Herbal Tea Extracts Inhibit Cytochrome P450 3A4 in Vitro

8/12/2019 Herbal Tea Extracts Inhibit Cytochrome P450 3A4 in Vitro

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Herbal tea extracts inhibit Cytochrome P450 3A4 in vitroSophie Dufaya, Alan Worsleya, Aymeric Monteilliera, Charlotte Avanzia, Jaclyn Syb, Ting Fat Nga,Jean-Michel Garciac, Man-Fai Lamd, Paul Vanhouttea and Ian C. K. Wonga

aDepartment of Pharmacology and Pharmacy, b Chemistry Department, The University of Hong Kong, dDepartment of Medicine, Queen MaryHospital Hong Kong and cInstitute Pasteur Korea, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea

Keywords

ciclosporin; Cytochrome 3A4; herbdrug

interaction; herbal teas; sirolimus; traditional

Chinese medicine

Correspondence

Sophie Dufay, Department of Pharmacology

and Pharmacy, The University of Hong Kong,

21 Sassoon Roads, Pokfulam, Hong Kong

SAR.

E-mail:[email protected]

Received August 21, 2013

Accepted March 23, 2014

doi: 10.1111/jphp.12270

Abstract

Objectives Ciclosporin and sirolimus, two immunosuppressive agents with

narrow therapeutic windows, are mainly metabolized by Cytochrome 3A4

(CYP3A4). A clinical case of toxic blood levels of these drugs after the consump-

tion of a 24-flavours tea was reported. This study aims to identify the causative

ingredients of the 24-flavour herbal tea in the inhibition of CYP3A4 metabolism.

Methods Two commercially available 24-flavour tea products purchased in Hong

Kong and the six plant constituents were tested for their CYP3A4 inhibitoryeffects utilizing an in-vitro fluorometric assay.

Key findings Of the commercially available teas available in Hong Kong, the most

potent inhibitory effect was observed with the tea consumed in the initial clinical

case. Of the six universal constituents, chrysanthemum exhibited the greatest

inhibitory effect, with an IC50 of 95.7 g/ml. Dandelion, liquorice and bishops

weed have IC50 of 140.6, 148.4 and 185.5 g/ml, respectively. Field mint and Japa-

nese honeysuckle have weaker inhibitory effect on CYP3A4 with IC50 of 1153.3

and 1466.3 g/ml.

Conclusions This study confirms the possible implication of herbal tea constitu-

ents in the inhibition of ciclosporin and sirolimus CYP3A4 metabolism. Com-

bined usage of herbal teas with drug should be closely monitored.

Introduction

The use of natural medicines has increased in western

countries primarily due to the belief that they are associated

with fewer adverse reactions. A survey performed in the

United States in 19981999 on ambulatory patients showed

that 16% of drug users concurrently used some form of

herbal supplement.[1] This number was even higher for

patients with long-term disease. A survey performed in the

United States in 2001 showed that 26% of patients with

human immunodeficiency virus (HIV) consumed herbalremedies at the same time as their HIV treatment.[2] This

number increases to more than one third of cancer patients

in two European surveys.[3,4] The impact of combined thera-

peutic drug, natural medicine use has been evaluated in a

clinical survey conducted over 804 patients from six outpa-

tient clinics in the United States. It showed that 40% of

herbal users were at risk of herbdrug interaction, with 7%

of them effectively reporting adverse reactions.[5] However,

in most cases, potential interactions between the different

treatments were not evaluated.

With the increased consumption of herbal remedies con-

comitant to drug treatment, the potential risk of herbdrug

interaction and sparse scientific evaluation, more scientific

data are needed on assessment of the safety of such

combination.[610] In this article, a scientific investigation of

a clinical case with suspected herbdrug interaction is pre-

sented. Experiments were designed to mimic real case situa-

tion where most of in-vitro studies published failed.

A clinical case from a regional nephrology unit (QueenMary Hospital, Hong Kong) reported a tenfold increase in

the blood level of two immunosuppressants (ciclosporin A

and sirolimus) in a patient with a stabilized renal allograph

for 10 years.[11] A full patient history revealed ingestion of a

commonly consumed herbal tea (24-flavour tea,)

during the three previous days, and it was suggested to be

the possible causative agent of the increased levels of the

two immunosuppressant drugs. Herbal teas are part of daily

life in Hong Kong and China. Many different kinds of

herbal teas can be found under different forms. They are

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sold as ready-to-drink infusions in teashops at street

corners, but also are available as dried plant mixtures

(which are subsequently prepared at home) or freeze-dried

granules of infused decoctions. They are drunk as a bever-

age for many suggested reasons, primarily as a tonic and to

improve health both physically and mentally and to preventillness.

