Herbal tea extracts inhibit Cytochrome P450 3A4 in vitro.

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Research Paper

Journal of Pharmacy And Pharmacology

Herbal tea extracts inhibit Cytochrome P450 3A4 in vitro Sophie 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 a Department of Pharmacology and Pharmacy, bChemistry Department, The University of Hong Kong, dDepartment of Medicine, Queen Mary Hospital Hong Kong and cInstitute Pasteur Korea, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea

Keywords ciclosporin; Cytochrome 3A4; herb–drug 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 consumption 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 inhibitory effects 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 μg/ml. Dandelion, liquorice and bishop’s weed have IC50 of 140.6, 148.4 and 185.5 μg/ml, respectively. Field mint and Japanese honeysuckle have weaker inhibitory effect on CYP3A4 with IC50 of 1153.3 and 1466.3 μg/ml. Conclusions This study confirms the possible implication of herbal tea constituents in the inhibition of ciclosporin and sirolimus’ CYP3A4 metabolism. Combined 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 1998–1999 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 herbal remedies 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 therapeutic drug, natural medicine use has been evaluated in a clinical survey conducted over 804 patients from six outpatient clinics in the United States. It showed that 40% of herbal users were at risk of herb–drug 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 concomitant to drug treatment, the potential risk of herb–drug interaction and sparse scientific evaluation, more scientific data are needed on assessment of the safety of such combination.[6–10] In this article, a scientific investigation of a clinical case with suspected herb–drug interaction is presented. Experiments were designed to mimic real case situation where most of in-vitro studies published failed. A clinical case from a regional nephrology unit (Queen Mary 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, 廿四味茶) 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

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Inhibitory effect of herbal teas on CYP3A4

Sophie Dufay et al.

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 beverage for many suggested reasons, primarily as a tonic and to improve health both physically and mentally and to prevent illness. A comprehensive literature review, utilizing the search terms 24 flavour teas (廿四味茶), tea, herbal teas, herbal infusion, interaction, herbal remedies, Chinese medicine, cyclosporin and sirolimus, was realized. From these papers, two clinical cases were observed by Nowack et 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) while both taking ciclosporin. Both patients showed an increase in ciclosporin level following their respective herbal tea consumption. The authors suggested that the responsible agents were probably orange peel, or other citrus ingredients and possibly chamomile.[12] However, without herbal tea composition, 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 herb–drug interaction 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 composition of 24-flavour tea. As there is not set formula of 24-flavour tea, composition may vary between manufacturers, and therefore 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 inhibitory effect on the cytochrome of these 20 plants, little or no information appeared available. The most reported interaction was the inhibition of Cytochrome 3A4 (CYP3A4) by citrus fruit consumption (in particular, grapefruit, which however 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 gastrointestinal 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 2

of the gastrointestinal tract.[13] To access to liver cytochromes, chemical compounds need to be lipophilic to pass through the gut barrier. However, these chemical compounds 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 much higher concentration. However, p-gp has been associated with competitive and short-lived inhibition effect, whereas CYP3A4 has a potential 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 doctor’s hypothesis on the identical tea consumed by the patient in the clinical case and another commercially 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 highperformance liquid chromatography hyphenated with photodiode array (HPLC-PDA).

Materials and Methods Preparations of water extract of herbal remedies and ‘24-flavour’ tea ‘24-flavours’ tea (廿四味茶) 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 Pharmacopoeia (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 specimens were deposited and kept in chemistry department. The plants studied were chrysanthemum (Chrysanthemum morifolium Ramat., 2014-SGD-01), dandelion (Taraxacum mongolicum Hand-Mazz., 2014-SGD-02), Japanese honeysuckle (Lonicera japonica Thunb., 2014-SGD-03), bishop’s weed (Houttuynia cordata Thunb., 2014-SGD-04), liquorice (Glycyrrhiza ularensis Fisch., 2014-SGD-05) and field mint (Mentha haplocalyx Briq., 2014-SGD-06). The ‘24-flavours’ teas were prepared according to the recommendations given by the street tea store/wholesaler or by the manufacturers’ instructions. One example of ‘24flavours’ tea (廿四味茶) consumed by the patient in the


Sophie Dufay et al.

