief of symptoms
enzymes by drugs, their metabolites, and natural
3
4
35, 36
products
; for instance, goldenseal (Hydrastis
5
,
6
9
7, 8
, cardiovascular diseases
,
canadensis), liquorice (Glycyrrhiza glabra), valerian
10
37
(Valeriana officinalis) , St. John's wort (Hypericum
1
1
38, 39
to complement
perforatum)
, and medicinal plants used in
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Traditional Chinese medicine
metabolites, such as polyphenols
.
Secondary
1
2, 13
28, 41, 42
.
and
4
3
In 2012, the National Health Interview Survey
carried out a study revealing that the use of natural
products was the most common complementary
health approach among adults . In this context,
among CAM, the use of medicinal plants stands out
, and their use combined with conventional drugs
exposes users to the risk of herb-drug interactions
. Unfortunately, the assumption that these folk
remedies are always safe encourages their
alkaloids
are also described as CYP enzyme
inhibitors. However, due to the enormous plant
biodiversity of the planet, most medicinal plants
have been poorly studied, and little is known about
3
4
4
their chemical constituents .
1
4
The regulatory agencies Food and Drug
Administration (FDA, USA) and European Medicines
Agency (EMA), provide guidelines concerning the
1
5
interactions involving CYP enzymes and emphasize
1
6, 17
32, 45
consumption without professional guidance
.
the need for further studies
. Thus, this work
Herb-drug interactions are a complex process that
can occur with multiple medications targeting
different metabolic pathways, and involving
reports the investigation of the standardized
extracts of the following nine medicinal plants
regarding their potential to lead herb-drug
interactions mediated by CYP3A4 and CYP2D6
enzymes: Bauhinia forficata Link., Cecropia glaziovii
Sneth., Cimicifuga racemosa L., Cynara scolymus L.,
Echinacea sp., Ginkgo biloba L., Glycine max (L.)
Merr., Ilex paraguariensis A. St.-Hil, and Matricaria
recutita L. Midazolam and metoprolol, that are
CYP3A4 and CYP2D6 substrates, respectively, were
used to analyze the potential interactions
employing human recombinant enzymes. The
extracts that inhibited more expressively these
enzymes activities were characterized by Ultra-
performance Liquid Chromatographic-tandem Mass
Spectrometry (UPLC-MS/MS) to detect their major
phytoconstituents and to explore their involvement
in the detected CYP3A4 and CYP2D6 inhibition.
1
8-
different compounds present in herbal products
2
0
.
These processes are not yet completely
understood, although extensive research on this
subject has been conducted in the last years to
provide more information about the way these
interactions occur, and their impact on
1
8,
pharmacological treatments and patients’ health
2
0-22
.
Herb-drug interactions usually arise when
medicines and products prepared with medicinal
plants are administered orally, which is the patients’
preferred route of administration due to the
convenience of use. However, this route exposes the
xenobiotics to first-pass metabolism , a process
involving several enzymes, including the
cytochrome P450 (CYP), a superfamily of enzymes
that metabolize 70-80% of conventional drugs in
clinical use. These enzymes are responsible for the
biotransformation, controlling the plasma and
tissue levels of the bioactive compounds, and they
play an essential role in drug-drug and herb-drug
2
3
2. MATERIALS AND METHODS
2.1 Materials
2.1.1 Enzyme and chemicals
CYP3A4 Baculosomes™ Plus Reagent, rHuman was
®
purchased from Thermo
Fisher Scientific
2
4-27
interactions
.
(Waltham, MA, USA). Cytochrome P450 2D6 human
enzyme (E9413), Trizma base, NaCl, nicotinamide
Inhibition or induction of CYP enzymes may modify
the bioavailability or other pharmacokinetic
parameters of drugs, which can lead to therapeutic
failure and/or toxic effects, especially with drugs
adenine
ethylenediaminetetraacetic
dinucleotide
phosphate
acid
(NADPH),
(EDTA),
trifluoracetic acid (TFA), dimethyl sulfoxide
(DMSO), ketoconazole (≥ 99% purity), and
quinidine (> 99% purity) were purchased from
Sigma-Aldrich (Darmstadt, Hessen, Germany).
Metoprolol tartrate (≥ 98% purity) and midazolam
(> 99% purity) were provided by HenriFarma
Produtos Químicos e Farmacêuticos (Cambuci, SP,
Brazil) and Nortec Química (Rio de Janeiro, RJ,
Brazil), respectively. All HPLC grade solvents were
obtained from Merck (Darmstadt, Hessen,
Germany). Information concerning the tested
extracts is shown in Table 1.
2
8, 29
with a narrow therapeutic index
.
Among the CYP enzymes, CYP3A4 is the most
abundant in the body, with high expression in the
liver and intestine, particularly in the jejunum and
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0, 31
ileum
. CYP3A4 enzyme metabolizes about half
of the available drugs, and it is modulated by several
compounds, such as ketoconazole, verapamil,
rifampicin, and clarithromycin
major drug-metabolizing enzyme is CYP2D6, which
catalyzes the metabolism of approximately one-
third of the available drugs . The inhibition of this
isoenzyme may lead to potential adverse
32, 33
. The second
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Table 1. Standardized medicinal plant extracts.
a
Medicinal plant
Family
Asteraceae
Asteraceae
Urticaceae
Asteraceae
Ranunculaceae
Aquifoliaceae
Ginkgoaceae
Fabaceae
Common name
Globe artichoke
Chamomile
Red embaúba
Coneflower
Black cohosh
Yerba mate
Ginkgo
Chemical markers
Chlorogenic acid 0.5%
Flavonoids 0.16%
Chlorogenic acid 0.11%
Tannins 1.62%
Cynara scolymus L.