A comprehensive literature review, utilizing the search

terms 24 flavour teas (), tea, herbal teas, herbal

infusion, interaction, herbal remedies, Chinese medicine,

cyclosporin and sirolimus, was realized. From these papers,

two clinical cases were observed by Nowacket al. (2005), in

which increased plasma levels of ciclosporin were observed in

patients concurrently consuming herbal tea products. These

studies showed two renal transplant patients drinking herbal

teas (one of whom had consumed chamomile tea, and the

other one consumed a mixture called wild fruit tea drink

containing rose hip extract, hibiscus extract and lemon) whileboth taking ciclosporin. Both patients showed an increase in

ciclosporin level following their respective herbal tea con-

sumption. The authors suggested that the responsible agents

were probably orange peel, or other citrus ingredients and

possibly chamomile.[12] However, without herbal tea composi-

tion, there was a lack of evidence that the 24-flavour tea

drunk by our patient could be linked with these two clinical

cases. With regard to the narrow therapeutic window of

ciclosporin and sirolimus, and the fact that renal transplanted

patients are advised to consume a large volume of fluids to

improve renal graft function, the impact of herbdrug inter-

action on transplant patients might be significant due to the

risk of drug nephrotoxicity.To investigate the described clinical case and possible causes

for observed elevation in immunosuppressant agents, the first

step was to obtained detailed information about the composi-

tion of 24-flavour tea. As there is not set formula of 24-flavour

tea, composition may vary between manufacturers, and there-

fore 22 different brands of 24-flavour tea were sought to

establish a list of those 20 plants present in the majority of

these products. When screened into the literature for inhibi-

tory effect on the cytochrome of these 20 plants, little or no

information appeared available. The most reported interac-

tion was the inhibition of Cytochrome 3A4 (CYP3A4) by

citrus fruit consumption (in particular, grapefruit, whichhowever is not included into any 24-flavour tea formula).

Sirolimus and ciclosporin are mainly metabolized by

CYP3A4 and are substrates of p-glycoprotein (p-gp) in the

small intestine. These two sites might be responsible for the

increased level of ciclosporin and sirolimus. P-gp is an

effective efflux transporter of the luminal surface of gastro-

intestinal epithelial cells and opposes the absorption of

some molecules like sirolimus and ciclosporin. CYP3A4 is

the most abundant cytochrome enzyme in the liver, but it is

also present in sustentative amounts in the enteric mucosa

of the gastrointestinal tract.[13] To access to liver cyto-

chromes, chemical compounds need to be lipophilic to pass

through the gut barrier. However, these chemical com-

pounds are water-soluble (infusion of the tea), then it is

most likely that inhibitory effect will occur at the gut level

where chemical compounds will be available at a muchhigher concentration.

However, p-gp has been associated with competitive and

short-lived inhibition effect, whereas CYP3A4 has a poten-

tial for a prolonged inhibitory effect.[14] Also, CYP3A4 is

involved in the metabolism of more than 50% of the drugs

on the market,[13] so with regard to the mechanism but also

the impact it was decided to evaluate the inhibitory effect of

CYP3A4 in vitro. As 24-flavour tea composition varies, the

investigation of its inhibitory effect in vitro was performed

to confirm the doctors hypothesis on the identical tea con-

sumed by the patient in the clinical case and another com-

mercially available product to avoid anecdotal conclusions.The four commonly found plant within the list of 20 (two

third of the 22 brands of tea), and two widespread plants,

included in more than one third of the 22 brand were tested

in vitro for their inhibitory effect on CYP3A4. A literature

search for the chemical constituents of those plants tested

was performed, revealing a whole series of compounds.

Where those compounds identified were commercially

available, these were purchased and used as standards

against those plant extracts isolated after analysis by high-

performance liquid chromatography hyphenated with

photodiode array (HPLC-PDA).

Materials and MethodsPreparations of water extract of herbalremedies and 24-flavour tea

24-flavours tea () and the identified constituent

plants were purchased from a traditional Chinese medicine

wholesaler in Hong Kong. Taxonomic authentication of the

plants has been realized with regard to Chinese Pharmaco-

poeia (year 2010, English version) by an expert of chemistry

department involved in Hong Kong Chinese Materia Medica

Standards at the University of Hong Kong. Voucher speci-

mens were deposited and kept in chemistry department. The

plants studied were chrysanthemum (Chrysanthemummorifolium Ramat., 2014-SGD-01), dandelion (Taraxacum

mongolicum Hand-Mazz., 2014-SGD-02), Japanese honey-

suckle (Lonicera japonica Thunb., 2014-SGD-03), bishops

weed (Houttuynia cordata Thunb., 2014-SGD-04), liquorice

(Glycyrrhiza ularensis Fisch., 2014-SGD-05) and field mint

(Mentha haplocalyxBriq., 2014-SGD-06).