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 freezedried after filtration (number 1). The second ‘24-flavours’ tea (廿四味茶) (number 2) presented was purchased as a mixture of crude plant material and 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 Büchner 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 μl/ 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 incubation time for each step was 10 min at 37°C. 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 nm for 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 concentration 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 triplicate. 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 plants extract 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 purchased 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 Infinity 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 performed on the Agilent Prep-C18 Scalar Column 4.6 × 250 mm, 5 μm 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 chromatographic analysis was performed using the following aqueous mobile phase (phase A) gradient with a flow rate


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Table 1

Sophie Dufay et al.

Doses and indications of plants commonly found into ‘24-flavours’ tea

English name/Latin name

Chinese name

Posology

Therapeutic indication

[15]

Chrysanthemum/Chrysanthemum morifolium Dandelion/Taraxacum mongolicum

菊花

10–15 g

蒲公英

9–15 g[15] 9–30 g[17]

Japanese honeysuckle/Lonicera japonica

金银花

10–30 g[17] 10–60 g[15]

Bishop’s weed/Houttuynia cordata

鱼腥草

15–60 g[17]

Liquorice/Glycyrrhiza ularensis

甘草

Field mint/Mentha haplocalyx

薄荷

5–15 g i.e. 200–800 mg of glycyrrhizin[15] 1–15 g (standard dose 3 g)[17] 1–8 g[17]

of 1 ml/min: 0 min, 95%; 0–10 min, 75%; 10–20 min, 75%; 20–36 min, 50%; 36–40 min, 18.4%; and 40–60 min, 18.4%.

Table 2

It is used as an anti-inflammatory agent, and for the treatment of headache, cold, vertigo and conjunctivitis.[16] Upper respiratory tract infection, acute tonsillitis, pharyngitis, gastritis, enteritis, general fatigue, relief of cholecystitis, abdominal pain, pyoderma, snakes bites.[15,17] 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] It is used as an anti-inflammatory, antidote and diuretic agent for the treatment of pulmonary infection, bronchitis, asthma oliguria and carbuncle.[16] It is used as general tonic, an anti-inflammatory, mucolytic, expectorant and analgesic agent for the treatment of gastrointestinal and respiratory disorders.[16,17] It is used to promote bile secretion, antiseptic and spasmolytic action. It is used for gastrointestinal disorder, upper respiratory infection and biliary disorder.[17] IC50 of 24-flavours teas (廿四味茶) on CYP3A4

Results

24-flavours tea

Concentration of the daily dose (g/l)

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.[15–17] The daily dose 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 historical 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 daily dose 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 bishop’s weed (Houttuynia cordata) and field mint (Mentha haplocalyx). But bishop’s weed (Houttuynia cordata) daily

24-flavours tea n°1 24-flavours tea n°2

5.2 10.8

4

IC50 (μg/ml)

% of extract to reach IC50 concentration.

190.9 337.1

3.7 3.1

posology is 15–60 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 recommended 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 recommendation 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 available products where a customer prepares the 24-flavour tea themselves, 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.


Sophie Dufay et al.


Table 3 IC50 of CYP3A4 by chrysanthemum (Chrysanthemum morifolium), dandelion (Taraxacum mongolicum), Japanese honeysuckle (Lonicera japonica), Bishop’s weed (Houttuynia cordata), liquorice (Glycyrrhiza ularensis) and field mint (Mentha haplocalyx)

Chinese name

Concentration of the daily dose of 15 g of plants (g/l)

菊花

13.3

蒲公英

10

140.6

金银花

15

1466.3

鱼腥草

7

185.5

甘草

10

148.4

薄荷

9

1153.3

Plants

English name/Latin name Chrysanthemum/ Chrysanthemum morifolium Dandelion/Taraxacum mongolicum Japanese Honeysuckle/Lonicera japonica Bishop’s weed/Houttuynia cordata Liquorice/Glycyrrhiza ularensis Field mint/Mentha haplocalyx

