Author’s Accepted Manuscript

Bush mint (Hyptis suaveolens) and spreading

hogweed (Boerhavia diffusa) medicinal plant
extracts differentially affect activities of CYP1A2,
CYP2D6 and CYP3A4 enzymes

Nicholas Ekow Thomford, Kevin Dzobo, Faustina

Adu, Shadreck Chirikure, Ambroise Wonkam,
Collet Dandara www.elsevier.com/locate/jep

PII: S0378-8741(17)31943-8
DOI: http://dx.doi.org/10.1016/j.jep.2017.09.023
Reference: JEP11035
To appear in: Journal of Ethnopharmacology
Received date: 17 May 2017
Revised date: 15 September 2017
Accepted date: 18 September 2017
Cite this article as: Nicholas Ekow Thomford, Kevin Dzobo, Faustina Adu,
Shadreck Chirikure, Ambroise Wonkam and Collet Dandara, Bush mint ( Hyptis
suaveolens) and spreading hogweed (Boerhavia diffusa) medicinal plant extracts
differentially affect activities of CYP1A2, CYP2D6 and CYP3A4 enzymes,
Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.09.023
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Bush mint (Hyptis suaveolens) and spreading hogweed (Boerhavia diffusa)
medicinal plant extracts differentially affect activities of CYP1A2, CYP2D6
and CYP3A4 enzymes
Nicholas Ekow Thomford a, b, Kevin Dzobo c, d
, Faustina Adu b, Shadreck Chirikuree,
Ambroise Wonkam a, Collet Dandara a*.
a
Pharmacogenomics and Drug metabolism Research Group, Division of Human Genetics,
Department of Pathology & Institute for Infectious Disease and Molecular Medicine, Faculty of
Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Cape Town, South
Africa
b
School of Medical Sciences, University of Cape Coast, Cape Coast, PMB, Ghana
c
ICGEB, Cape Town component, Faculty of Health Sciences, University of Cape Town, Anzio
Road, Observatory 7925, Cape Town, South Africa
d
Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Anzio
Road, Observatory 7925, Cape Town, South Africa
e
Department of Archaeology, University of Cape Town, Cape Town, Rondebosch 7701, South
Africa
* Correspondence: collet.dandara@uct.ac.za; Tel.: +27-214-066-506; Fax: +27-216-502-
010
Abstract
Ethno-pharmacological relevance: Hyptis suaveolens (L) Poit and Boerhavia diffusa Linn are
medicinal herbal plants commonly found in the tropics and sub-tropics. They are used to treat various
conditions among them boils, dyslipidaemia, eczema, malaria, jaundice and gonorrhoea. Thus, the
herbal medicinal extracts are now found as part of some commercial herbal formulations. There has
not been adequate characterisation of these medicinal herbs on their effects on drug metabolising
enzymes.

Aim of the study: To investigate the effects of extracts of Hyptis suaveolens (HS) and Boerhavia
diffusa (BD) on activity of drug metabolizing enzymes, CYP1A2, CYP2D6 and CYP3A4, as well
predict their potential for herb-drug interaction. A secondary aim was to identify constituent
compounds such as polyphenolics, in the crude extract preparations of Hyptis suaveolens and
Boerhavia diffusa and measure them for activity.

Materials and methods: CYP450 inhibition assays using recombinant CYP450 (rCYP) and
fluorescence screening employing individual isozymes (CYP1A2, CYP2D6 and CYP3A4) were used
to determine reversible- and time-dependent inhibition (TDI) profiles of extracts of Hyptis suaveolens
and Boerhavia diffusa. Inhibition kinetic parameters, Ki and Kinact were also estimated. UPLC-MS
employing a Synapt G2 (ESI negative) coupled to a PDA detector was used to identify polyphenolic
compounds in crude extracts of Hyptis suaveolens and Boerhavia diffusa.

Results: The inhibitory potency of Hyptis suaveolens and Boerhavia diffusa extracts varied among
the different enzymes, with CYP1A2 (3.68 ± 0.10µg/mL) being the least inhibited by HS compared to
CYP2D6 (1.39 ± 0.01 µg/mL) and CYP3A4 (2.36 ± 0.57 µg/mL). BD was most potent on CYP3A4
(7.36 ± 0.94 µg/mL) compared to both CYP2D6 (17.79 ± 1.02 µg/mL) and CYP1A2 (9.48 ±
0.78µg/mL). Extracts of Hyptis suaveolens and Boerhavia diffusa exhibited TDIs on all CYPs. The
most prominent phenolic candidates identified in both medicinal herbs using UPLC-MS analysis
included caffeic acid, rutin, quercetin, citric acid, ferulic acid and gluconic acid. These phenolic
compounds are thought to potentially give HS and BD their therapeutic effects and inhibitory

1
characteristics affecting CYP450 activities. In vivo predictions showed the potential for HS and BD
extracts to cause significant interactions if co-administered with other medications.

Conclusions: The study reveals that crude aqueous extracts of HS and BD potentially inhibit drug
metabolising isozymes CYP1A2, CYP2D6 and CYP3A4 in a reversible and time-dependent manner.
Thus care should be taken when these extracts are co-administered with drugs that are substrates of
CYP1A2, CYP2D6 and CYP3A4.