The 24-flavours teas were prepared according to the rec-

ommendations given by the street tea store/wholesaler or by

the manufacturers instructions. One example of 24-

flavours tea () consumed by the patient in the

Sophie Dufayet al.Inhibitory effect of herbal teas on CYP3A4

2014 Royal Pharmaceutical Society,Journal of Pharmacy and Pharmacology,, pp. 2


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above-mentioned clinical report was purchased as an

aqueous infusion (the commonest form available in Hong

Kong). The entire content of one bottle (250 ml) was freeze-

dried after filtration (number 1).

The second 24-flavours tea () (number 2) pre-

sented was purchased as a mixture of crude plant materialand was processed as recommended by the manufacturer.

A sample of 15 g of each plant material was weighed and

extracted with 150 ml of distilled water and boiled for 1 h.

The plant solid residues were then removed and further

extracted with the same procedure. Both water extracts were

combined and vacuum-filtered on Bchner with paper filter

(Advantec 90 mm; Toyo Roshi Kaisha Ltd, Tokyo, Japan).

The combined filtrates were freeze-dried (freeze dryer,

IlShin, IlShin Europe B.V., Ede, The Netherlands) overnight

and removed from the machine when extracts were totally

dried. The resulting powders were weighed and stored in

airtight individual brown glass bottles, for light protection.

CYP3A4 enzyme assay

Different methods can be used to evaluate inhibitory effects

on cytochromes: human liver slices, human hepatocytes,

microsomes and human cytochromes. Human cytochrome

technique was selected with regard to reliability to assess an

inhibitory effect on a given cytochrome, as it is isolated

from others. CYP3A4 inhibition was measured using the

fluorometric Cytochrome P450 CYP3A4/DBF Inhibition

Kit (BD Supersomes, BD Biosciences, Le Pont de Claix,

France) following manufacturer recommendations. Briefly,

incubations were conducted in a reaction volume of 200 l/well in 96-well microlitre plate, as described below.

The procedure was performed in two stages. During the

first step, NADPH co-factor mix was incubated with tested

samples (plants/teas or ketoconazole as positive control

inhibitor). During the second step, the enzymatic reaction

was started by addition of the enzyme (CYP3A4) and its

substrate (dibenzylfluoresceine, DBF) solution. The incuba-

tion time for each step was 10 min at 37C. Finally, NaOH

(2N) was added to stop the enzyme reaction, followed by

measure of the fluorescence in Cary Ellipse plate reader

fluorometer (Agilent Technology, Santa Clara, CA, USA).

The wavelengths set were 485 nm for excitation and 538 nmfor emission, specific of fluorescein, which is the metabolite

of DBF used as substrate of CYP3A4.

To determine the concentration at which 50% of the

CYP3A4 activity is inhibited (IC50), the liquid herbal

extracts concentration was expressed as the quantity in

gram of dry extract per litre. Eight points of threefold serial

dilutions of each sample were performed directly in the

96-well plates, starting from a concentration of 10.0 mg/ml.

The logarithms of these values were plotted onto the

graphs. Each compound was tested in triplicate. Inhibition

curves were plotted from the log values of the samples con-

centration versus the percentage inhibition calculated.

Ketoconazole was used as positive control, while negative

control wells contained all constituents of the reaction

except the inhibitors (ketoconazole, teas or plants).

Data analysis

Extracts of plants and 24-flavour teas will be tested in tripli-

cate. Means and standard deviation of each inhibitory effect

measured were calculated. All three analyses will be used to

determine IC50. The calculation of IC50 values for CYP3A4

inhibition values was carried out by nonlinear regression

analysis using REGTOX program freely available on

Normale Superieure website (http://www.normalesup.org/

vindimian/fr_index.html). It was also used for the fitting

of the dose-response sigmoid curves.

Analysis of 24-flavour teas and plantsextract by HPLC-PDA analysis

Chemicals

Different chemical compounds thought to be possible

causative agents of enzyme inhibition were identified in the

herbal extracts of 24-flavour teas and plants. When available

commercially with purity higher than 98%, standards were

used for identification by HPLC-PDA analysis: oleanolic

acid, ursolic acid, hesperidin and naringenin were pur-

chased from Sigma-Aldrich (St Louis, MO, USA); quercetin

from Cayman Chemical Company (Ann Arbor, MI, USA),

glycyrrhizin ammonium was bought from Calbiochem(EMD Chemicals Inc, San Diego, CA, USA, affiliated to

Merck, Darmstadt, Germany) and hesperetin from Tokyo

Chemicals Industry Co Ltd (Tokyo, Japan).