IC50 (μg/ml) 95.7

The concentrations at which the CYP3A4 activity is inhibited by 50% (IC50) are presented in Table 2 for the two ‘24-flavours’ teas brands (廿四味茶), and in Table 3 for individual plants. Table 4 showed the means and the standard deviations of the different concentration points. Ketoconazole was used as a positive inhibitor. All plants showed inhibition activity at tested doses (15 g). The ‘24flavours’ tea number 1 showed the same order of magnitude of inhibitory effect than chrysanthemum (Chrysanthemum morifolium), dandelion (Taraxacum mongolicum) and bishop’s 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 (μg/ml). It was followed closely by dandelion (Taraxacum mongolicum), liquorice (Glycyrrhiza ularensis) and bishop’s weed (Houttuynia cordata), which were approximately1.5 times less potent than chrysanthemum. Field mint (Mentha haplocalyx) and Japanese honeysuckle (Lonicera japonica) were found to be 10–15 times less potent than chrysanthemum.

Analysis of 24-flavour teas and plants extract 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, chromatograms are extracted at 208 nm and 254 nm, and identification 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 identified in the plants extracts. Figure 2 showed the analysis of herbal extracts by HPLCPDA method. Quercetin was founded in Chrysanthemum morifolium (Figure 2a) and Houttuynia 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 compounds were present either in Lonicera japonica nor in the two brands of 24-flavour tea tested.

Discussion ‘24-Flavours’ teas (廿四味茶) 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 in an attempt to confirm the doctor’s 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 concentration 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 compounds. As the formulation of 24-flavour tea varies, it was important to try to identify the causative(s) plants included into the formula.

Individual plants tested Chrysanthemum morifolium/Chrysanthemum Chrysanthemum morifolium showed the most potent inhibitory 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


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(b)

100

100

80 60 40 20 0 –20 0

1

2

3

4

% Inhibition of Cytochrome 3A4 activity

40 20 0

100

100 80 60 40 20 0 –20

60

120


(d)

120

80

0 1 2 3 4 –20 Log (24 herbal tea 2 extract concentration) in µg/ml

–40 Log (24-flavour tea 1 extract concentration) in µg/ml (c)

120


120


(a)

Sophie Dufay et al.

0

1

2

3

4

80 60 40 20 0 0

–20

Log (Chrysanthemum extract concentration) µg/ml (e)

(f)

4

120



60 40 20 0 0

1

2

3

4

–40 Log (Japanese Honeysuckle extract concentration) in µg/ml (h) 120 100 80 60 40 20 0 0

0.5

1

1.5

2

2.5

3

3.5

4

Log (Licorice extract concentration) in µg/ml

80 60 40 20 0 0

–20


(g) % Inhibition of Cytochrome 3A4 activity

3

100

80

–20

2

Log (Dandelion extract concentration) in µg/ml

100

–20

1

1

2

3

4

Log (Bishop’s weed extract concentration) in µg/ml 120 100 80 60 40 20 0 0

1

2

3

4

Log (Field mint extract concentration) in µg/ml

Figure 1 Percentage of inhibition of CYP3A4 activity by the two different 24-flavours teas (廿四味茶). (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) Bishop’s weed (Houttuynia cordata), (g) Liquorice (Glycyrrhiza ularensis) and (h) Field mint (Mentha haplocalyx).

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Table 4


Means and standard deviations of Cytochrome 3A4 activity

Concentration

24-flavour tea 1 drunk by the patient (number 1)

μg/ml

Mean

SD

Mean

SD

10000 3333.3 1111.1 370.4 123.5 41.2 13.7 4.6

100.3 99.0 93.5 71.5 38.4 17.1 16.9 −18.2

1.4 3.6 6.2 9.2 9.3 10.7 5.4 1.2

96.5 92.9 80.5 48.0 21.1 15.7 −2.8 −5.5

1.7 5.0 4.8 1.4 4.0 8.0 10.2 0.5

Chrysanthemum morifolium

Concentration

Another brand of 24-flaour tea (number 2)