Matricaria recutita L.
Cecropia glaziovii Sneth.
Echinacea sp.
Cimicifuga racemosa L.
Ilex paraguariensis A. St.-Hil.
Ginkgo biloba L.
Triterpene glycosides 0.77%
Chlorogenic acid 6.75%
Flavonoid glycosides 25.8%
Flavonoid expressed as quercetin 2%
Isoflavone 40.5%
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Bauhinia forficata Link.
Glycine max (L.) Merr.
Orchid tree
Soy
Fabaceae
a
These materials are referred as standardized extracts and are conformed to the international analytical requirements.
2
.2 Methods
The reactions were performed using microsomes
prepared from CYP2D6 human and yeast CYP-
reductase expressed in Saccharomyces cerevisiae.
The reaction mixtures containing metoprolol (80
µM), with or without quinidine or the extracts and 1
pmol of Cytochrome P450 2D6 human enzyme,
were pre-incubated at 30°C for 5 min. After, 600 µM
NADPH were added to initiate the reactions. The
reaction mixtures (50 μL) were incubated at 30°C
for 10 min and the reactions were quenched by
adding 1 µL of TFA 50%. The samples were vortexed
for 30 s and placed in an ice bath for 1 min.
Afterwards, cold acetonitrile (ACN, 49 µL) was
added to the mixtures, vortexed for 30 s and
centrifuged for 5 min at 9,400 x g and the
supernatants were collected for UPLC-MS analyses.
Quinidine 0.08 µM was used as CYP2D6 inhibitor.
2.2.1 Drugs and extracts preparation
Stock solutions of midazolam (1 mM) and
ketoconazole (100 µM) were solubilized in dimethyl
sulfoxide (DMSO) and diluted in a mixture of 50 mM
Tris (pH 7.4) plus 1 mM EDTA at 125 µM and 50 µM,
respectively. Stock solutions of metoprolol (1 mM)
and quinidine (0.5 µM) were solubilized in a mixture
of 50 mM Tris (pH 7.4) plus 1 mM EDTA. The stock
solutions of standardized medicinal plant extracts
(
3
(
1,000 µg/mL) were solubilized in PBS (pH 7.4) plus
mM MgCl for CYP3A4 reactions or 50 mM Tris
pH 7.4) plus 1 mM EDTA for CYP2D6 reactions. All
2
solutions were filtered through 0.22 µm membranes
and used for the enzymatic reactions. To prepare
the inhibition curves, the extracts were diluted at
the concentrations used for the curves in their
reaction mixtures. For the dereplication studies, I.
paraguariensis extract was solubilized in methanol:
water (1:1 v/v) at 500 µg/mL, and C. glaziovii in
ultrapure water at 100 and 200 µg/mL. Samples
were injected in the UPLC-MS/MS system described
below.
2.2.3 Inhibition curves of the extracts
The inhibition curves were determined only for the
extracts that showed the most expressive enzymatic
inhibition (C. glaziovii and I. paraguariensis) and
were statistically significant (p < 0.05). The extracts
were diluted and tested at 500, 250, 125, 62.5, and
31.25 µg/mL, and the enzyme inhibition assays
were performed according to the enzyme reaction
conditions described above.
2
2
.2.2 Enzyme reaction conditions
.2.2.1 CYP3A4
The reactions were performed using microsomes
prepared from insect cells infected with
recombinant baculovirus containing a human CYP
isozyme as well as a human cytochrome P450
reductase. Human cytochrome b5 is commercially
included in the preparation.
The reaction mixtures containing midazolam (4
µM), with or without ketoconazole or the extracts
and 1 pmol of CYP3A4 Baculosomes™ Plus Reagent,
were pre-incubated at 37°C for 5 min. After, 960 µM
NADPH were added to initiate the reactions. The
reaction mixtures (50 μL) were incubated at 30°C
for 20 min and the reactions were quenched by
adding 50 μL of cold methanol. The samples were
vortexed for 30 s and placed in an ice bath for 1 min.
Afterwards, the samples were centrifuged for 5 min
at 9,400 x g and the supernatants were collected for
UPLC-MS analyses. Ketoconazole 2.0 μM was used
as CYP3A4 inhibitor.
2.2.4 Instrumentation of UPLC-MS/MS analyses
The analyses were performed using a liquid
chromatography equipment (Acquity-UPLC™)
coupled to a photodiode array detector (PDA) and a
high-resolution mass spectrometer (Xevo G2 QTof
model – Waters ) equipped with an electrospray
ionization source (ESI) operating in positive (ESI+)
and negative (ESI-) ionization modes. The
separation of samples and C. glaziovii dereplication
was achieved on a C18 column 1.7 µm, 2.1 x 100 mm
Kinetex (Phenomenex ). For I. paraguariensis
extract dereplication, a C18 column, 2.6 µm 2.1 x
®
®
®
®
150 mm Kinetex (Phenomenex ) was used. All data
were processed by using the software MassLynx
V4.1.
2.2.5 Liquid chromatography conditions for the
analyses of midazolam, metoprolol, and their
metabolites
2.2.2.2 CYP2D6
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Liquid chromatography analyses were performed
hold at 95% (B) for 1.5 min, and returned to 2-98%
(B) for 30 sec. After that, the UPLC column was
maintained in the initial conditions (2-98%, B-A)
during 4 min for re-equilibration.