Chromatographic system

The chromatographic system was composed of an HPLC

apparatus from Agilent 1260 Infinity Quaternary LC System

(Agilent Technologies Ltd), which includes helium degasser

(1260 Infinity Standard degasser), a quadratic pump (1260

Infinity binary pump VL) and an auto sampler (1260 Infin-

ity Standard Autosampler). This system was piloted by the

Open LAB CDS Chemstation software version B. 04.03.Detection was done with a photodiode array detector (1260

Infinity Diode Array Detector VL+).

Chromatographic analysis of plasma samples were per-

formed on the Agilent Prep-C18 Scalar Column 4.6

250 mm, 5 m from Agilent (Bellafonte, PA, USA). The flow

rate was set at 1.0 ml min. A gradient of phase A (water

with 0.1% of phosphoric acid) and phase B (acetonitrile)

was used to separate all the chemical compounds. The chro-

matographic analysis was performed using the following

aqueous mobile phase (phase A) gradient with a flow rate

Sophie Dufay et al. Inhibitory effect of herbal teas on CYP3A4

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of 1 ml/min: 0 min, 95%; 010 min, 75%; 1020 min, 75%;

2036 min, 50%; 3640 min, 18.4%; and 4060 min, 18.4%.

Results

The experiments were designed to mimic closely the observed

clinical situation as reported in the case. For this reason, the

doses tested were close to those recommended by the herbal

tea manufacturers. Table 1 represents the usage and those

doses recommended for the six most common plants found

in 24-flavours teas identified in Hong Kong.[1517] The dailydose for the six plants investigated ranges between 1 g and

60 g. With regard to the recommended dose of each plant, it

was decided to extract 15 g with water. All of these plants

have some reported effect upon respiratory disease, as

explained in the chart below, and this may justify their his-

torical incorporation into 24-flavours teas (Table 1). The

plants described were extracted by water in the manner

described to obtain all those hydrophilic compounds that

would be present after preparation by the commercial tea

shop or in the domestic setting.

Between 2 g and 4 g of freeze-dried powder was obtained

for each tea/plant samples. The concentration of the dailydose was calculated using the weight of the freeze-dried

powder divided by the amount of water used (300 ml).

To evaluate the inhibitory potency of the different plants, the

starting dose of 10 g/l was chosen for all in-vitro experiments.

Tables 2 and 3 present the concentration of a daily dose of

herbal tea or plant (weight in grams of extract obtained after

freeze-dried procedure divided by the extraction volume of

water). They are all higher than 10 g/l except for bishops

weed (Houttuynia cordata) and field mint (Mentha

haplocalyx). But bishops weed (Houttuynia cordata) daily

posology is 1560 g, as a dose of 15 g lead to a concentration

of 7 g/l, hence a dose of 20 g will reach 10 g/l. For field mint

(Mentha haplocalyx), 15 g is twice higher than the higher rec-

ommended dose. This means that a dose of 10 g/l is a realistic

clinical gut concentration.

CYP3A4 enzyme assay

The enzymatic testing of the 24-flavours teas was performed

at concentrations that reflected adult human doses. The rec-

ommendation made by Hong Kong herbal street sellers of

24-flavour tea is that an adult consumes the entire content of

one bottle as a single dose. Similarly those commercially avail-

able products where a customer prepares the 24-flavour teathemselves, the instructions are that the full bag of plants

(approximate weight 250 g) should be used for one infusion

with water, resulting in a total volume after processing of

250 ml of beverage. Consequently, 250 ml of infused products

was reduced via freeze drying to dry residues.

The sigmoid curves of the percentage of inhibition of

CYP3A4 as a function of the concentration obtained after

curve fitting by REGTOX program are presented in

Figure 1. All of them showed inhibitory effect when 15 g of

plants were used.

Table 1 Doses and indications of plants commonly found into 24-flavours tea

English name/Latin name Chinese name Posology Therapeutic indication

Chrysanthemum/Chrysanthemum

morifolium

1015 g[15] It is used as an anti-inflammatory agent, and for the treatment

of headache, cold, vertigo and conjunctivitis.[16]

Dandelion/Taraxacum

mongolicum

915 g[15] Upper respiratory tract infection, acute tonsillitis, pharyngitis,

gastritis, enteritis, general fatigue, relief of cholecystitis,abdominal pain, pyoderma, snakes bites.[15,17]

930 g[17]

Japanese

honeysuckle/Lonicera

japonica

1030 g[17] It is used as an antibacterial and anti-inflammatory agent for

the treatment of abscess, laryngitis, pharyngitis, infection of

upper respiratory tract, dysentery, common cold and

fever.[15,16]