Taraxacum mongolicum

μg/ml

Mean

SD

Mean

SD

10000 3333.3 1111.1 370.4 123.5 41.2 13.7 4.6

95.0 99.3 91.0 67.4 51.7 32.4 12.9 7.1

0.2 0.9 0.7 1.7 17.7 8.8 1.1 4.4

97.7 99.7 87.1 60.2 42.6 25.6 17.1 −9.3

0.2 1.2 3.9 5.8 7.9 0.3 5.6 6.9

Lonicera japonica

Concentration

Houttuynia cordata

μg/ml

Mean

SD

Mean

SD

10000 3333.3 1111.1 370.4 123.5 41.2 13.7 4.6

88.6 71.8 37.7 18.5 7.5 4.5 0.9 −2.7

4.7 1.9 3.7 8.2 16.3 7.9 1.9 14.8

105.3 107.1 102.6 68.8 26.2 28.2 14.9 8.0

1.6 1.7 2.3 5.0 10.6 24.6 3.4 18.0

Concentration

Glycyrrhiza ularensis

Mentha haplocalyx

μg/ml

Mean

SD

Mean

SD

10000 3333.3 1111.1 370.4 123.5 41.2 13.7 4.6

101.5 97.5 90.5 65.3 35.7 32.3 −2.5 18.7

0.3 3.4 0.3 4.8 6.1 11.6 6.3 23.1

92.0 88.3 60.6 35.3 17.2 15.0 0.7 2.4

1.1 1.8 1.0 2.1 9.2 0.2 1.3 1.3

SD, standard deviation.

Chrysanthemum moriflorium have been thought to be involved in causing an increase in ciclosporin serum levels by grapefruit juice:[14,18–20] hesperetin,[21] kaempferol[22] and quercetin.[21–23] 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 and quercetin,[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 standard, 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 78.4 μm), whereas ursolic acid has no inhibitory effect (IC50 > 500 μm).[26] Glycyrrhiza ularensis/Liquorice The inhibitory effect of Glycyrrhiza 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 1–2% 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 655 μm, 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 μm), liquiritigenin 7,4’-digucoside (IC50 = 17 μm) and 4hydroxyguaiacol apioglucoside (IC50 = 20 μm).[27] Houttuynia cordata/Bishop’s 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, 15–60 g. It has been reported that Houttynia cordata contains quercetin,[30–36] 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 evaluated in our study has also been reported with the use of peppermint oil on CYP3A4.[38] Hesperetin, hesperidin,


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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) Bishop’s weed (Houttuynia cordata). (e) Liquorice (Glycyrrhiza ularensis). (f) Field mint (Mentha haplocalyx).

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Sophie Dufay et al.


oleanolic and ursolic acid were identified in the sample analysed 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 liver microsomes.[26] Naringenin has been identified to be one compound of Mentha haplocalyx species 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 Wacher et 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 330 μl 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 constituents, menthol and menthyl acetate, as reversible, partially mixed CYP3A4 inhibitors in liver microsome test.[42] Maliakal et 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 μg/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 inhibitory 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.

References 1. Kaufman DW et al. Recent patterns of medication use in the ambulatory adult population of the United States

The analysis of plant extracts by HPC-PDA helped demonstrate the presence of chemical compounds (quercetin, hesperidin, hesperetin and oleanolic acid) possibly responsible 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 used plants 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 possible herb–drug interaction when no information was available in the literature. The in-vitro experiments on CYP3A4 described above utilizing aqueous extracts at representative clinical 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 of Taraxacum mongolicum, Chrysanthemum 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 chromatograms to test potency of their inhibitory effect. Doctor should ask for herbal consumption in front of unexplained drug toxicity and put in place clinical surveillance programme especially for patients treated with immunosuppressant. Similar attitude should be adopted with other drugs known to interact with grapefruit juice: antiarrhythmics, calcium channel blockers, statins, cytotoxic agents, carbamazepine or some antipsychotics. Further scientific evaluations of potential herb–drug interaction are needed.

Declarations Funding This study has been funded by the Department of Pharmacology and Pharmacy of the University of Hong Kong.