For I. paraguariensis extract, the mobile phase was
consisted by a gradient of 0.1% of aqueous formic
acid (A) and methanol (B) at constant flow rate of
0.5 mL/min. This gradient was programmed as
follows: Initial condition 15-85% (B-A); a linear
gradient from 15-25% (B) for 2 min; 25-60% (B) for
2-4 min; 60-70% (B) for 4-9 min, and 70-15% (B)
for 9-10 min. After that, the UPLC column was
maintained in the initial conditions (15-85%, B-A)
during 2 min for re-equilibration.
using a mobile phase consisting of a mixture of 0.1%
aqueous formic acid (A) and acetonitrile (B) in
gradient elution mode at a flow rate of 0.4 mL/min.
The solvent program steps were set for midazolam
and its metabolite in a gradient elution mode as
follows: initial conditions 10-90% (B-A); a linear
gradient from 10-50% (B) for 6 min and hold at 50%
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(
B) for 30 sec. After that, the UPLC column was
maintained in the initial conditions (10-90%, B-A)
during 1.5 min for re-equilibration before the next
injection.
The solvent program steps were set for metoprolol
and its metabolites in a gradient elution mode as
follows: 0-3 min of 2-98% (B-A); a linear gradient
from 2-90% (B) for 3 min and hold at 90% (B) for 1
min. After that, the UPLC column was maintained in
the initial conditions (2-98%, B-A) during 5 min for
re-equilibration before the next injection.
The total run time including equilibration was 8 min
and 12 min for samples containing midazolam and
metoprolol, respectively.
2.2.8 Spectrometric analyses of the medicinal
plant extracts
For C. glaziovii dereplication analyses, the mass
spectrometer parameters were set as follows: ESI+
and/or ESI-, capillary voltage of 3.0 kV; source block
temperature of 90°C; desolvation temperature of
300°C; nebulizer nitrogen flow rate of 200 L/h;
desolvation nitrogen gas flow of 900 L/h. MS/MS
analyses were performed using a collision 28 eV
(m/z 300-400) and 30 eV (m/z > 400).
For I. paraguariensis dereplication analyses, the
mass spectrometer parameters were set as follows:
For ESI-, capillary voltage of 2.5 kV; source block
temperature of 150°C; desolvation temperature of
500°C; nebulizer nitrogen flow rate of 150 L/h;
desolvation nitrogen gas flow of 1,000 L/h. For ESI+,
capillary voltage of 3.5 kV; source block
temperature of 90°C; desolvation temperature of
400°C; nebulizer nitrogen flow rate of 30 L/h, and
desolvation nitrogen gas flow of 900 L/h were set.
MS/MS analyses were performed using a collision
energy ramp 10-30 eV.
The volume of sample injected were 4 µL for
midazolam reaction samples and
2 µL for
metoprolol reaction samples. The autosampler was
kept at 6°C, and the column temperature was held at
3
5°C or 40°C for midazolam and metoprolol
reaction samples, respectively.
Precision, accuracy, and selectivity parameters
were evaluated for the validation of the analytical
4
6
methods
.
2
.2.6 Spectrometric analyses of midazolam,
metoprolol, and their metabolites
For midazolam and its metabolite, the mass
spectrometer parameters were set as follows: ESI+,
capillary voltage of 3.0 kV; source block
temperature of 90°C; desolvation temperature of
For C. glaziovii, mass scanning ranged from m/z 80
to 1,000, and for I. paraguariensis from m/z 50 to
1,200 with a scan time of 1.0 sec. Argon was used as
the collision gas.
4
00°C; nebulizer nitrogen flow rate of 10 L/h; and
desolvation nitrogen gas flow of 350 L/h.
Spectrometric analyses were performed using
sampling cone 50 V and collision energy 28 eV.
For metoprolol and its metabolites, the mass
spectrometer parameters were set as follows: ESI+,
capillary voltage of 2.0 kV; source block
temperature of 90°C; desolvation temperature of
2.2.9 Statistical analyses
All data were analyzed using Microsoft Excel 2016
®
and GraphPad Prism 6 and were obtained from
three independent experiments. For the substrate
saturation curves, data were expressed as mean ±
3
00°C; nebulizer nitrogen flow rate of 200 L/h; and
desolvation nitrogen gas flow of 900 L/h.
Spectrometric analyses were performed using
sampling cone 40 V and collision energy 30 eV. For
both, mass scanning ranged from m/z 100 to 1,000
with a scan time of 1.0 sec. Argon was used as the
collision gas and leucine enkephalin as a reference
compound to determine the masses accurately.
standard derivation (SD). Values of K
were determined by non-linear regression and
Michaelis-Menten equation (V = Vmax [S]/ (K + [S]).
m
and Vmax
m
Data on the potential changes in CYP3A4 and
CYP2D6 enzymatic activity caused by the tested
medicinal plant extracts were expressed as mean ±
standard derivation (SD) and analyzed by one-way
ANOVA followed by the post-hoc Dunnett's test
(confidence interval 95%). The inhibition curve
data of the extracts were expressed as mean ±
standard derivation (SD) and analyzed by non-
linear regression.
2
.2.7 Liquid chromatographic conditions for the
analyses of the medicinal plant extracts
For C. glaziovii extract, the mobile phase was a
gradient consisted by 0.1% of aqueous formic acid
(
A) and acetonitrile (B) at constant flow rate of 0.4
mL/min programmed as follows: 0-2 min of 2-98%
B-A); a linear gradient from 2-95% (B) for 12 min;
3. RESULTS
(
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3.1 Characterization of drug substrates and
their metabolites
their metabolites. The metabolites OH-metoprolol
and O-demethylmetoprolol coeluted, and no further
separation was observed after several attempts.
Since these molecules present similar polarity
features and both result from CYP2D6 metabolism,
the formed peak corresponding to these two
metabolites was used for quantification.