1060 g[15]

Bishops weed/Houttuynia

cordata

1560 g[17] It is used as an anti-inflammatory, antidote and diuretic agent

for the treatment of pulmonary infection, bronchitis, asthma

oliguria and carbuncle.[16]

Liquorice/Glycyrrhiza ularensis 515 g i.e. 200800 mg

of glycyrrhizin[15]It is used as general tonic, an anti-inflammatory, mucolytic,

expectorant and analgesic agent for the treatment of

gastrointestinal and respiratory disorders.[16,17]115 g (standard

dose 3 g)[17]

Field mint/Mentha haplocalyx 18 g[17] It is used to promote bile secretion, antiseptic and spasmolytic

action. It is used for gastrointestinal disorder, upperrespiratory infection and biliary disorder.[17]

Table 2 IC50 of 24-flavours teas () on CYP3A4

24-flavours tea

Concentration of

the daily dose (g/l)

IC50

(g/ml)

% of extract to

reach IC50

concentration.

24-flavours tea n1 5.2 190.9 3.7

24-flavours tea n2 10.8 337.1 3.1




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The concentrations at which the CYP3A4 activity is

inhibited by 50% (IC50) are presented in Table 2 for the

two 24-flavours teas brands (), and in Table 3 for

individual plants. Table 4 showed the means and the stand-

ard deviations of the different concentration points.

Ketoconazole was used as a positive inhibitor. All plants

showed inhibition activity at tested doses (15 g). The 24-

flavours tea number 1 showed the same order of magnitude

of inhibitory effect than chrysanthemum (Chrysanthemum

morifolium), dandelion (Taraxacum mongolicum) and bis-

hops weed (Houttuynia cordata), and twice than that of the

24-flavours tea number 2.

The most potent plant, with respect to inhibitory effect

on CYP3A4, was found to be Chrysanthemum morifolium

with an IC50 of 95.7 (g/ml). It was followed closely by

dandelion (Taraxacum mongolicum), liquorice (Glycyrrhiza

ularensis) and bishops weed (Houttuynia cordata), which

were approximately1.5 times less potent than chrysan-

themum. Field mint (Mentha haplocalyx) and Japanese

honeysuckle (Lonicera japonica) were found to be 1015

times less potent than chrysanthemum.

Analysis of 24-flavour teas and plantsextract by HPLC-PDA analysis

Chemical compounds previously reported into literature to

be present in some of the plant tested and commercially

available with excellent purity were searched in the herbal

extracts used for the CYP3A4 assay. Hesperidin, quercetin,

naringenin, hesperetin, glycyrrhiza, oleanolic and ursolic

acid were effectively separated by HPLC-PDA method. As

maxima of compounds UV spectrum are different, chro-

matograms are extracted at 208 nm and 254 nm, and iden-

tification will be done with regard to retention time and

UV spectra, which improve the sensitivity of the detection

but also the specificity of identification method in

such complex matrix. Retention time were hesperidin(16.8 min), quercetin (29.6 min), naringenin (33.4 min),

hesperetin (34.5 min), glycyrrhizin (36.0 min), oleanolic

acid (55.0 min) and ursolic acid (55.7 min).

With regard to the retention time of standards and their

spectrum, some known chemical compounds were identi-

fied in the plants extracts.

Figure 2 showed the analysis of herbal extracts by HPLC-

PDA method. Quercetin was founded in Chrysanthemum

morifolium(Figure 2a) andHouttuynia cordata(Figure 2d).

Oleanolic acid and ursolic acid were founded in Taraxacum

mongolicum(Figure 2b) and Mentha hoplocalyx(Figure 2f).

Glycyrrhizin was identified in Glyccyrrhyzia ularensis(Figure 2e). Hesperidin and hesperetin were found in

Mentha haplocalyx (Figure 2f). None of the tested com-

pounds were present either in Lonicera japonica nor in the

two brands of 24-flavour tea tested.

Discussion

24-Flavours teas ()

Testing the 24-flavours tea itself was important to evaluate

the total inhibitory effect of the herbal teas and in particular

the one drunk by the patient described in the initial clinical

case. The two brand of 24-flavour tea showed inhibitory

effect on CYP3A4 at clinical doses. This testing was done inan attempt to confirm the doctors hypothesis that the

24-flavour tea was responsible for the inhibitory effects of

CYP3A4. However, none of the seven compounds screened

by HPLC-PDA were identified in the 24-flavour tea. It

might be due to the limit of detection of the analytical

method, which does not allow identification of the low level

of chemical compound even if they are present at concen-

tration at which they show inhibitory effect (data not

shown). Also, as the tea is a mixture of different plant, the

amount of each of them might be low and the total effect

might be due to a synergistic effect of inhibitory com-

pounds. As the formulation of 24-flavour tea varies, it wasimportant to try to identify the causative(s) plants included

into the formula.

Individual plants tested

Chrysanthemum morifolium/Chrysanthemum

Chrysanthemum morifoliumshowed the most potent inhibi-

tory effect on CYP3A4 among the six tested plant extracts.

Out of the seven standards tested, quercetin was founded in

the sample analysed. Three flavonoids also present in

Table 3 IC50 of CYP3A4 by chrysanthemum (Chrysanthemum

morifolium), dandelion (Taraxacum mongolicum), Japanese honey-

suckle (Lonicera japonica), Bishops weed (Houttuynia cordata), liquo-

rice (Glycyrrhiza ularensis) and field mint (Mentha haplocalyx)

Plants Concentration of

the daily doseof 15 g of

plants (g/l)

IC50

(g/ml)English name/Latin name

Chinese

name

Chrysanthemum/

Chrysanthemum

morifolium

13.3 95.7

Dandelion/Taraxacum

mongolicum

10 140.6

Japanese

Honeysuckle/Lonicera

japonica

15 1466.3

Bishops weed/Houttuynia

cordata

7 185.5

Liquorice/Glycyrrhiza

ularensis

10 148.4

Field mint/Mentha haplocalyx 9 1153.3




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%I

nhibitionof

Cytochrome

3A4activity

%I

nhibitionofCytochrome

3A4activity

%I

nhibitionofCytochrome

3A4activity

%I

nhibitionofC

ytochrome

3A4activity

%I

nhibitionofC

ytochrome

3A4activity

%I

nhibitionofC

ytochrome

3A4activity

%I

nhibitionofC

ytochrome

3A4activity

%I

nhibitionof

Cytochrome

3A4activity

120

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Log (24-flavour tea 1 extract concentration) in g/ml

Log (Chrysanthemum extract concentration) g/ml

Log (Japanese Honeysuckle extract concentration) in g/ml

Log (Licorice extract concentration) in g/ml

Log (24 herbal tea 2 extract concentration) in g/ml

Log (Dandelion extract concentration) in g/ml

Log (Bishops weed extract concentration) in g/ml

Log (Field mint extract concentration) in g/ml

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 1 Percentage of inhibition of CYP3A4 activity by the two different 24-flavours teas (). (a and b), and the six individual plants

bought in Western Market in Hong Kong Island, (c) Chrysanthemum ( Chrysanthemum morifolium), (d) Dandelion (Taraxacum mongolicum), (e)

Japanese honeysuckle (Lonicera japonica), (f) Bishops weed (Houttuynia cordata), (g) Liquorice (Glycyrrhiza ularensis) and (h) Field mint (Mentha

haplocalyx).




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Chrysanthemum moriflorium have been thought to be

involved in causing an increase in ciclosporin serum levels

by grapefruit juice:[14,1820] hesperetin,[21] kaempferol[22] and

quercetin.[2123] Quercetin was found slightly more potent

than kaempferol in the inhibition of human liver

microsomes.[24]

Taraxacum mongolicum/Mongolian dandelion

The inhibitory effect of Taraxacum mongolicum can be

linked to the presence of hesperetin, hesperidin andquercetin,[25] which are thought to be partially responsible

for the effects of grapefruit juice on increasing the

bioavailability of ciclosporin.[19,20,24] When analysing the

extract by HLPC-PDA with hesperetin and quercetin stand-

ard, none of these three compounds were identified, which

might be due to the HPLC-PDA limit of detection. Ursolic

acid and oleanolic acid were newly reported as being

present in Taraxacum mongolicum. Oleanolic acid has been

reported to have inhibitory effect on CYP3A4 (IC50 of

78.4m), whereas ursolic acid has no inhibitory effect

(IC50 >500 m).[26]

Glycyrrhiza ularensis/Liquorice

The inhibitory effect ofGlycyrrhiza ularensis found in this

study has already been reported for a dose of 0.02 mg/ml. [27]

Another liquorice species,Glycyrrhiza glabra, has shown to

inhibit CYP3A4 in human liver microsome experiments

with IC50 in a range of 12% of the full strength, which was

classified as a potent inhibitor by Budzinski et al.[28]

Glycyrrhiza ularensis has been reported to contain

glycyrrhizin, liquiritin and liquiritin aposide like all

Glycyrrhiza species.[29] Glycyrrhizin was identified in the

sample analysed by HPLC-PDA. Glycyrrhizin was reported

to have no inhibitory effect (IC50> 1.2 mM). However,liquiritin and liquiritin aposide have IC50 of 57 and

655m, respectively, on human CYP3A4.[27]

Tsukamoto has also identify three other compounds

in Glycyrrhiza ularensis that have potent inhibitory

effect on human CYP3A4: (3R)-vestitol (IC50=3.2 m),

liquiritigenin 7,4-digucoside (IC50= 17 m) and 4-

hydroxyguaiacol apioglucoside (IC50 =20 m).[27]

Houttuynia cordata/Bishops weed

The inhibitory effect was less potent but might be much

more important if the dose used in the test is increased with

regard to recommended posology, 1560 g. It has been

reported that Houttynia cordata contains quercetin,[3036]

which was also identified in the sample used in this current

research. Quercetin is thought to be involved in the increase

of ciclosporin bioavailability with the consumption of

grapefruit juice.[20,24,37]

Mentha haplocalyx/Field mint

The mild inhibitory effect of Mentha haplocalyx evalu-

ated in our study has also been reported with the use of

peppermint oil on CYP3A4.[38] Hesperetin, hesperidin,

Table 4 Means and standard deviations of Cytochrome 3A4 activity

Concentration

24-flavour tea

1 drunk by the

patient (number 1)

Another brand of

24-flaour tea

(number 2)

g/ml Mean SD Mean SD

10000 100.3 1.4 96.5 1.7

3333.3 99.0 3.6 92.9 5.0

1111.1 93.5 6.2 80.5 4.8

370.4 71.5 9.2 48.0 1.4

123.5 38.4 9.3 21.1 4.0

41.2 17.1 10.7 15.7 8.0

13.7 16.9 5.4 2.8 10.2

4.6 18.2 1.2 5.5 0.5

Concentration

Chrysanthemum

morifolium

Taraxacum

mongolicum


10000 95.0 0.2 97.7 0.2

3333.3 99.3 0.9 99.7 1.21111.1 91.0 0.7 87.1 3.9

370.4 67.4 1.7 60.2 5.8

123.5 51.7 17.7 42.6 7.9

41.2 32.4 8.8 25.6 0.3

13.7 12.9 1.1 17.1 5.6

4.6 7.1 4.4 9.3 6.9

Concentration

Lonicera

japonica

Houttuynia

cordata


10000 88.6 4.7 105.3 1.6

3333.3 71.8 1.9 107.1 1.7

1111.1 37.7 3.7 102.6 2.3

370.4 18.5 8.2 68.8 5.0

123.5 7.5 16.3 26.2 10.6

41.2 4.5 7.9 28.2 24.6

13.7 0.9 1.9 14.9 3.4

4.6 2.7 14.8 8.0 18.0

Concentration

Glycyrrhiza

ularensis

Mentha

haplocalyx


10000 101.5 0.3 92.0 1.1

3333.3 97.5 3.4 88.3 1.8

1111.1 90.5 0.3 60.6 1.0

370.4 65.3 4.8 35.3 2.1

123.5 35.7 6.1 17.2 9.241.2 32.3 11.6 15.0 0.2

13.7 2.5 6.3 0.7 1.3

4.6 18.7 23.1 2.4 1.3

SD, standard deviation.




8/13

(a) -Chrysanthemum morifolium(Chrysanthemum)

(b) -Taraxacum mongolicum (Dandelion)

mAU

350

300

250

200

150

100

50

0

mAU

350

300

250

200150

100

50

0

mAU

35

30

25

2015

10

5

0

mAU

35

30

25

20

15

10

5

0

10 20 30 40 50 60 min

min10 20 30 40 50 60

Figure 2 HPLC-PDA analysis of plant extracts to identify chemical compounds. Chromatograms were extracted at 208 (ursolic acid and oleanolic

acid) and 254 nm (hesperidin, quercetin, naringenin, hesperetin and glycyrrhizin). (a) Chrysanthemum (Chrysanthemum morifolium). (b) Dandelion

(Taraxacum mongolicum). (c) Japanese honeysuckle (Lonicera japonica). (d) Bishops weed (Houttuynia cordata). (e) Liquorice (Glycyrrhiza ularensis).

(f) Field mint (Mentha haplocalyx).