– the Slone survey. JAMA 2002; 287: 337–344. 2. Duggan J et al. Use of complementary and alternative therapies in HIV-infected patients. AIDS


Patient Care STDS 2001; 15: 159– 167. 3. Engdal S et al. Identification and exploration of herb-drug combinations used by cancer patients. 11


4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

12

Integr Cancer Ther 2009; 8: 29– 36. Molassiotis A et al. Use of complementary and alternative medicine in cancer patients: a European survey. Ann Oncol 2005; 16: 655–663. Bush TM et al. Adverse interactions between herbal and dietary substances and prescription medications: a clinical survey. Altern Ther Health Med 2007; 13: 30–35. Shi S, Klotz U. Drug interactions with herbal medicines. Clin Pharmacokinet 2012; 51: 77–104. Hu Z et al. Herb-drug interactions: a literature review. Drugs 2005; 65: 1239–1282. Izzo AA, Ernst E. Interactions between herbal medicines and prescribed drugs: an updated systematic review. Drugs 2009; 69: 1777–1798. De Lima Toccafondo Vieira M, Huang SM. Botanical-drug interactions: a scientific perspective. Planta Med 2012; 78: 1400–1415. Mukherjee PK et al. Botanicals as medicinal food and their effects on drug metabolizing enzymes. Food Chem Toxicol 2011; 49: 3142–3153. Kwan LP et al. Acute drug toxicity related to drinking herbal tea in a kidney transplant recipient. Ren Fail 2014; 36: 309–312. Nowack R, Nowak B. Herbal teas interfere with cyclosporin levels in renal transplant patients. Nephrol Dial Transplant 2005; 20: 2554–2556. Ohno Y et al. General framework for the prediction of oral drug interactions caused by CYP3A4 induction from in vivo information. Clin Pharmacokinet 2008; 47: 669–680. Seden K et al. Grapefruit-drug interactions. Drugs 2010; 70: 2373–2407. World Health Organization. Medicinal Plants in China: A Selection of 150 Commonly Used Species. Singapore: World Health Organization, Regional Office for the Western Pacific, 1989. Tang W, Eisenbrand G. Handbook of Chinese Medicinal Plants: Chemistry, Pharmacology, Toxicology, Vol. 1. Weinheim, UK: Wiley-VCH, 2011. Hempen C-H, Fischer T. A Materia Medica for Chinese Medicine: Plants,

Sophie Dufay et al.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Minerals, and Animal Products. London: Elsevier Health Sciences, 2009. Lin SP et al. Citrus grandis peel increases the bioavailability of cyclosporine and tacrolimus, two important immunosuppressants, in rats. J Med Food 2011; 14: 1463–1468. Fujita T et al. Comparative evaluation of 12 immature citrus fruit extracts for the inhibition of Cytochrome P450 isoform activities. Biological & Pharmaceutical Bulletin 2008; 31: 925–930. Ho PC et al. Inhibition of human CYP3A4 activity by grapefruit flavonoids, furanocoumarins and related compounds. J Pharm Pharm Sci 2001; 4: 217–227. Wang YJ et al. Studies on chemical constituents in Huangjuhua (flowers of Chrysanthemum morifolium). Zhongguo Zhong Yao Za Zhi 2008; 33: 526–530. Lai JP et al. Identification and characterization of major flavonoids and caffeoylquinic acids in three Compositae plants by LC/DADAPCI/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 848: 215–225. Miyazawa M, Hisama M. Antimutagenic activity of flavonoids from Chrysanthemum morifolium. Biosci Biotechnol Biochem 2003; 67: 2091– 2099. Miniscalco A et al. Inhibition of dihydropyridine metabolism in rat and human liver microsomes by flavonoids found in grapefruit juice. J Pharmacol Exp Ther 1992; 261: 1195–1199. Shi S et al. Identification of antioxidants from Taraxacum mongolicum by high-performance liquid chromatography-diode array detection-radical-scavenging detectionelectrospray ionization mass spectrometry and nuclear magnetic resonance experiments. J Chromatogr A 2008; 31: 1–2. Kim KA et al. Inhibition of Cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes. Life Sci 2004; 74: 2769–2779.