The in vitro herb-drug interactions mediated by
CYP3A4 and CYP2D6 enzymes were evaluated using
standardized medicinal plants extracts and the
drugs midazolam and metoprolol, which are well-
known as substrates of CYP3A4 and CYP2D6
3
2, 47
enzymes, respectively
. It is relevant to highlight
Table 2 presents the data concerning the molar
mass and retention times determined by UPLC-
MS/MS for midazolam and metoprolol, their
metabolites as well as their molecular formulas.
These results are similar to those previously
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that the interactions detected using these substrates
are useful for predicting the occurrence of
interactions with other drugs metabolized by
CYP3A4 and CYP2D6. A comprehensive list of drugs
that are substrates for these enzymes is available at
36, 49
described in the literature
.
the
SuperCYP
database
To select the drug substrate concentrations to be
used for the evaluation of potential herb-drug
interactions involving CYP3A4 and CYP2D6
recombinant enzymes, the substrate saturation
curves were obtained for midazolam (Fig. 1D) and
metoprolol (Fig. 1H). The kinetic parameters of drug
4
8
http://bioinformatics.charite.de/supercyp/
.
This study employed the following criteria to
validate the analytical methods: precision - RSD <
1
5%; accuracy - % recovery between 80-120%; and
specificity, which was assessed by injecting the
reaction mix into the UPLC-MS equipment. The
results are available in the Supplementary Material
m
metabolization in vitro (K and Vmax) are also
displayed in Table 2.
(
Table S1 and Fig. S1).
Fig.1 shows chromatograms and spectra obtained
by UPLC-MS/MS for midazolam, metoprolol, and
Figure 1. Drug probes and metabolites detected by UPLC-MS and substrate saturation curves data. A. Midazolam 4 µM
peak 2) and metabolite OH-midazolam (peak 1) chromatogram. B. OH-midazolam peak extracted (peak 1) and mass
(
spectra (m/z 203.0364). C. Chromatogram of midazolam peak extracted (peak 2) and mass spectra (m/z 291.1178). D.
Substrate saturation curve - metabolites formation rates (pmol/min/pmol of CYP3A4) in relation to midazolam
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substrate concentrations (µM). E. Metoprolol 80 µM (peak 2) and metabolites OH-metoprolol/O-demethylmetoprolol
(peak 1) chromatogram. F. OH-metoprolol/O-demethylmetoprolol peak extracted (peak 1) and mass spectra (m/z
2
84.1832/254.1729). G. Chromatogram of metoprolol peak extracted (peak 2) and mass spectra (m/z 268.1926). H.
Substrate saturation curve - metabolites formation rates (pmol/min/pmol of CYP2D6) in relation to metoprolol
substrate concentrations (µM). The curves were automatically fitted using nonlinear regression and the Michaelis–
Menten equation; values are presented as mean ± SD.
Table 2. Drug substrates and metabolites characteristics.
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Enzyme
CYP3A4
CYP2D6
Chemical characteristics
Substrates
Mass detected
Midazolam
291.1178 g/mol
4.17 min
Metoprolol
268.1926 g/mol
5.70 min
Retention time
Molecular formula
18
C H
13CIFN
3
15 3
C H25NO
OH-metoprolol /
O-demethylmetoprolol
284.1832/ 254.1729 g/mol
5.08 min
Metabolites
OH-midazolam
Mass detected
Retention time
Molecular formula
203.0364 g/mol
4.05 min
18
C H
13CIFN
3
O
15 4 3
C H25NO /C14H23NO
Kinetics characteristics
a
K
V
m
3.33 ± 1.50 µM
1.50 ± 0.254 pmol/min/pmol of
enzyme
71.88 ± 19.46 µM
31.64 ± 4.629 pmol/min/pmol of
enzyme
a
max
a
Data represent the mean ± SD and were determined by nonlinear regression and the Michaelis-Menten equation.
3
.2 Modulation of CYP3A4 and CYP2D6 activity
by the medicinal plant extracts
After determining the value of k
CYP3A4 - Midazolam
1
50
00
50
0
m
for each substrate
used, the enzyme activity modulation analyses were
performed. Potential modifications in CYP2D6 and
CYP3A4 enzymatic activity were analyzed using the
standardized medicinal plant extracts at 500 µg/mL.
Ketoconazole (2.0 µM) and quinidine (0.08 µM)
were used as controls since these drugs are selective
inhibitors of CYP3A4 and CYP2D6, respectively, and
are recommended by the FDA for in vitro
1
**
**
***
**** ****
****
A
****
****
****
M
M
IP


CSc
MR
CG
EQ
CR
GB
BF
GM
Keto
2
Control
4
3
2
metabolism studies
.
CYP2D6 - Metoprolol
Both drug enzyme-specific inhibitors, ketoconazole
and quinidine, significantly (p < 0.001) inhibited the
production of the respective enzymatic metabolites
when compared to the control reaction (Fig. 2),
demonstrating the proper performance of these
inhibitors under the established enzymatic reaction
conditions.
1
50
100
50
0
B
**
**
M
M
IP


CSc
MR
CG
EQ
CR
GB
BF
GM
A significant reduction (p < 0.05) of OH-midazolam
production was detected when the CYP3A4 enzyme
was incubated with most of the extracts tested and
compared to the control reaction. The production of
this metabolite was inhibited by all extracts except
the soy extract (G. max) (Fig. 2A). On the other hand,
CYP2D6 was significantly (p < 0.05) inhibited only
by red embaúba (C. glaziovii) extract (Fig. 2B).
0.08
Control 80Quin
Figure 2. Modulation of CYP3A4 and CYP2D6
activity in the presence of standardized medicinal
plant extracts (500 µg/mL). A. CYP3A4 enzymatic
reactions - % OH-midazolam formation versus samples
tested. B. CYP2D6 enzymatic reactions - % OH-
metoprolol/O-demethylmetoprolol formation versus
samples tested. Data are expressed as percentages (%)
relative to controls. Data were analyzed by two-way
ANOVA followed by the post-hoc Dunnett's test
(
confidence interval 95%) and compared to the
controls. Data were obtained from three independent
experiments (mean ± SD). BF - Bauhinia forficata; CG -
Cecropia glaziovii; CR - Cimicifuga racemosa; CSc -
Cynara scolymus; EQ - Echinacea sp; GB - Ginkgo biloba;
GM - Glycine max; IP - Ilex paraguariensis; Keto –
ketoconazole; and MR - Matricaria recutita; Quin –
Quinidine; ** (p <0.01); **** (p <0.0001).
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3.3 Determination of CYP3A4 and CYP2D6
inhibition curves
peaks were identified for each extract. Tables 3 and
4 show the characterization of these compounds as
well as their retention times, m/z peaks, molecular
formulas, fragments, and common names.
CYP3A4 and CYP2D6 inhibition curves were
determined for the extracts that demonstrated the
most relevant inhibition of CYP3A4 (C. glaziovii and
I. paraguariensis) and CYP2D6 (C. glaziovii) activity,
and their IC50 values were calculated. Fig. 3, reveals
that the C. glaziovii extract inhibited CYP2D6
activity with an IC50 value of 396.0 µg/mL. For
CYP3A4 reactions, C. glaziovii and I. paraguariensis
presented IC50 values equal to 102.1 µg/mL and
1
1
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0
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1
24.2 µg/mL, respectively.
CYP3A4 - Cecropia glaziovii inhibition curve
1
20
00
1
80
A
B
C
60
IC50 = 102.1 g/mL
4
0
0
0
2
0
100
200
300
400
500
600
Extract concentration (g/mL)
CYP2D6 - Cecropia glaziovii inhibition curve
120
100
80
60
IC50 = 396.0 g/mL
4
0
0
0
2
0
100
200
300
400
500
600
Extract concentration (g/mL)
CYP3A4 - Ilex paraguariensis inhibition curve
Figure 4. Chromatograms of (A) Cecropia glaziovii
(CG) (ESI-), (B) Ilex paraguariensis (IP) (ESI+), and (C)
Ilex paraguariensis (IP) (ESI-) obtained by UPLC-
MS/MS for their major compounds. The identification
of each numbered peak can be seen in Tables 3 and 4.
The complete chromatograms of the extracts and
blanks of the solvents used in the analysis can be seen
in Supplementary Material (Fig. S2 and S3).
1
20
00
1
80
60
IC50 = 124.2 g/mL
40
20
0
0
100
200
300
400
500
600
Extract concentration (g/mL)
4
. DISCUSSION
4
.1 Drug probes and K
Concerning the determination of the K
the values obtained were close to those reported by
m
values
Figure 3. CYP3A4 and CYP2D6 inhibition curves. A and
B C. glaziovii and C. I. paraguariensis. Data were
analyzed by non-linear regression and represent each
m
parameter,
5
0, 51
other similar studies with CYP3A4
enzymes
, CYP2D6
extract concentrations (µg/mL) versus
%
of
5
2
, and their respective substrates
metabolites formation normalized to the control. Data
were obtained from three independent experiments.
midazolam and metoprolol. Based on both substrate
saturation curves, the concentrations of 4 μM for
midazolam and 80 μM for metoprolol were chosen
for the subsequent in vitro herb-drug interaction
assays. These concentrations are close to the values
3.4
Identification
of
the
major
phytoconstituents of Cecropia glaziovii and Ilex
paraguariensis by UPLC-MS/MS
m
of K , and they enabled the detection of drug
In order to establish a relationship with the in vitro
findings and the chemical composition of the
extracts that presented the most relevant inhibitory
effects of CYP3A4 and CYP2D6 activity, the major
compounds of C. glaziovii and I. paraguariensis
extracts were identified by UPLC-ESI-QTof-MS/MS.
Fig. 4 presents the chromatograms in ESI+ and ESI-.
For C. glaziovii and I. paraguariensis, nine major
substrates and their metabolites by UPLC-MS.
4
.2 Modulation of CYP3A4 and CYP2D6 activity
by the standardized medicinal plant extracts
All tested extracts inhibited CYP3A4 activity to some
degree, except soy extract (G. max) (Fig. 2A). Other
authors have reported the inhibition of CYP3A4 by
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several xenobiotics. For example, Ashour and
profile similar to that found for the ketoconazole
inhibitor (Fig. 2A). No reports on the inhibition of
CYP3A4 activity using I. paraguariensis extract was
found in the literature. However, the inhibition of
CYP2E1 activity has already been reported and, in
this case, the extract of yerba mate also protected
coworkers (2017) verified that of the 57 Traditional
Chinese Medicine extracts tested, all inhibited
4
0
CYP3A4 activity . The inhibition of this enzyme by
most of the extracts tested may be associated with
the enzyme conformational flexibility and its ability
to bind to large and structurally diverse compounds,
7
3
against ethanol-induced liver injury
.
which increases the chances of their modulation 53
.
From the extracts tested, I. paraguariensis stands
out because it is used in the preparation of
traditional beverages known as chimarrão, mate
0
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On the other hand, in this study, only the extract of
red embaúba (C. glaziovii) inhibited CYP2D6 activity
(
Fig. 2B).
and tererê, especially in southern and southeastern
74, 75
regions of Brazil, Argentina, and Uruguay
. In
4
.2.1
Red embaúba (Cecropia glaziovii)
addition, this extract showed a relatively low IC50
value when compared to the other samples tested.
Therefore, herb-drug interactions with this plant
can cause important effects on the bioavailability of
Important pharmacological effects were reported
for red embaúba , such as anti-inflammatory,
antioxidant , hepatoprotective , antiviral
anti-acid secretion
vasorelaxant , and antihypertensive activities,
which have been credited to the presence of
catechins, procyanidins, flavonoids, and chlorogenic
5
4
5
5
56
56, 57
,
5
8
,
hypoglycemic and
drugs metabolized by CYP3A4 due to the chronic
5
9
60
74
and considerable amount consumed of this drink
.
Thus, it would be important to evaluate in vivo
interactions involving the beverages prepared with
this plant.
5
4
and caffeic acids
.
The extract of red embaúba inhibited the CYP3A4
and CYP2D6 activity similarly when compared to
ketoconazole (2.0 µM) and quinidine (0.08 µM)
4.2.3
Other medicinal plant extracts tested
The CYP3A4 activity was inhibited (p < 0.05) weakly
by the other extracts tested, and it was not inhibited
by the soy extract. According to the available
literature, all extracts evaluated herein contain
(
both > 50% of the metabolite production),
respectively (Fig. 2). To the best of our knowledge,
there are no previous reported studies about the
modulation of CYP3A4 and CYP2D6 activity by C.
glaziovii. However, in a recent paper published by
our research group, it was reported that the
treatment of Caco-2 cells with 100 µg/mL of C.
glaziovii for 48 h induced the expression of CYP3A4
when assessed via a gene reporter assay, while the
76-82
phenolic compounds
. The HPLC-UV analyses
performed with the standardized medicinal plant
extracts tested showed peaks in the UV spectra,
confirming the presence of phenolic compounds
(data not shown).
Thus, the polyphenols may be responsible for the
inhibition of CYP3A4 activity observed to a lesser
extent for C. scolymus (globe artichoke) M. recutita
(chamomile), Echinacea sp. (echinacea), G. biloba
(ginkgo), and B. forficata (orchid tree) extracts.
Similar results have been reported for CYP3A4 and
Echinacea sp. (echinacea), C. racemosa (black
cohosh), and G. biloba (ginkgo) extracts. For
Echinacea purpurea, a weak inhibition of CYP3A4
activity was detected using different enzyme
1
8
CYP2D6 expression was not significantly altered
.
This increase in the CYP3A4 expression could be
associated with the feedback in response to a
previous inhibition of this enzyme activity since the
analysis was performed 48 h after Caco-2 treatment.
Also, in this case, the simultaneous activity of
different compounds on biochemical and molecular
pathways cannot be excluded due to the complexity
of the chemical composition of the extracts. For
example, several polyphenolic compounds inhibit
CYP3A4 activity, while others act on the nuclear
receptors such as PXR (Pregnane X Receptor) to
induce the expression of CYP enzymes, including
8
3
substrates . C. racemosa inhibited CYP3A4 activity,
and this effect was attributed to the triterpene
8
4
glycosides present in this plant . G. biloba also
8
5
caused a slight inhibition of CYP3A4 activity . Thus,
the inhibition of CYP3A4 activity by these medicinal
plant extracts corroborate the reports in the
literature.
6
1, 62
that of CYP3A4
.
4
.2.2 Yerba mate (Ilex paraguariensis)
Another tested sample that significantly inhibited
CYP3A4 activity was the extract of yerba mate.
Several pharmacological effects have been
attributed to this plant, such as: antiatherosclerotic
Concerning the G. max (soy) extract, despite the
presence of polyphenols, such as isoflavonoids, no
alterations on the metabolism of midazolam were
observed herein, which have been stated previously
63
64, 65
61
,
anticonvulsant, neuroprotective
, anti-
. Thus, qualitative and quantitative variations of
66
67
68
inflammatory
cardioprotective , and hypolipidemic activities.
Additionally, this plant is employed to prevent or
,
anti-obesity
,
antiviral
,
the compounds present in medicinal plants may also
be crucial for the final observed effects.
69
70
Most of the extracts tested on CYP2D6 activity did
not alter the metabolism of metoprolol. However, a
study performed with the essential oil of M. recutita
and their isolated compounds described that the
CYP2D6 activity was less affected by the isolated
compounds than by the crude oil. Nevertheless, in
7
1
treat osteoporosis and to reduce oxidative stress
7
2
.
In this work, the yerba mate extract significantly
inhibited (p < 0.001) the production of OH-
midazolam metabolite and showed an inhibition
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the same study, few isolated compounds inhibited
strongly inhibited CYP2D6 activity 88. The inhibition
of other CYP enzymes activity, such as CYP1A1,
CYP2C9, and CYP3A4, by catechin-3-O-gallate,
gallocatechin-3-O-gallate, and epigallocatechin-3-O-
8
6
other CYP enzymes, such as CYP1A2 and CYP3A4
.
In the same way, Gurley and coworkers (2008) did
not detect significant effects in vivo on CYP2D6
8
7
92
activity caused by E. purpurea and C. racemosa
.
gallate have already been described . For this
The same finding was observed in vitro for C.
reason, it seems that catechins, which are abundant
in C. glaziovii (Table 3), may be responsible, at least
in part, for the inhibition detected.
3
7
racemosa . Furthermore, in vivo assays conducted
with a soy extract (G. max) containing isoflavones
did not affect the pharmacokinetics and
1
1
1
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2
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2
2
2
2
2
2
2
2
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3
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The
phytochemical,
pharmacological,
and
8
8
pharmacodynamics of metoprolol
.
pharmacokinetic properties of C. glaziovii are still
scarcely studied. However, some of its constituents
have been investigated concerning potential herb-
drug interactions. An in silico investigation showed
that the C-glycosylflavonoids, orientin, and
isoorientin, did not inhibit CYP2D6 activity, while
No relevant information concerning the CYP2D6
inhibitory activity in vitro of the other tested
extracts is available in the literature. Indeed, to the
best of our knowledge, the in vitro evaluation of the
potential inhibitory effects of CYP2D6 activity
caused by C. scolymus, I. paraguariensis, and B.
forficata extracts is being reported here for the first
time.