Graphical Abstract

Key words: CYP450, Herb-drug interaction, time-dependent inhibition, Ki, Hyptis suaveolens,
Boerhavia diffusa, flourimetric assay, medicinal herb, polyphenolic compounds, UPLC-MS

1.0 Introduction
Herbal medicine use is increasingly becoming popular in the treatment of non-communicable
diseases (Chintamunnee and Mahomoodally, 2012;Hughes et al., 2015) in both developed
and developing countries. To this extent, remedies are sprouting in pharmacies, chemical
stores and supermarkets where special shelves are created for ‘natural products’ (Ekor, 2014).
Medicinal plants have potential therapeutic value and are an integral part of the health care
system. Due to ineffectiveness of single molecules or drugs, multiple drug therapies are a
common practice especially among patients presenting with multiple diseases such as
diabetes, cancer, HIV/AIDS, malaria and hypertension (Nadler et al., 2003; HemaIswarya
and Doble, 2006). Each of the conditions may need a different drug intervention, thus,
concurrent intake of medicinal herbs with prescribed therapeutic agents (Graham et al., 2008;
Chen et al., 2012; Agbabiaka et al., 2016) becomes a possibility, sometimes leading to
adverse effects.

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Unlike conventional drugs, medicinal herbs contain mixtures of phytochemicals, each of
which could be biologically active and capable of interacting with conventional/allopathic
drugs pharmacokinetically and/or pharmacodynamically (Morris and Zhang, 2006; Křížková
et al., 2009). Such interactions mainly involve inhibition of enzyme activities and induction
of expression of genes coding for drug metabolising enzymes, especially the cytochrome
P450 (CYP450) enzymes (Doligalski et al., 2012). CYP450 is a mixed-function oxygenase
system involved in the metabolism of both endogenous and exogenous substrates. Exogenous
substrates include drugs, alcohols, anti-oxidants, organic solvents, environmental pollutants
and chemicals whose metabolism facilitates their excretion from the body (Isin and
Guengerich, 2007). There are several CYP450s, however, CYP1A2, CYP2D6 and CYP3A4
are responsible for the metabolism of 10% (Zanger and Schwab, 2013), 20% -25%
(Ingelman-Sundberg, 2005) and 50% (Fujita, 2004) of all prescribed medications,
respectively. Due to the multi-phyto-constituent nature of medicinal herbs, it is estimated that
the risk of herb-drug interaction (HDI) occurring is high (Fugh-Berman and Ernst, 2001;
Izzo, 2005). This presents a public health concern taking into account the growing number of
patients concurrently taking medicinal herbs with conventional medicines (Djuv, 2013).
Concomitant administration of medicinal herbs and conventional medications has a potential
to affect drug metabolism and significantly increase the risk of serious adverse reactions
(Budzinski et al., 2000; Chen et al., 2012). There are reports of interactions between Gingko
biloba and efavirenz, an antiretroviral drug, (Naccarato et al., 2012), Hibiscus sabdariffa and
chloroquine, an antimalarial (Mahmoud et al., 1994) and Gingko biloba and warfarin, an
anticoagulant (Matthews, 1998), due to their effects on drug metabolising enzymes,
particularly CYP450s. There is little research on the effects of Hyptis suaveolens and Boerhavia
diffusa on drug metabolising enzymes and yet these medicines are widely used.

Hyptis suaveolens (L.) Poit (HS), commonly known as bush mint, bush tea or pignut is an
ethno-botanically important medicinal plant belonging to the family Lamiaceae. Hyptis
suaveolens found in countries such as Brazil, Venezuela, Ecuador , United States
,Bangladesh, China and India in Asia, Cameroon, Congo, Benin, Ghana, Kenya, Nigeria,
Sudan and Togo (Jesus et al., 2013). Hyptis suaveolens is used in the treatment of various
ailments and disease conditions including boils, dyslipidaemia, eczema, malaria, diabetes
mellitus, cancer and hepatitis (Danmalam et al. 2009; Mishra et al., 2011; Ghaffari et al.,
2012). Boerhavia diffusa Linn (BD), commonly known as spreading hogweed, red hogweed
or pigweed is an herbaceous member of the family Nyctaginaceae mostly distributed in the

3
tropics and subtropics. It has a long history of use by indigenous people as a natural
medicinal herb. Boerhavia diffusa is a wild perennial herb found in different terrestrial
habitats, ranging from managed grass lands, wastelands, agro-eco systems to large forest
gaps. It has been reported in countries such Ghana, Togo, Nigeria, Congo, Benin, Guinea,
Cote d’Ivoire, South Africa, and India (Mishra et al., 2014). It is used traditionally for the
treatment/management of anaemia, heart troubles, palpitations, jaundice, gonorrhoea,
pulmonary tuberculosis and cancer (Hamayun et al., 2006) (Biswas et al., 2010).

The present study aimed to evaluate the effects of Hyptis suaveolens and Boerhavia diffusa
extracts on inhibition profiles of CYP1A2, CYP2D6 and CYP3A4 using recombinant
CYP450 enzymes and contribute to knowledge regarding their potential interactions with
conventionally used drugs. Enzyme kinetics were used to determine the inhibitory
mechanisms and predict potential interactions with commonly used substrates of the three
CYP450s. Phytofingerprinting was performed to evaluate the profile of possible
phytocompounds in both Hyptis suaveolens and Boerhavia diffusa extracts.

2.0 Materials and methods

2.1 Chemicals and reagents
Black costar 96-well plates from ThermoFischer Scientific (Pittsburgh, PA, USA) were used
for reversible-, time-dependent inhibition and kinetic reactions. α- naphthoflavone,
furafylline, quinidine, ketoconazole and erythromycin were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Vivid® Blue screening kits and vivid® substrates, 7-benzyl-
oxymethyloxy-3-cyanocoumarin, (BOMCC-cat. no. P2975) and 7- ethoxy-methloxy-3-
cyanocoumarin (EOMCC-cat.no. P3024) were obtained from Life Technologies, (Grand
Island, NY, USA). CYP1A2 (cat. no. P2863), CYP2D6 (cat. no. P2972) and CYP3A4 (cat.
no. P2858) blue screening kit included baculosome (respective isozymes and NADPH-P450
reductase); regeneration system (part no. P2878; glucose-6- phosphate, glucose-6-phosphate
and 30 U/mL Glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate, pH 8.0)
and NADP+ (part no. P2879; 10 mM NADP+ in 100 mM potassium phosphate, pH 8.0) were
used for the study. Ultra-Pure double distilled and deionized water was obtained from Milli-Q
plus water purification system (Millipore, Bedford, MA, USA). Formic acid of spectroscopic
grade and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Extraction of plant material extraction