9/13

(c) - Lonicera japonica(Honeysuckle)

350

300

250200

150

100

50

0

350

300

250

200

150

100

50

0

10 20 30 40 50 60 min

min

(d) - Houttuynia cordata (Bishops weed)

350

mAU

mAU

300

250

200

150

120

100

80

60

40

20

0

100

50

0

10 20 30 40 50 60

Figure 2 Continued




10/13

(e) - Glycyrrhiza ularensis(Licorice)

mAU

350

300

250

200

150

100

50

0

350

300

250

200

150

100

50

0

10 20 30 40 50 60min

min

(f) - Mentha haplocalyx (Field mint)

mAU

350

300

250

200

150

100

50

0

mAU

350

300

250

200

150100

50

0

10 20 30 40 50 60

Figure 2 Continued




11/13

oleanolic and ursolic acid were identified in the sample ana-

lysed by HPLC-PDA. Hesperetin and hesperidin have been

described in the literature as being responsible for the

inhibitory effect on CYP3A4, and thus increased the

metabolism of ciclosporin.[19,20,24] Oleanolic acid has also

been reported to have such effect when tested on livermicrosomes.[26] Naringenin has been identified to be one

compound ofMentha haplocalyxspecies but was not found

in this study possibly because of the low sensitivity of the

method. It was thought to be one of the compounds

responsible for the grapefruit juice interaction with

ciclosporin.[19,37,39,40]

Some in-vivo studies conducted by Wacheret al. revealed

a significant increase of about threefold in ciclosporin peak

plasma concentrations and area under curve when a dose of

100 mg/kg of peppermint oil was administered in rats. [41]

The in-vitro liver microsomes test showed peppermint oil

was a less potent CYP3A4 inhibitor than ketoconazole,while a fourfold increase in dose was required to elicit the

same degree of inhibition on ciclosporin metabolism in

human liver microsomes than in rats.[41] Another in-vivo

trial by Dresser et al. showed significant increase in area

under curve and peak plasma concentration of felodipine,

mainly metabolized by CYP3A4, in 12 subjects challenged

with 330l of peppermint oil in 300 ml water.[42] The mean

increase was 140% compared with subjects with water

only.[42] With unchanged half-life, it was suggested that the

preparation predominantly affect the enteric enzymes.[42]

The in-vitro test identified peppermint oil and its constitu-

ents, menthol and menthyl acetate, as reversible, partially

mixed CYP3A4 inhibitors in liver microsome test.[42]

Maliakalet al. have reported no inhibitory effect but this

was after chronic treatment for 4 weeks with peppermint tea

(2% m/v).[43]

Lonicera japonica/Japanese honeysuckle

Lonicera japonica showed some mild inhibitory effect on

CYP3A4 (IC50 =1466 g/ml) compared with the other five

plants. Pao et al. have tested water extracts of Lonicera

japonica at three different concentrations (2, 4 and 6 mg/

ml) on human liver microsomes and did not see any inhibi-

tory effect.[44] This might be explained by the usage of liver

microsomes technique, containing all liver cytochromes,compared with individual cytochrome testing. Also, none of

the seven chemical compounds screened by HPLC-PDA

were identified into Lonicera japonica, possibly due to the

low sensitivity of HPLC-PDA method.

The analysis of plant extracts by HPC-PDA helped dem-

onstrate the presence of chemical compounds (quercetin,

hesperidin, hesperetin and oleanolic acid) possibly respon-

sible of inhibitory effect on CYP3A4 in some of the herbal

extracts studied. The implication of chemical compounds

previously reported in the literature in some of the usedplants but not identified in this study cannot be ruled out as

their concentration at which they exert their inhibitory

effect might be lower than the ability of the HPLC-PDA

method to identify them.

Conclusions

This article was aimed to present a methodology mimicking

closely clinical situation to investigate clinical case of pos-

sible herbdrug interaction when no information was avail-

able in the literature. The in-vitro experiments on CYP3A4

described above utilizing aqueous extracts at representativeclinical doses have demonstrated that 24-flavours teas and

the six mostly common plants have potential to inhibit the

metabolism function of CYP3A4.

The inhibitory effects ofTaraxacum mongolicum, Chry-

santhemum morifolium and Houttuynia cordata can most

likely be explained by the presence of specific chemical

compounds that has already been proposed to play a role

in the interaction of grapefruit juice with ciclosporin

(hesperetin, kaempferol, quercetin and hesperidin) but also

by the presence of other compounds, ursolic acid and

oleanolic acid. Further work is needed to purify and identify

other compounds present in the plant as shown on chro-

matograms to test potency of their inhibitory effect.

Doctor should ask for herbal consumption in front of

unexplained drug toxicity and put in place clinical surveil-

lance programme especially for patients treated with immu-

nosuppressant. Similar attitude should be adopted with

other drugs known to interact with grapefruit juice: antiar-

rhythmics, calcium channel blockers, statins, cytotoxic

agents, carbamazepine or some antipsychotics. Further sci-

entific evaluations of potential herbdrug interaction are

needed.

DeclarationsFunding

This study has been funded by the Department of Pharma-

cology and Pharmacy of the University of Hong Kong.

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Herbal Tea Extracts Inhibit Cytochrome P450 3A4 in Vitro