9
4
isovitexin and vitexin did
.
Additionally,
chlorogenic acid also inhibited CYP2D6 activity in
9
5
vitro . Thus, isovitexin, vitexin and/or chlorogenic
acid could count for the inhibitory effects of CYP2D6
activity observed for C. glaziovii.
4.3
Identification
of
the
major
phytoconstituents of Cecropia glaziovii and Ilex
paraguariensis by UPLC-MS/MS
Interestingly, the inhibition of CYP2D6 activity by I.
paraguariensis was not observed, although this
extract is rich in chlorogenic acid. Nonetheless,
further studies with these isolated metabolites are
required to determine their CYP2D6 inhibition
profiles.
The major secondary metabolites found in the
standardized extract of C. glaziovii were catechin,
epicatechin, caffeoylquinic acid, procyanidins, and
C-glycosylflavonoids (vitexin, isoorientin and
isovitexin) (Table 3), which have been previously
Although these secondary metabolites are known to
be CYP inhibitors, it is relevant to note that the
verified enzymatic inhibitory effects may not refer
just to a single compound. Moreover, additive or
synergistic effects of the components of the extracts
tested on CYP enzymes activity cannot be ruled out
54, 89
described
.
For the standardized extract of I. paraguariensis, the
major compounds identified were caffeoylquinic
acid, caffeine, theobromine, quinic acid, and rutin
(
Table 4), which have also been previously reported
7
5, 90
96
.
.
In this way, the extracts of C. glaziovii, and I.
paraguariensis are well known to be rich in
polyphenolic compounds, and the potential of these
secondary metabolites to inhibit CYP3A4 activity
has been described before indicating that they may
be responsible for the inhibition detected in this
61, 91
work
. These effects, associated with the
phenolic groups, can be due to their interactions
with the amino groups of the enzyme (arginine,
lysine, and histidine) forming hydrogen and ionic
4
0, 62
bonds at the enzyme active sites
.
Some catechins have been described as CYP3A4
9
2
inhibitors , and they could also be responsible for
the inhibition caused by C. glaziovii. Likewise, I.
paraguariensis and C. glaziovii present chlorogenic
acid in their chemical composition, which could
have contributed to the inhibition of CYP3A4
activity, since this compound was described as an
9
3
inhibitor of such activity
.
The flavonoid rutin, which is one of the major
compounds of I. paraguariensis extract, also acts as
a CYP3A4 inhibitor, showing high stability within
40
the active enzymatic sites
.
Concerning the inhibition of CYP2D6 activity, an in
silico study investigated the CYP2D6 activity
inhibitory potential of 43 polyphenolic compounds,
including catechin, epicatechin, epigallocatechin-3-
O-gallate, and gallocatechin-3-O-gallate, which
i
showed low values of K (< 6.88 µM), suggesting they
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2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 3. Characterization of the major phytoconstituents of Cecropia glaziovii (red embaúba) standardized extract.
Peak
number
t
R
Expt.
(m/z)
Molecular
formulas
Fragments (m/z)
Common names
a
(min)
ESI (-)
1
2
0.53
4.53
195.0530
353.0890
[C
[C16
6
H
H
11
O
7
-H]-
-
Gluconic acid
Caffeoylquinic acid
Procyanidin B2, B3, B4, B5, or
B7
-
18
O
9
-H]
191.0592
0
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2
3
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2
3
4
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7
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9
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1
2
3
4
5
6
12-H]-
3
4
5
4.75
4.86
5.26
577.1353
289.0742
447.0953
[C30
H
26
O
125.0261, 151.0405, 245.0858, 289.0742, 407.0716
109.0289, 123.0424, 125.0261, 137.0147, 151.0379, 188.0458, 203.0714
327.0648, 357.0666
[C15
H
14
O
6
-H]-
Catechin or Epicatechin
Isoorientin or
Luteolin-8-C-glucoside
Vicenin-2
Schaftoside or vicenin-1
Isovitexin or vitexin
[C21H20O11-H]-
-
6
7
8
5.37
5.45
5.56
593.1514
563.1394
431.1021
[C27H30O15-H]
[C26H28O14-H]
[C21H20O10-H]
119.0354, 243.0324, 269.0422, 293.0456, 353.0698, 413.0833, 473.1130
293.0491, 311.0591, 323.0561, 341.0707, 353.0621, 413.0916, 443.1084
161.0260, 282.0510, 283.0594, 311.0591, 341.0669, 342.0569
281.0479, 283.0628, 311.0555, 323.0561, 341.0659, 354.0707, 413.0916,
431.1021
-
-
9
5.96
473.1130
[C22H22O11-H]-
Acetylvitexin
aCorresponding peaks can be seen in the chromatograms of Fig. 4.
Table 4. Characterization of the major phytoconstituents of Ilex paraguariensis (yerba mate) standardized extract.
Peak
number
t
R
Expt.
(m/z)
Molecular formula
Fragments (m/z)
Common names
a
(min)
ESI (+)
+ H]+
b
1
2
5,48
6,67
181,0734
195,0886
[C
[C
7
H
8
N
N
4
O
2
163.0607; 138.0664; 122.0582; 108.0562
138.0664; 123.0430; 110.0723
Theobromine
+
b
8
H
10
4
O
2
+ H]
Caffeine
ESI (-)
1
2
2.70
5,60
191.0550
353.0873
[C
7
H
12
O
6
- H]-
93.0353; 109.0303;127.0399; 171.0258
179.0343; 135.0445
Quinic acid
5-O-caffeoylquinic acid
-
b
[C16
[C15
[C16
[C25
[C25
[C27
H
H
H
H
H
H
18
18
18
O
9
- H]
- H]-
- H]-
3
23.0784; 281.0677; 251.0589; 221.0482;
79.0370
179.0343; 173.0451; 135.0445
3
5.80
341,0887
O
9
6-O-caffeoylglucose
1
4
5
6
7
6,34
7,1
7,42
7,54
353,0873
515,1195
515,1195
609,1490
O
9
4-O-Caffeoylquinic acid or 3-O-Caffeoylquinic acidb
3,4-di-O-caffeoylquinic acid or 3,5-di-O-caffeoylquinic acid
4,5-di-O-caffeoylquinic acid
-
24
O
24
O
30
O
12 - H]
12 - H]
16 - H]
191.0550; 179.0343; 173.0451; 135.0445
191.0550; 179.0370; 173.0451; 135.0445
301.0358
-
-
b
Rutin
a
Corresponding peaks can be seen in the chromatograms of Fig. 4.
b
Identified with authentic standards
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. CONCLUSION
and CYP2D6 activity. Thus, many other natural
products containing the same phytoconstituents
present in those extracts may affect drug
bioavailability and result in herb-drug interactions.
Further mechanistic and in vivo experiments are
required to fully assess their safety profile.
Likewise, the role of intestinal metabolism of
natural compounds, in which the microbiota
actively participates, should also be investigated for
a better correlation with the results obtained in vitro
and in vivo.
In summary, this study suggests that the
concomitant use of C. glaziovii and/or I.
paraguariensis extracts with xenobiotics (ex. drugs,
isolated compounds, other medicinal plant extracts)
metabolized by CYP3A4 or CYP2D6 may interfere in
vivo with the xenobiotic’s metabolism.
Due to the chemical composition of these medicinal
plant extracts, the polyphenolic compounds appear
to play an important role in the inhibition of CYP3A4
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AUTHOR INFORMATION
Corresponding Author
Watanabe, K. (2016) Inhibitory effects of eight green tea
catechins on cytochrome P450 1A2, 2C9, 2D6, and 3A4
activities. J Pharm Pharm Sci 19, 188-97.
Cláudia Maria Oliveira Simões,
claudia.simoes@ufsc.br.
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Niwatananun, K., Narongchai, S., Kusirisin, W. (2011)
Inhibitory effects of caffeic acid ester analogues on free
radicals and human liver microsome CYP1A2 activities.
Med Chem 7, 99-105.
FUNDING INFORMATION
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CNPq (MCTI) - Grant number 305878/2016-6.
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94) Narayanaswamy, R., Isha, A., Wai, L. K., Ismail, I. S.
2016) Molecular docking analysis of selected
ACKNOWLEDGEMENTS
The authors would like to thank the team of
Pharmacognosy Laboratory (Department of
Pharmaceutical Sciences, CCS, UFSC) for donating
the standardized extracts of C. glaziovii, I.
paraguariensis, and B. forficata, and for enabling the
analyses of UPLC-MS/MS. The authors also
acknowledge VitalFarm and Vita Essência
pharmacies for making available the standardized
extracts of C. racemosa, C. scolymus, Echinacea sp., G.
biloba, G. max and M. recutita. We are grateful to Dr.
Vitor Clasen Chaves for technical assistance.
Clinacanthus nutans constituents as xanthine oxidase,
nitric oxide synthase, human neutrophil elastase, matrix
metalloproteinase 2, matrix metalloproteinase 9 and
squalene synthase inhibitors. Pharmacogn Mag 12, S21-6.
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A., Al-Dahbi, N. A., Duraipandiyan, V. (2015) Inhibition
potential of Moringa oleifera Lam. on drug metabolizing
enzymes. Indian J Tradit Know 14, 6.
(
96) Caesar, L. K., Cech, N. B. (2019) Synergy and
antagonism in natural product extracts: when 1 + 1 does
not equal 2. Nat Prod Rep 36, 869-88.
ABBREVIATIONS
[S]: Substrate concentration; BF: Bauhinia forficata;
CAM: Complementary and Alternative Medicine; CG:
Cecropia glaziovii; CR: Cimicifuga racemosa; CSc:
Cynara scolymus; CYP: Cytochrome P450 enzymes;
ASSOCIATED CONTENT
Supporting Information
Table with the parameters used for the validation of
analytical methods and the chromatograms of the
reaction mix blank. (Table 1 and Fig. S1, i.e., Word).
DMSO:
Dimethyl
Sulfoxide;
EDTA:
Ethylenediaminetetraacetic acid; EMA: European
Medicines Agency; EQ: Echinacea sp; FDA: Food and
Drug Administration; IC : Half maximal inhibitory
Complete chromatograms of the solvent and
Cecropia glaziovii extract solution with numerical
indications of peaks corresponding to the major
compounds identified (Fig. S2, i.e., Word).
5
0
concentration; IP: Ilex paraguariensis; Keto:
ketoconazole; K : Michaelis constant; MR:
m
Matricaria recutita; NADPH: Nicotinamide Adenine
Dinucleotide Phosphate; p: p value; PBS: Phosphate-
buffered Saline; Quin: Quinidine; SD: Standard
Complete chromatograms of the solvent and Ilex
paraguariensis extract solution with numerical
indications of peaks corresponding to the major
compounds identified (Fig. S3, i.e., Word).
Deviation; t : Retention time; TFA: Trifluoro acetic
R
acid; V: Velocity; Vmax: Maximal velocity.
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