4
The method of extraction of the plant material was similar to our previously published
protocol (Thomford et al., 2016). Briefly, herbal practitioners assisted in obtaining Hyptis
suaveolens (UCC/BS/687) and Boerhavia (UCC/BS/688) leaves and the plants were
authenticated by a practicing botanist, Mr. Aggrey Fynn, from the University of Cape Coast.
Specimen vouchers were deposited at the University’s herbarium. Air dried powdered plant
material was used in the extraction with water as the solvent to mimic the indigenous mode of
extraction. Filtrates were freeze-dried and stored at -20ºC until needed for use. The yield of
the extract was 6.5% (w/w) for Hyptis suaveolens and 11.24% (w/w) for Boerhavia diffusa
relative to dried starting materials.

2.3 UPLS-MS analysis of phenolic compounds

Analysis of phenolic compounds in the extracts was performed using a Waters Acquity
UPLC system from Waters Corporation (Milford, MA, USA) with an Acquity BEH C18
column (2.1 mm × 100 mm, 1.7 µm particle size) incorporating a binary pump, vacuum
degasser, auto-sampler, column oven and Micromass Xevo tandem quadrupole mass
spectrometric detector (QTOF Xevo G2; Waters micromass, Manchester, UK) equipped with
ESI (negative) probe. Gradient elution was performed at a flow rate of 0.1 mL/min
throughout at injection volumes of 10 µL. Gradient parameters were adjusted by
systematically changing the percentage organic modifier at initial conditions, and/or the
isocratic hold period at initial conditions, and/or gradient steepness. Mobile phase A was
7.5% formic acid in water and mobile phase B acetonitrile. The gradient started with 1% B
isocratically for 1 min followed by a linear increase to 28% at 22 min, 40% at 22.5 min and
100% at 23 min. Column clean-up at 100% B then followed for 1 min followed by re-
equilibration for 4 min at a total run-time of 29 min. Electrospray mass spectra data were
recorded on a negative ionization mode for a mass range m/z 100 to m/z 1500 at a collision
energy of 50 V. Low injection volumes were used to provide responses for major constituents
within the linear range. The instrument was operated in negative ionization mode and
calibrated using a sodium formate solution. Data were acquired in resolution mode and
MS/MS scanning mode and processed using MassLynx v.4.1 software (Waters). For
MS/MS experiments, the trap collision energy (CE) was set to obtain sufficient fragmentation
for selected precursor ions (30/45V). The eluent was split 3:1 prior to introduction into the
ionization chamber. The injection volume was 10 µL and UV-Vis spectra were acquired over
220-400 nm at 20 Hz. The accurate mass and composition for the precursor ions and for

5
fragment ions were determined using MassLynx v.4.1 software (Waters) that was
incorporated with the instrument. Using a non-targeted approach, gradient parameters were
utilized by systematically adjusting solvents to be able to determine as many compounds as
possible. Using fragmentation data and available databases such as KNApSAcK metabolite
database (Shinbo et al., 2006; Akiyama et al., 2008) and MOTO database (Moco et al., 2006),
possible compounds that may be found in the extracts were proposed.

2.4 CYP450 inhibition Assay

2.4.1 Reversible inhibition profile
Reversible inhibition activities of human CYP isoforms CYP1A2, CYP2D6 and CYP3A4 by
extracts of H. suaveolens and B. diffusa was carried out using a flourimetric assay as
described previously by Thomford et al. (2016). Two-point screening using two different
concentrations was initially used to determine the maximum inhibitory effect of the extracts
on the CYP450 isozymes. Conditions of the experimental assays including wavelengths are
shown in Table 1. The formation of fluorescent metabolite was measured to monitor
enzymatic activity at a wavelength of 405/460 nm using a Varian Cary eclipse (SSN
instruments, Set Point Technology, South Africa) with Advanced reads software. Known
standard inhibitors suggested by the manufacturer were used to validate the assay.

Table 1: Experimental conditions involved in fluorogenic assay

[E] Substrate [S] Standard inhibitor Fluorescence Fluorescent product
(nM) (µM) filter
CYP1A2 5 EOMCC 5 α-naphthoflavone/ Ex: 405 7-hydroxy-3-
furafylline nm/Em:460 nm cyanocoumarin
CYP2D6 10 EOMCC 10 quinidine Ex: 405 7-hydroxy-3-
nm/Em:460 nm cyanocoumarin
CYP3A4 5 BOMCC 5 ketoconazole/ Ex: 405 7-hydroxycoumarin
erythromycin nm/Em:460 nm
7-benzyl-oxymethyloxy-3-cyanocoumarin (BOMCC); 7- ethoxy-methloxy-3-cyanocoumarin (EOMCC),
enzyme concentration [E]; Substrate concentration [S]

Fluorescent read data was exported into an excel spreadsheet and the residual activity which
is a measure of the metabolite formed relative to the control was calculated using the
equation below.

………… (1)

6
Residual activity expressed as percentages was plotted against log transformed concentrations
of the extracts and known standard inhibitors. A sigmoid curve employing non-linear
regression analysis in GraphPad® Prism version 5.0 (GraphPad® Software Inc., San Diego,
CA, USA) was used to calculate the IC50.

2.4.2 Time-Dependent Inhibition (TDI) Potency using IC50 curve shift

Time-dependent inhibition (TDI) assay was determined using the curve-shift technique
according to Berry and Zhao (Berry and Zhao, 2008). Conditions of TDI assays are similar to
those shown in Table 1. The difference between the reversible and TDI assays is the pre-
incubation step of 30 minutes with NADPH to allow for interaction between the inhibitor
(extract or standard) and enzyme. Similar to the reversible inhibition, measurement of
fluorescent metabolite formation was used to monitor enzymatic activity measured at a
wavelength of 405/460 nm using a Varian Cary eclipse (SSN instruments, Set Point
Technology, South Africa) with Advanced reads software.

Fold shift for TDI classification was calculated using the equation below

…………… (2)

2.4.3 Determination of kinetics of inactivation

The method employed is a slight modification from the reversible inhibition and TDI assays
originally published by Krippendorff et al. (Krippendorff et al., 2009) with the detailed
protocol and further modified in our previous paper (Thomford et al. 2016). The modification
in this methodology is the measurement of fluorescent metabolite formation at 5-minute
intervals for 30 minutes.

Natural logarithm of the percentage remaining activity was then plotted against the pre-
incubation times at each concentration to obtain the slope (Kobs) where Kobs refers to the rate
constant describing the inactivation at each inhibitor concentration. The inhibitor
concentration required for half-maximal inactivation, Ki and the maximal rate of inactivation,
Kinact were then estimated by using non-linear regression analysis (GraphPad® Software Inc.,
San Diego, CA, USA) using the equation below.

…………………… (3)

Kinact is the maximal rate of inactivation; Ki is the inhibitor concentration required for half-

7
maximal inactivation; and [I] is the pre-incubation concentration of inhibitor (extract).

2.6 In vivo HDI prediction

Risk ranking of herbal extracts of Hyptis suaveolens and Boerhavia diffusa to potentially
cause in-vivo herb-drug interaction (HDI) was predicted using published guidelines for drug
interactions (Bjornsson et al., 2003; Zhang et al., 2009). The putative gastrointestinal tract
(GIT) concentration which served as the inhibitor concentration [I] gut was estimated using
the equation below

………………….. (4)

The % yield was used to estimate the bioavailable concentration knowing that herbal
medicines also have differential bioavailability’s (Abourashed, 2013). For soluble phyto-
constituents that are likely to interact with liver CYP450s, the maximum hepatic input
concentration [I]in was calculated using the equation below

[I]in = [I]gut + ka•Fa•D/Qh …………………… (5)

on the assumption that there was complete absorption of at least one bioavailable
phytochemical from the herbal extracts into the portal vein (Fa = 1.0), the values of the
absorption rate constant (ka), hepatic blood flow rate (Qh) were assumed to be 0.1 min-1 and
1450ml/min respectively (Obach et al., 2007).

2.7 Statistical analysis

All values are expressed as Mean ± SEM. Comparisons for significance were performed
using unpaired t-test with p< 0.05 considered statistically significant.

3.0 Results
3.1 UPLC-MS analysis of Hyptis suaveolens (HS) and Boerhavia diffusa (BD) extracts
identified several polyphenols.
Peaks shown in chromatogram as depicted in Figs. 1 and 2 were tentatively characterized and
identified in Table 2 and 3 respectively with their ESI-MS/MS data and fragmentations listed.
Tentatively identified compounds from HS (Table 2) included gluconic acid, caffeic acid,
rutin and quercetin. Tentatively identified compounds for BD (Table 3) also included
gluconic acid, citric acid and ferulic acid. Selected structures of these polyphenols are
assigned and assigned to peaks in the chromatograms.

8
 Fig.1: UPLC–MS chromatogram of crude aqueous extracts of Hyptis suaveolens (HS). (I) Gluconic acid (II)
Caffeic acid (III) Rutin (IV) Isoquercetin (V) Rosmarinic acid (VI) Ethyl caffeate

Table 2: Characterization of tentatively identified compounds in Hyptis suaveolens extract

by UPLC-MS.
Peak Rt [M-H]- Formula/Elemental Ppm UV MS/MS fragmentation Proposed
(min) composition (nm) compound
1 1.30 195.0520 C6H12O7 1.0 177,159,160 Gluconic acid
2 1.60 341.1097 C12H22O11 3.8 335, 330 Sucrose
3 2.24 191.0193 C6H8O7 0.5 111,127,129 Citric acid
4 06.71 197.0453 C9H10O5 1.5 230,280 190,97,87 Syringic acid
5 11.29 179.0343 C9H8O4 -0.6 230,321 161,135 Caffeic acid
6 13.59 417.1390 C18H26O11 -1.7 417,407,385,399 Oleoside
dimethyl ester
7 16.14 597.1233 C29H26O14 -1.8 359,295,179 Yunnaneic acid
8 16.42 609.1455 C27H30O6 -0.2 230,348 301,271 Rutin
9 16.85 463.0859 C21H20O12 -3.9 230,348 301,271, 179,151 Isoquercetin
10 17.67 505.0939 C30H18O8 3.2 447,505 Protohypericin
11 18.23 719.1625 C36H32O16 2.1 538,521,359 Sagerinic acid
12 19.13 359.0767 C18H16O8 0.0 230,329 197,179,161,151,135,133 Rosmarinic acid
13 19.58 599.1382 C29H28O14 -3.2
14 22.63 301.0343 C15H10O7 -1.7 295, 285, 301 Quercetin
15 23.23 207.0663 C11H12O4 2.9 230 179,133 Ethyl caffeate

9
Fig.2: UPLC–MS chromatogram of crude aqueous extracts of Boerhavia diffusa (BD). (I) Ferulic acid (II)
Citric acid (III) Syringetin 3-rutinoside (IV) Quercetin 3,7-dimethyl ether 4’-glucoside

Table 3: Characterization of tentatively identified compounds in Boerhavia diffusa extract by

UPLC–MS.
Peak Rt [M-H]- Formula/Elemental ppm UV MS/MS Assignment
(min) composition (nm) fragmentation
1 1.06 272.9587 CH6O16 3.3
2 1.29 195.0509 C6H12O7 2.1 230 177,159 Gluconic acid
3 1.51 177.0405 C6H10O6 3.4 Gluconolactone
4 1.67 209.0662 C7H14O7 0.5 Seduheptulose
5 2.43 191.0192 C6H8O7 0.0 269 111,127,129 Citric acid
5 2.63 203.0195 C7H8O7 1.5 284 167,97,124,141
6 6.96 315.0711 C13H16O9 -1.6 230 295 4-(ß-D/L-glucosyl)-3-
hydroxy-benzoic acid
7 10.40 417.1030 C17H22O12 -0.7 230 Citrofoline B epimer
8 10.78 325.0923 C15H18O8 0.0 230,325 315,311 (Z)-4-coumaric acid-4-
O-α/ß-glucopyranoside
9 11.48 369.0827 C16H18O10 1.4 230 207,192 Fraxin
10 11.53 287.1491 C14H24O6 -1.4 230
11 11.84 193.0499 C10H10O4 -1.0 230 165, 179,193 Ferulic acid
12 11.99 371.0970 C16H20O10 -2.2 230 Deacetylasperuloside
13 12.18 387.1650 C18H28O9 -1.3 230
14 15.85 787.2629 C60H36O2 -1.0
15 16.64 815.2257 C35H44O22 1.3 Syringetin 3-rutinoside-
7-glucoside
16 16.75 449.2026 C20H33O11 0.7
17 17.97 623.1611 C28H32O16 -0.2 230 Isorhamnetin 3-O-
rutinoside
18 19.53 653.1730 C29H34O17 1.8 230,270 Syringetin 3-rutinoside
19 22.13 491.1183 C23H24O12 -1.4 230,339 270,328 Quercetin 3,7-dimethyl
ether 4’-glucoside

10
3.2 3.2 Hyptis suaveolens (HS) and Boerhavia diffusa (BD) extracts exhibit strong inhibitory
effects on CYP450 enzymes.
Aqueous extracts of HS and BD were investigated for their capability to inhibit CYP450
enzyme activities by determination of reversible IC50 values, TDI potency and inactivation
kinetics. A summary of the data is given in Table 4. HS and BD exhibited strong inhibitory
effects on the three CYP450 enzymes studied. The inhibitory potency of HS on CYP1A2,
CYP2D6 and CYP3A4 were 3.68 ± 0.10µg/mL, 1.39 ± 0.01µg/mL and 2.36 ± 0.57 µg/mL,
respectively, while the inhibitory potency of BD was 9.48 ± 0.78µg/mL, 17.79 ± 1.02µg/mL
and 7.36 ± 0.94 µg/mL, respectively. However, the inhibitory potency for each of the two
medicinal herbs on each of the three CY450 enzyme, were weaker than those of respective
positive inhibitors, α-naphthoflavone for CYP1A2 (0.09 ± 0.01 µM), quinidine for CYP2D6
(0.42 ± 0.07 µM) and ketoconazole for CYP3A4 (0.02 ± 0.00 µM). HS was the most potent
inhibitor for the three enzymes, CYP1A2, CYP2D6 and CYP3A4. It is also observed that due
to longer incubation time, there was a slight apparent activation on CYP3A4 observed for HS
which is a possible phenomenon that can occur. However, this needs to be confirmed with
other CYP inhibition study systems to rule out wrong interpretation due to high fluorescence
readings with HLMs and HPLC-UV or LC-MS/MS analysis.

Table 4: Inhibition of CYP-catalysed reactions by herbal extracts

CYP1A2 CYP2D6 CYP3A4

Hyptis suaveolens
(Mean ± SEM) µg/mL 3.68 ± 0.10 1.39 ± 0.01 2.36 ± 0.57
Boerhavia diffusa
(Mean ± SEM) µg/mL 9.48 ± 0.78 17.79 ± 1.02 7.36 ± 0.94
Positive inhibitor
(Mean ± SEM) (µM) 0.09 ± 0.01 0.42 ± 0.07 0.02 ± 0.00
NB: Positive inhibitors, α-naphthoflavone for CYP1A2, quinidine for CYP2D6 and ketoconazole for CYP3A4

The potency of HS and BD to cause TDI was determined using IC50 curve-shift and this is
shown in Fig.3 A potential TDI will cause a shift in the sigmoid curve to the left after pre-
incubation with NADPH. A ratio of no pre-incubation with NADPH and pre-incubation with
NADPH of greater or equal to 1.5 was considered significant for a potential time-dependent

11
inhibition of CYP450 activity by the extract using the classification of Berry and Zhao (Berry
and Zhao, 2008).

Fig.3: IC50 curve shift for Time Dependent Inhibition (TDI) determination. Hyptis suaveolens (HS) and
Boerhavia diffusa (BD) at various concentrations were incubated with and without NADPH for 30 min.
Percentage residual activity compared to control for no pre-incubation (closed circles) and 30 min pre-
incubation (closed squares) is shown. (A, B): CYP1A2; (C, D): CYP2D6; (E, F): CYP3A4

12
Fig.4: IC50 curve shift for Time Dependent Inhibition (TDI) of known TDI and non-TDIs of CYP1A2, CYP2D6
and CYP3A4. (A) Furafylline (CYP1A2); (B) Quinidine (CYP2D6); (C) Erythromycin (CYP3A4). Percentage
residual activity compared to control for no pre-incubation (closed circles) and 30 min pre-incubation (closed
squares) is shown.

The assay was validated with furafylline a known CYP1A2 TDI compound, quinidine (non-
TDI compound for CYP2D6) and erythromycin (TDI compound for CYP3A4) as shown in
Fig.4. Extracts of HS and BD exhibited a TDI potency (dashed line) with an IC50 ratio of
greater than 1.5 on CYP1A2, CYP2D6 and CYP3A4 (Fig. 5). TDI assessment of HS on
CYP1A2, CYP2D6 and CYP3A4 were 1.7, 1.8 and 2.0, respectively while BD TDI was 2.1,
2.0 and 6.0, for these three enzymes. TDI assessment of positive inhibitors were furafylline,

13
CYP1A2 (2.6), quinidine, CYP2D6 (0.7) and erythromycin, CYP3A4 (1.6) validating the
assay.

CYP2D6
CYP1A2

BD BD

HS
HS

FUR

QND
-NF

5
0.

0.

1.

1.

2.

2.
0

5
0.

0.

1.

1.

2.

2.

-IC50 /+IC50 -IC50 /+IC50

CYP3A4

BD

HS

ERT

KTZ
0

-IC50 /+IC50

Fig.5: TDI potency classification of HS and BD extracts on CYP1A2, CYP2D6 and CYP3A4 using the IC50
curve shift. A ratio of ≥ 1.5 is considered significant enough for an extract to be classified as a TDI candidate.
FUR-furafylline; α-NF-alpha naphthoflavone; QND-quinidine; KTZ-ketoconazole; ERT-erythromycin

3.3 Hyptis suaveolens and Boerhavia diffusa inactivated CYP1A2, CYP2D6 and CYP3A4
Inactivation kinetics of CYP1A2, CYP2D6 and CYP3A4 by extracts of HS and BD were
estimated in a concentration- and time- dependent manner and followed pseudo first order
kinetics (Thelingwani et al., 2009). The inactivation time course is shown in Figs 6 to 8. Non-
linear regression analysis was then used to estimate the inactivation constants Ki and Kinact for
the two extracts on CYP450 enzyme activities. Inactivation kinetic parameters for CYP1A2
(Fig. 6) by Hyptis suaveolens and Boerhavia diffusa extracts were: Ki; 0.92 µg/mL and 2.02
µg/mL, 0.019 min-1, 0.012 min-1, respectively. Inactivation kinetic parameters of CYP2D6
(Fig. 7) were: Ki; 0.75 µg/mL and 0.57 µg/mL, Kinact; 0.015 min-1 and 0.014 min-1,

14
respectively. Kinetics parameters of inactivation for CYP3A4 (Fig.8) were Ki; 0.25 µg/mL
and 4.37 µg/mL, Kinact; 0.008 min-1 and 0.038 min-1, respectively.

Fig.6: Inactivation kinetics of CYP1A2 by extracts of Hyptis suaveolens (HS) and

Boerhavia diffusa (BD). CYP1A2 was pre-incubated with varying concentrations of HS and BD and assayed for
residual activity as described in materials and methods. (A, C): Kobs value was determined for respective
concentrations and non-linear fitted regression analysis used to estimate Ki and Kinact (B, D)

15
Fig.7: Inactivation kinetics of CYP2D6 by extracts of Hyptis suaveolens (HS) and
Boerhavia diffusa (BD). CYP2D6 was pre-incubated with varying concentrations of HS and BD and assayed for
residual activity as described in materials and methods. (A, C): Kobs value was determined for respective
concentrations and non-linear fitted regression analysis used to estimate Ki and Kinact (B, D)

16
Fig.8: Inactivation kinetics of CYP3A4 by extracts of Hyptis suaveolens (HS) and
Boerhavia diffusa (BD). CYP3A4 was pre-incubated with varying concentrations of HS and BD and assayed for
residual activity as described in materials and methods. (A, C): Kobs value was determined for respective
concentrations and non-linear fitted regression analysis used to estimate Ki and Kinact (B, D)

17
 3.4 Potential significant herb-drug interaction predictions
In vivo HDI predictions using in vitro data was carried out according to published guidelines
(Bjornsson et al., 2003; Zhang et al., 2009) for CYP1A2, CYP2D6 and CYP3A4. The
bioavailable concentration was calculated using the % yield and the inhibitor concentration
estimated as such (Table 5) under the assumption that at least one phytochemical is soluble
and absorbed.

Table 5: Estimated calculation of herbal medicine concentration in the gut

Estimated
Recommended Putative GIT Bioavailable
Herbal Dose Concentration Concentration
Herbal Extracts % Yield (Single; mg) (µg/mL) (µg/mL)
Hyptis suaveolens 6.51 400 1600 104.16
Boerhavia diffusa 11.24 200 800 89.92

Under these assumptions, the ratio of the inhibitor concentrations to Ki was used to risk rank
the extracts for the respective CYPs. For reversible inhibitors, the categorization of drug
interactions based on [I]/Ki is widely used and acceptable in the drug discovery and
development process where classification of in vivo drug interactions is based on likely
([I]/Ki > 1), possible (1 > [I] /Ki > 0.1) and remote (0.1 > [I] /Ki) (Zhang et al., 2009). It was
predicted there could be a significant in vivo HDI (Table 6). Also, comparing the obtained
IC50 values to the inhibitor concentration, it was observed that the values were four times
lower than the bioavailable concentration and therefore should adequate amounts enter
systemic circulation could interact with the respective CYPs. For TDIs, λ/Kdeg risk ranking
also indicated the likelihood of HDI by HS and BD as mechanism based inhibitors (Table 7)
(Ito et al., 2004).

Table 6: In vivo prediction of HDI from in vitro data for CYP1A2, CYP2D6, and CYP3A4

Predicted %
Inhibitor inhibition
concentration
[I]gut (µg/mL) IC50 Ki *Risk of
(µg/mL) (µg/mL) [I]gut/Ki HDI
CYP1A2
Hyptis
104.16 0.92
suaveolens 3.68 113.22 Likely 99.12
Boerhavia
89.92 2.02
diffusa 9.48 44.51 Likely 97.80

18
 CYP2D6
Hyptis
104.16 0.75
suaveolens 1.39 138.88 Likely 99.29
Boerhavia
89.92 0.57
diffusa 17.79 157.75 Likely 99.37
CYP3A4
Hyptis
104.16 0.25
suaveolens 2.36 416.64 Likely 99.76
Boerhavia
89.92 4.37
diffusa 7.36 20.58 Likely 95.37
Note: HDI, herb-drug interaction, inhibitor concentration = estimated bioavailable concentration (µg/mL), * the likelihood of
a clinically relevant interaction occurring when these herbal extracts are taken assumes that the % yield serves as the
bioavailable fraction which was used in estimating the bioavailable concentration in the gut and if there is complete
absorption, [I]/Ki > 1 (possible), 1 > [I] /Ki > 0.1 (possibly) and 0.1 > [I] /Ki (remote)

Table 7: in vivo prediction of irreversible CYP1A2, CYP2D6 and CYP3A4 inactivation by extracts

Parameters Hyptis suaveolens Boerhavia diffusa

CYP1A2
Concentration [I]in (µg/mL) 141.77 108.73
Kinact (min-1) 0.019 0.012
Ki (µg/mL) 0.92 2.02
λ[I] 0.018 0.012
Gut (ƛ/kdeg) 60.81 40.54
HDI risk Likely Likely
CYP2D6
Concentration [I]in (µg/mL) 141.77 108.73
Kinact (min-1) 0.015 0.014
Ki (µg/mL) 0.75 0.57
λ[I] 0.015 0.014
Gut (ƛ/kdeg) 66.37 61.95
HDI risk Likely Likely
CYP3A4
Concentration [I]in (µg/mL) 141.77 108.73
Kinact (min-1) 0.008 0.038
Ki (µg/mL) 0.25 4.37
λ[I] 0.007 0.036
Gut (ƛ/kdeg) 14.55 74.84
Hepatic (ƛ/kdeg) 21.81 112.15
HDI risk Likely Likely
λ= (Kinact × [I])/ (Ki + [I]); intestinal kdeg; CYP1A2 (0.000296/min;); CYP2D6 (0.000226/min;); CYP3A4 (0.000481/min;)
hepatic kdeg (CYP3A4) =0.000321/min λ/Kdeg > 1 (possible), 1 > λ/Kdeg > 0.1 (possibly) and 0.1 > λ/Kdeg (remote) (Obach et
al., 2007)

19
4.0 Discussion

In situations of co-morbidities, multi-drug and multi-target therapy is a reality which has

brought along with it, the co-use of medicinal herbs with conventional drugs (Pan et al.,
2014). Medicinal herb use is largely promoted by many factors but mostly the lack of
adequate cures for many non-communicable diseases for example among cancers where
conventional therapy effectiveness in some cases is less than 30% (Bhattaram et al., 2002;
Graham et al., 2008) and perceptions of their effectiveness due to being natural with lack of
associated side effects (Moreira et al., 2014). However, there is inadequate data concerning
the effects of medicinal herbs on the expression and activity of drug metabolising enzymes
which are part of the cornerstone to drug response. Components of medicinal herbs are
substrates of drug metabolising enzymes, and thus, have their pharmacokinetic (PK) and
pharmacodynamics (PD) modalities affected in situations of co-administration (He et al.,
2010). The constituent phytochemicals in medicinal herbs, individually are affected by
different PK and PD factors which similarly affect conventional drugs through inhibition or
induction (Izzo, 2005). Inhibition or induction of drug metabolising enzymes may affect
medicinal herbs’ and conventional drugs’ pharmacological activity and lead to sub
therapeutic levels or increase plasma concentration leading to adverse drug reactions (ADRs)
(Sørensen, 2002; Venkataramanan et al., 2006). The current study evaluated in vitro,
inhibition of cytochrome P450 enzymes by the two medicinal herbs, Hyptis suaveolens and
Boerhavia diffusa and their UPLC-MS profiles. This screening is useful in understanding
herb-drug interaction potential of these medicinal herbs through three important DMEs,
CYP1A2, CYP2D6 and CYP3A4.

UPLC-MS analysis of crude extracts revealed the presence of more than 15 polyphenols
candidates in each medicinal herb which likely explains their therapeutic effects and also
their drug interaction potential since polyphenols have been implicated in HDIs
(Vijayakumar et al., 2014). Some of the polyphenol compounds identified include citric acid,
gluconic acid, quercetin, isoquercetin, caffeic acid, ferulic acid, rutin, syringic acid and
rosmarinic acid. Caffeic acid and its ester rosmarinic acid act as multipurpose active
polyphenols and have been found to possess promising therapeutic potential against

20
conditions such as inflammation, cancer, infection and neurodegeneration (Chung et al.,
2004; Hossan et al., 2014;Murtaza et al., 2015). Quercetin and isoquercetin, are reported to
possess a wide range of biological effects including anti-carcinogenic, anti-inflammatory,
antiviral as well as attenuating lipid peroxidation and platelet aggregation (Portillo, 2011;
Maalik et al., 2014; Li et al., 2016). Ferulic acid exhibits a variety of biological activities
such as antioxidant, anti-inflammatory, antimicrobial, anti-allergic, hepatoprotective,
anticarcinogenic, antithrombotic, increase sperm viability, antiviral and vasodilatory actions
(Alam et al., 2013; Kumar and Pruthi, 2014). Similarly, polyphenols such as caffeic acid,
citric acid and quercetin have been implicated in interaction with CYP450 activity through
inhibition or induction (Baer-Dubowska et al., 1998; Surapaneni et al., 2014). However, it
remains to be determined, which of the polyphenols in these compounds are important for the
observed activities.

Hyptis suaveolens and Boerhavia diffusa extracts caused a concentration- and time-dependent
inhibition of CYP1A2, CYP2D6 and CYP3A4 enzyme activities, similar to what was
observed for Hypoxis obtusa (African potato) and Dicoma anomala commonly among
African populations for conditions like colic, diarrhoea, dysentery, toothache, fever,
haemorrhoids and HIV/AIDS (Gwaza et al., 2009). The reversible inhibitory potency of a test
compound can be classified according to its IC50 values as potent/strong (IC50 ≤20µg/mL or
10µM), moderate (IC50 20 -100 µg/mL or 10-50µM) or weak (IC50 ≥100µg/mL or ≥50µM)
(Kong et al., 2011). Reversible inhibition profiles indicated a potent inhibitory effect on
CYP450 activities by both extracts. The implication for HDI is that there is a possibility of
enzyme recovery should there be enough drug to compete out the herbal extracts. However, it
was reported that for most herbal remedies even with recommended dosages, patients
overdose, go beyond the stipulated duration or even use them outside their traditional
purposes (Phua et al., 2009). Reversible inhibition of DMEs is not an ideal situation for a
patient but once there is a possibility of enzymes recovery, it can be managed. For a new
chemical entity that exhibits reversible inhibition, it is important to evaluate the inhibition
potency over long term use.
The inhibitory potency of a medicinal herbal extract can also be affected by dosing duration
either through increase or decrease in interaction potential with the formation of inhibitory
metabolites and mechanism based inhibition (Grimm et al., 2009). This phenomenon is
referred to as time-dependent inhibition (TDI). This study therefore, also evaluated time-

21
dependent inhibition of CYP1A2, CYP2D6 and CYP3A4 enzyme activities using the IC50-
curve shift as employed by Berry and Zhao (Berry and Zhao, 2008) where an IC50- fold
decrease of ≥ 1.5 is considered significant enough for a time-dependent inhibition. It was
observed that, the inhibitory potency of extracts of HS and BD increased with time after TDI
assessment thus making them potential TDI candidates. Hyptis suaveolens and Boerhavia
diffusa extracts exhibited a TDI potency of 1.7 and 2.1 on CYP1A2, 1.8 and 2.0 on CYP2D6
and 2.0 and 6.0 on CYP3A4, respectively. Orally administered medicinal herb products are
metabolized by gut flora extrahepatically before being absorbed into the systematic
circulation (Wang et al., 2011). This is similar to observations in commonly used medicinal
herbs such as Hypoxis hermecollidea, Lessertia frutescens and Echinacea purpurea which
have been shown to present with inhibitory potency on CYP450 enzymes including CYP1A2,
2D6 and 3A4 in a reversible and time-dependent manner (Awortwe et al., 2013). Due to the
involvement of CYP1A2, CYP2D6 and CYP3A4 in drug metabolism of many commonly
used drugs and their wide substrate base, the reversible- and time-dependent inhibitory
profile of HS and BD presents a public health concern for patients likely to co-medicate with
drugs that are substrates of these CYPs and who could potentially experience clinically
significant HDIs.

Further evaluations of inhibitory parameters, Ki and Kinact values were carried out for HS and
BD extracts. These inhibitory parameters also indicated that HS and BD extracts were potent
inhibitors of CYP1A2, CYP2D6 and CYP3A4 activities. Using industry guidelines for drug
interactions, predictions were made for the possibility of in vivo HDI occurring under the
assumption that at least one phytochemical will be absorbed. It was observed that multi-
phytoconstituted extracts of HS and BD can cause significant in vivo HDIs acting as
mechanism-based inhibitors which can lead to clinically significant outcomes if co-used with
substrates of CYP1A2, CYP2D6 and CYP3A4. These are predictions, thus, for stronger
conclusions, in vivo HDI effects of herbals can best be studied in human subjects which
allows taking into consideration polymorphisms in genes coding for other enzymes
participating in minor pathways of metabolism of respective drugs or medicinal herb
constituents. Ultimately, for commercially available herbal products, this information (where
available) could be used on product information labels that will lead to minimisation of risk.

5.0 Conclusion

22
This study has shown that the commonly used herbal medicines, Hyptis suaveolens and
Boerhavia diffusa present with reversible- and time-dependent inhibitory effects on CYP1A2,
CYP2D6 and CYP3A4 activities. Given the pharmacogenomics importance of these
enzymes, attention should be paid to the use of these medicinal herbs in patients are also on
treatment with therapeutic drugs that are substrates of CYP1A2, CYP2D6 and CYP3A4.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

The authors would like to express their gratitude to the National Research Foundation
(NRF) of South Africa for support under an Indigenous Knowledge Systems (IKS) Research Grant for
funding the research. The views and opinions expressed are not those of the NRF but of the authors of
the material published. Nicholas Ekow Thomford was awarded a Faculty of Health Sciences,
University of Cape Town (UCT) postgraduate publication incentive (PPI) and fellowship.

Author contributions:
Nicholas Ekow Thomford (Nicholas.thomford@uct.ac.za), Kevin Dzobo
(kd.dzobo@uct.ac.za), Faustina Adu (a.faustina@uccsms.edu.gh), Shadrack Chirikure
(shadrack.chirikure@uct.ac.za), Ambroise Wonkam (Ambroise.wonkam@uct.ac.za) and
Collet Dandara (collet.dandara@uct.ac.za) contributed to the study. All authors listed
contributed and participated in the manuscript preparation. Nicholas Ekow Thomford, Collet
Dandara and Faustina Adu performed the laboratory, data analysis and interpretation. The
draft manuscript was written by Nicholas Ekow Thomford and Collet Dandara. Valuable
comments were received from Kevin Dzobo, Faustina Adu, Shadreck Chirikure and
Ambroise Wonkam. Shadreck Chirikure confirmed the traditional uses of the medicinal
plants.

23
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