27. Tsukamoto S et al. CYP3A4 inhibitors isolated from licorice. Biol Pharm Bull 2005; 28: 2000–2002. 28. Budzinski JW et al. An in vitro evaluation of human Cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures. Phytomedicine 2000; 7: 273–282. 29. Kondo K et al. Constituent properties of licorices derived from Glycyrrhiza uralensis, G. glabra, or G. inflata identified by genetic information. Biological & pharmaceutical bulletin 2007; 30: 1271–1277. 30. Cho EJ et al. The inhibitory effects of 12 medicinal plants and their component compounds on lipid peroxidation. Am J Chin Med 2003; 31: 907– 917. 31. Chou SC et al. The constituents and their bioactivities of Houttuynia cordata. Chem Pharm Bull 2009; 57: 1227–1230. 32. Meng J et al. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromatography-diode array detectorelectrospray mass spectrometry. Fitoterapia 2009; 80: 468–474. 33. Meng J et al. Study on chemical constituents of flavonoids in fresh herb of Houttuynia cordata. Zhongguo Zhong Yao Za Zhi 2006; 31: 1335–1337. 34. Meng J et al. Establishment of HPLCDAD-MS fingerprint of fresh Houttuynia cordata. Chem Pharm Bull 2005; 53: 1604–1609. 35. Xu X et al. Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection. Talanta 2006; 68: 759–764. 36. Zhang TT et al. [Study on HPLC fingerprint of flavonoids from Houttuynia cordata by comparing with fingerprint reference]. Zhong Yao Cai 2009; 32: 687–690. 37. Dahan A, Altman H. Food-drug interaction: grapefruit juice augments drug bioavailability–mechanism, extent and relevance. Eur J Clin Nutr 2004; 58: 1–9.


Sophie Dufay et al.

38. Unger M, Frank A. Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major Cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction. Rapid Commun Mass Spectrom 2004; 18: 2273–2281. 39. Dorman HJ et al. Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. J Agric Food Chem 2003; 51: 4563–4569.


40. Bailey DG et al. Grapefruit juice-drug interactions. Br J Clin Pharmacol 1998; 46: 101–110. 41. Wacher VJ et al. Peppermint oil enhances cyclosporine oral bioavailability in rats: comparison with D-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS) and ketoconazole. J Pharm Sci 2002; 91: 77–90. 42. Dresser GK et al. Evaluation of peppermint oil and ascorbyl palmitate as inhibitors of Cytochrome P4503A4 activity in vitro and in


vivo. Clin Pharmacol Ther 2002; 72: 247–255. 43. Maliakal PP, Wanwimolruk S. Effect of herbal teas on hepatic drug metabolizing enzymes in rats. J Pharm Pharmacol 2001; 53: 1323–1329. 44. Pao LH et al. Herb-drug interaction of 50 Chinese herbal medicines on CYP3A4 activity in vitro and in vivo. Am J Chin Med 2012; 40: 57–73.

13

Mechanism-based inhibition of Alantolactone on human cytochrome P450 3A4 in vitro and activity of hepatic cytochrome P450 in mice.

Inhibitory effect of salvianolate on human cytochrome P450 3A4 in vitro involving a noncompetitive manner.

Characterization of ritonavir-mediated inactivation of cytochrome P450 3A4.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase-cytochrome P450 3A4 in nanodiscs.

In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4.

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

Selected tea and tea pomace extracts inhibit intestinal α-glucosidase activity in vitro and postprandial hyperglycemia in vivo.

Cytochrome P450 enzyme mediated herbal drug interactions (Part 2).

Cytochrome P450 enzyme mediated herbal drug interactions (Part 1).

Polymorphisms and phenotypic analysis of cytochrome P450 3A4 in the Uygur population in northwest China.

Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

Mechanism-based inactivation of human cytochrome P450 3A4 by two piperazine-containing compounds.

Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions.

Inhibitory mechanisms of celastrol on human liver cytochrome P450 1A2, 2C19, 2D6, 2E1 and 3A4.

Metabolic behavior prediction of pazopanib by cytochrome P450 (CYP) 3A4 by molecular docking.

Effect of cholesterol on the structure of membrane-attached cytochrome P450 3A4.

Nano-sized cytochrome P450 3A4 inhibitors to block hepatic metabolism of docetaxel.

Ritonavir analogues as a probe for deciphering the cytochrome P450 3A4 inhibitory mechanism.

Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4.

Cytochrome P450 3A4*22, PPAR-α, and ARNT polymorphisms and clopidogrel response.

A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine.

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras.