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Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies
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Tao Hu ⇑, Xuelin Zhou, Lin Wang, Penelope M.Y. Or, John H.K. Yeung, Yiu Wa Kwan, Chi Hin Cho
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School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
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a r t i c l e
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Article history: Received 23 November 2014 Received in revised form 20 January 2015 Accepted 6 February 2015 Available online xxxx Keywords: Tanshinones CYP2C19 Omeprazole Human liver microsomes Molecular docking
a b s t r a c t Objectives: This study aimed to investigate the effects of five tanshinones, the lipophilic components from Danshen (Salvia miltiorrhiza), on CYP2C19 activity in pooled human liver microsomes (HLMs). Methods: The effects of tanshinones on CYP2C19 activity were compared by enzyme inhibition study using omeprazole 5-hydroxylation in pooled HLMs. The inhibition constant (Ki) values and inhibition modes of effective tanshinones were evaluated by enzyme kinetic study. Molecular docking analysis was used to simulate the binding conformations of tanshinones to the active cavity of human CYP2C19. Results: Dihydrotanshinone and miltirone showed potent inhibitory effects on CYP2C19 activity in a concentration-dependent manner. Tanshinone I showed weaker inhibitory effect, whereas tanshinone IIA and cryptotanshinone had no inhibitory effect. Further enzyme kinetic study showed that the inhibition by dihydrotanshinone and miltirone was a mixed type. The effects of tanshinones were also confirmed by a molecular docking study. Besides, the ethanol extract of Danshen also showed a mixed type of inhibition, whereas the water extract had no inhibitory effect. Conclusions: The current findings demonstrate the inhibition of CYP2C19 activity by the ethanol extract of Danshen and its components tanshinones, implicating the potential herb–drug interactions between Danshen and therapeutic agents metabolized by CYP2C19 in clinical practice. Ó 2015 Published by Elsevier Ireland Ltd.
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1. Introduction
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Danshen, the dried root of Salvia miltiorrhiza Bunge, is widely used in Asian countries especially in China for the prevention and treatment of cardiovascular, cerebrovascular and hepatic diseases [1]. Danshen is often co-administrated with other drugs in order to improve therapeutic efficacies, thus, precaution must be taken for the possible herb–drug interactions. For instance, combination treatment of Danshen with warfarin, an anticoagulant, can give rise to gross anticoagulation and bleeding complications [2–4]. Tanshinones, the lipophilic components isolated from Danshen, are a series of abietane diterpenes [5]. Numerous studies have demonstrated the potential applications of tanshinones in a broad spectrum of diseases such as atherosclerosis, cardiac arrhythmias, hypertension, obesity, metabolic syndrome and cancer [6]. They are promising drug candidates for these diseases.
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⇑ Corresponding author at: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Fax: +86 852 2603 5139. E-mail address: [email protected] (T. Hu).
Thus, the potential herb–drug interactions between tanshinones with the current therapeutic agents should be evaluated at the early stage of drug development. Herb–drug interactions include pharmacodynamic and pharmacokinetic interactions. Pharmacokinetic interactions can change the absorption, distribution, metabolism and excretion of drugs, thereby affecting drug concentrations, therapeutic effects and toxicities in the body. Most of the pharmacokinetic interactions are mediated by the drug metabolizing enzymes in particular the cytochrome P450 (CYP) as well as membrane transporters [7]. Cytochrome P450 enzymes located in the liver and intestine are responsible for the biotransformation of drugs and other xenobiotics. Modulation of these enzymes can change the metabolism of drugs and their metabolites, leading to drug–drug interactions. Previous studies in our laboratory have demonstrated different inhibitory effects of Danshen extracts and several tanshinones on the activities of CYP1A2, 2C9, 2D6, 2E1 and 3A4, indicating their potential roles in metabolism-based herb–drug interactions [8–10]. However, the effects of these tanshinones on CYP2C19, another important CYP isoform, are still unknown.
http://dx.doi.org/10.1016/j.cbi.2015.02.006 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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Fig. 1. Chemical structures of tanshinones present in this study.
2.2. Preparation of Danshen extracts
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For the preparation of ethanol extract, 200 g Danshen root was minced and boiled in 250 mL 95% ethanol twice under reflex condition. Filtrate was collected and dried using a rotary evaporator with warming no more than 50 °C. The brown residue was redissolved in ethyl acetate and the ethyl acetate layer was collected and dried by a rotary evaporator. The reddish brown crystals obtained finally represented the ethanol fraction in which the percentage yield was about 1%. For water extract, the crude root of Danshen (200 g) was cut into small pieces and boiled in 250 mL distilled water in reflux for 1 h. Subsequently, the filtrate was collected and the residue was mixed with additional 250 mL distilled water and boiled for another one hour. The two filtrates were combined and allowed to cool at room temperature. Water in the filtrate was removed by freeze-drying and about 35 g (17.5% of yield) of the water extract was obtained. The content of individual tanshinones and phenolic acid in ethanol and water extracts was analyzed by HPLC-UV and listed in Table 1 as described in our previous study [12]. Miltirone was not detected in water and ethanol extracts used in this study as compared with other studies, probably due to the different batches of the herb.
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As the third member of human CYP2C subfamily, CYP2C19 is involved in the metabolism of various clinically important therapeutic agents, including proton pump inhibitors (e.g., omeprazole, lanzoprazole, pantoprazole and rabeprazole), tricyclic antidepressants (e.g., amitryptiline, imipramine and lomipramine), antiepileptics (e.g., diazepam, phenytoin, nordazepam and phenobarbital), warfarin, clopidogrel, propranolol and cyclophosphamide. Modulation of CYP2C19 activity can change the pharmacokinetics, safety and efficacy profiles of these drugs. Indeed, it has been reported that the herb–drug interactions between Danshen and warfarin were partly due to the effect of Danshen on the CYP2C19 activity [11]. As the bioactive components of Danshen, it is speculated that tanshinones could contribute to the Danshen-mediated herb–drug interactions. Therefore, in this study we focused on the inhibitory effects of five tanshinones, namely tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone (Fig. 1) on CYP2C19 activity in human liver microsomes [6]. Besides, we also compared the effects of ethanol and water extracts of Danshen, the most commonly used formulations in clinical practice. This study is pivotal to assess the potential herb–drug interactions between Danshen and some commonly used therapeutic agents in humans.
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2. Materials and methods
2.3. Omeprazole hydroxylation assay
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2.1. Materials
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Pooled human liver microsomes (Catalog Number: 452161) were obtained from GenTest Corporation (Woburn, MA, USA) and stored at 80 °C. All unspecified compounds were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA). Tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone were purchased from Chengdu Cogon Bio-tech Co., Ltd. (Sichuan, People’s Republic of China); Miltirone was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, People’s Republic of China). The purities of tanshinones were >98% as determined by HPLC-UV. The dried herb of Danshen (Batch No. 050822; from Sichuan Province, China) was supplied by Winsor Health Products Ltd. (Hong Kong, People’s Republic of China). After extraction, the extracts were validated with the authentic standards from the Hong Kong Jockey Club Institute of Chinese Medicine (Hong Kong, People’s Republic of China).
CYP2C19 activity was assessed by the formation of 5-hydroxyomeprazole from omeprazole, a probe substrate of CYP2C19, in pooled human liver microsomes (HLMs) [13]. Briefly, HLMs (0.8 mg/mL) were incubated with the NADPH-regenerating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, 0.4 U/mL glucose 6phosphate dehydrogenase and 3.3 mM MgCl2) and tanshinones in Tris/KCl buffer (pH 7.4) with a total volume of 250 lL. The incubation mixture was pre-incubated at 900 rpm and 37 °C for 5 min without HLMs, then the incubation was initiated by adding icecold HLMs into the mixture. After incubation for 1 h, the reaction was terminated by adding 250 lL ice-cold acetonitrile (ACN). Subsequently, all the incubation tubes were centrifuged at 10,000 rpm for 10 min to precipitate protein. The supernatant was collected and extracted with 250 lL ethyl acetate at 1400 rpm and 25 °C for 30 min. 10 lL propranolol (50 lg/mL in methanol) was added as the internal standard. Afterwards, the tubes were centrifuged at 10,000 rpm for 5 min and the organic layer was transferred into
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Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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T. Hu et al. / Chemico-Biological Interactions xxx (2015) xxx–xxx Table 1 Major components in water extract and ethanol extract of Danshen, as determined by HPLC-UV. (Data were present as mean ± SD, n = 3) [12]. Compounds
Tanshinone I Tanshinone IIA Cryptotanshinone Dihydrotanshinone Danshensu Salvianolic acid B
Water extract
Ethanol extract
Absolute value (lg/g)
Percentage (%)
10.4 ± 0.40 22.6 ± 0.51 34.6 ± 0.86 12.7 ± 0.46 3.1 ± 0.03 37.3 ± 0.50
(1.04 ± 0.04) 10 (2.26 ± 0.05) 10 (3.46 ± 0.09) 10 (1.27 ± 0.05) 10 0.31 ± 0.003 3.73 ± 0.05
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a glass tube for evaporation under a gentle stream of nitrogen. Eventually, the residue was re-suspended in 60 lL solvent (ACN:H2O = 1:2.4; pH 2.7, adjusted by H3PO4) for high-performance liquid chromatography (HPLC) analysis.
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2.4. HPLC analysis
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HPLC analysis of omeprazole and 5-hydroxyomeprazole was performed as previously described with minor modifications [14]. In brief, the Hewlett–Packard (HP) 1050 series pumping system with a multiple wavelength detector was used for the determination. Omeprazole, 5-hydroxyomeprazole and propranolol were separated using a reversed-phase Alltech Alltima C18 column (250 mm 4.6 mm, 5 lm), equipped with an Eclipse XDB-C18 guard column. The mobile phase consisted of 10 mM ammonium formate (pH 4), methanol, and ACN (60:25:15, v/v/v) at a flow rate of 1 mL/min. The wavelength of UV detection was 302 nm. The detection method was validated over the concentration range of 0.05–1 lg/mL (5-hydroxyomeprazole) and 5–100 lg/mL (omeprazole) with good intra-day and inter-day precision (relative standard deviation 100 lM >100 lM >100 lM 0.6 lM 26.9 lM 30.9 lg/mL >1000 lg/mL 0.2 lM
NDa NDa NDa 3.4 lM 7.0 lM 8.7 lg/mL NDa 0.9 lM
NDa NDa NDa Mixed type Mixed type Mixed type NDa Competitive
Not determined. Positive control.
Fig. 2. Inhibition of CYP2C19 activity in pooled human live microsomes by tanshinone I (A), tanshinone IIA (B), cryptotanshinone (C), dihydrotanshinone (D), miltirone (E), nootkatone (F), ethanol extract (G) and water extract (H) of Danshen. Data are expressed as mean ± SEM. (n = 3) ⁄p < 0.05 and ⁄⁄⁄p < 0.001 indicate significant difference as compared with the control group.
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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none, miltirone and ethanol extract (Fig. 3A–C), the plotted lines intersected at the point in the second quadrant, indicating that the inhibition was a mixed type. However, in the primary Lineweaver–Burk plot of nootkatone (Fig. 3D), the lines intersected on the y-axis, suggesting that the inhibition was competitive [19]. The inhibition by dihydrotanshinone and miltirone was a mixed type, in which the inhibitor binds to the enzyme regardless of whether the enzyme has already bound to the substrate. Whereas the inhibition of nootkatone was competitive type, in which both the inhibitor and the substrate compete for the same active site in the enzyme [20]. Thus, even though the dosage of the drug metabolized by CYP2C19 was increased, co-administrated tanshinones can still inhibit the enzyme activity due to their mixed inhibition types. This type of herb–drug interactions could be more clinically significant as one could not compensate the inhibitory action of the herbal medicine on the metabolic enzyme merely by increasing the dosages of therapeutic drugs in any human settings. Since the IC50 value depends on the experimental conditions, the value of a compound may vary between different reports [21]. In order to compare the potencies of tanshinones with other CYP2C19 inhibitors and predict the possible in vivo interactions, the inhibition constant (Ki) values of dihydrotanshinone and miltirone were determined by their secondary Lineweaver–Burk plots (Fig. 4), in which the x-intercept gave an estimate of the negative of Ki (–Ki) [22]. According to the results, the Ki value of nootkatone, a known CYP2C19 inhibitor, was 0.9 lM. This was consistent with the value (0.5 lM) indicated in the U.S. FDA guidance for drug interaction studies. The Ki values of dihydrotanshinone and miltirone were 3.4 lM and 7.0 lM, respectively.
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3.3. Molecular docking study
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The logarithm of binding free energies of all the test compounds was much lower than -7 (the threshold of positive binding), showing that they all can bind to the catalytic site of CYP2C19 (Table 3) [16]. The inhibition of tanshinones on the metabolism of omeprazole depended on whether their binding free energies were much lower than that of omeprazole or not. The structure of 0XV, an inhibitor existing in the crystal structure of CYP2C19, was
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re-generated by CORINA and re-docked into the crystal structure. As shown in Fig. 5A, the re-docked position of 0XV with binding free energy of 9.7 kcal/mol was comparable to the original one from the X-ray experiment, the related RMSD (root-mean-square deviation) value between docked pose and actual pose was 0.8 Å. It was also observed that the amino acid residues including Phe476, Thr-301, Ile-205, Ile-362, Leu-366, Ile-362 and Val-208 played key roles in this interaction as reported [23]. These results indicated that the simulation in this study was successful. Besides, the binding free energy ( 8.7 kcal/mol) of omeprazole also suggested its positive binding to the active cavity of CYP2C19 and the distances (>1.5 Å) of omeprazole and 0XV were not very close to Water601. The binding conformations of tanshinones were further analyzed. As shown in Fig. 5B–F, tanshinone I mainly interacted with Phe114, Val113, Ile205, Leu237, Gly296, Thr301, Ile362, Leu366 and Phe476 via van der Waals force, Asp293 and Ala297 via polarity, and Water601 via hydrogen interaction within a distance of 3 Å. Tanshinone IIA mainly interacted with Phe100, Leu102, Phe114, Ile205, Val208, Leu233, Ala297, Tr301 and Leu366 via van der Waals force, Gly296 via polarity, and Val208 via sigma-pi interaction within a distance of 3.8 Å, and Water601 via hydrogen interaction within a distance of 3.1 Å. Cryptotanshinone interacted withVal113, Phe114, Leu201, Asn204, Ile205, Leu237, Met240, Ala297, Leu366 and Phe476 via van der Waals force, and Asp293, Ala292, Gly296 via polarity. Dihydrotanshinone interacted with the amino acid residues including Val113, Ile205, Gly296, Leu366 and Phe476 via van der Waals force, as well as Ala292, Asp293 and Ala297 via polarity. Besides interaction with Asn107, Val208, Val113, Leu237, Ala297, Asp293, Thr301, Leu366 and Phe476 via van der Waals force, miltirone showed pi-pi interactions via Phe114 with a distance of 4 Å in the active cavity of CYP2C19. These van der Waals force and polarity mainly supported the interactions of tanshinones in the active cavity of CYP2C19.
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4. Discussion and conclusion
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CYP2C19 is responsible for the metabolism of various clinically important therapeutic agents, the modulation of CYP2C19 activity can change the pharmacokinetic properties of these drugs in clinic.
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Fig. 3. Primary Lineweaver–Burk plots for inhibitory effects of dihydrotanshinone (A), miltirone (B), ethanol extract of Danshen (C) and nootkatone (D) on omeprazole 5dihydroxylation mediated by CYP2C19 in pooled human liver microsomes. Data are expressed as mean (n = 3).
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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Fig. 4. Secondary Lineweaver–Burk plots for Ki values of dihydrotanshinone (A), miltirone (B), ethanol extract of Danshen (C) and nootkatone (D) on omeprazole 5-dihydroxylation mediated by CYP2C19 in pooled human liver microsomes. Data are expressed as mean (n = 3).
Table 3 Logarithm of binding free energies (kcal/mol) of tanshinones to the active cavity of human CYP2C19 (PDB code 4GQS) with or without considering the existence of conserved water molecule Water601. Compounds
Binding free energies (kcal/mol) Without Water601
Tanshinone I Tanshinone IIA Cryptotanshinone Dihydrotanshinone Miltirone 0XVa Omeprazole
9.4 9.2 9.1 9.6 9.3 9.7 8.7
With Water601 9.4 8.7 8.8 9.6 9.3 9.7 8.7
a
0XV: (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)-methanone, the positive control.
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For instance, it has been reported that CYP2C19 seems to play the most important role in the in vivo metabolic activation of clopidogrel, an antiplatelet agent [24]. A recent research work also demonstrated that grapefruit juice consumption could inhibit the metabolic activation of clopidogrel in healthy volunteers, mainly by inhibiting CYP2C19 in the liver and small intestine [25]. These results indicated the possible in vivo herb–drug interactions via inhibition of CYP2C19 in humans. The likelihood of an in vivo interactions is projected based on the [I]/Ki ratio where [I] represents the mean steady-state Cmax value for total drug following administration of the highest proposed clinical dose [26]. As reported, after a single-dose administration (1 g) of the Danshen ethanol extract in healthy volunteers, the Cmax values of dihydrotanshinone was 0.85 ng/mL [27]. The [I]/Ki value of dihydrotanshinone (Ki = 3.4 lM) was less than 0.1, thus, the in vivo inhibition on CYP2C19 by dihydrotanshinone alone seems to be remote. However, the same clinical study also revealed that treatment of the Danshen ethanol extract could
change the pharmacokinetic profiles of midazolam, a probe substrate of CYP3A4, indicating that the in vivo interactions between ethanol extract and CYP3A4 substrate drug did occur in humans [27]. As reported, tanshinone I, tanshinone IIA and cryptotanshinone also showed weak inhibitory effect on CYP3A4, dihydrotanshinone showed the most potent inhibitory effect with the Ki value of 2.1 lM in the in vitro incubation with human liver microsomes [18]. In this case, the [I]/Ki value of dihydrotanshinone was also less than 0.1. The discrepancy between this in vitro evaluation and the exact in vivo interactions presumably caused by the extensive tissue distribution of the tanshinones. Indeed, several in vivo studies in rats showed that after oral administration the concentrations of tanshinone IIA in the small intestine and liver were about 500 folds and 70 folds higher than its plasma concentration, respectively [28]. As the chemical structures of the tanshinones present in this study are very similar, they possibly show the similar tissue distribution in vivo. If this is the case, the concentration of dihydrotanshinone in the liver and intestine should be high enough to inhibit CYP2C19, leading to possible in vivo herb–drug interactions in humans. This is the first report on simulating drug binding in the catalytic site of CYP2C19 for CYP2C19-mediated drug–drug interactions by using a recently developed crystal structure of human CYP2C19. According to the molecular docking analysis, dihydrotanshinone was the strongest inhibitor with the lowest binding free energy ( 9.6 kcal/mol) among these tanshinones, its binding free energy was much lower than that of omeprazole ( 8.7 kcal/ mol). As compared with the in vitro CYP2C19 inhibition assay, dihydrotanshinone and miltirone showed the similar tendency in molecular docking study. However, when the existence of conserved water molecules in the active cavity of CYP2C19 was neglected, the binding free energies of tanshinone IIA and cryptotanshinone were not consistent with the in vitro inhibition assay. It was speculated that ligands should not be within the distance
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Fig. 5. Molecular docking analysis illustrates the favorable binding positions of all tanshinones with lowest binding free energy in the active cavity of human CYP2C19 (PDB code 4GQS) when considering the existence of Water601. The three-dimensional diagram (A) shows that re-docked position of 0XV with labeled amino residues is comparable to the experimental one in the crystal structure, indicating the high accuracy of this simulation with low RMSD. The two-dimensional diagrams demonstrate the interactions of tanshinone I (B), tanshinone IIA (C), cryptotanshinone (D), dihydrotanshinone (E) and miltirone (F) with the amino acid residues in the active cavity of CYP2C19. The numerals labeled above the lines indicate the distances between major functional residues and chemical groups.
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of 1.5 Å to the conserved water molecule [29]. Thus, binding conformations of each ligand within the distance of 1.5 Å to the conserved water molecule were further dismissed. During this procedure, Water601 was found to be one of the water molecules close to most of the conformations of tanshinone IIA and cryptotanshinone within the distance of 1.5 Å. After considering the existence of Water601, the lowest binding free energies of tanshinone IIA and cryptotanshinone were 8.7 kcal/mol and 8.8 kcal/mol, respectively. This meant that tanshinone IIA and cryptotanshinone should not be the inhibitor of CYP2C19, consistent with the results in the in vitro assay. Besides, water molecules within the binding site typically act by forming a hydrogen-bonded network, mediating the interactions between the ligand and the protein. Hence the
network may stabilize the complex formed with one ligand but not another (as seen in different tanshinones in this case), thereby contributing to differential binding as observed [30]. Although the binding free energy of tanshinone I was much lower than that of omeprazole, its in vitro inhibition activity was not so strong (IC20 = 4.0 lM). More parameters should be considered in the future docking study with tanshinone I. However, it is difficult to simulate the non-competitive binding of tanshinones in the active cavity of human CYP2C19 because only one active site of CYP2C19 was currently found, and allosteric site in the crystal structure of CYP2C19 was still not investigated. Furthermore, we also compared the inhibition of CYP2C19 activity by ethanol extract and water extract of Danshen. The
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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ethanol extract contains much more lipophilic tanshinones than those in the water extract in which the major components are hydrophilic danshensu and salvianolic acid B [12]. IC50 values of dihydrotanshinone and ethanol extract were 0.6 lM (0.17 lg/ mL) and 30.9 lg/mL, respectively. This indicates that dihydrotanshinone mostly contributes the inhibitory effect of ethanol extract, although its content is very low (0.93%) in the ethanol extract. Other miltirone-like compounds in the ethanol extract should also be considered for their potent activities. However, the water extract, danshensu and salvianolic acid B showed no inhibitory effect. Previous studies also demonstrated that the water extract had no inhibitory effect on the activities of CYP1A2, 2C9, 2D6, 2E1 and 3A4 [8]. Therefore, these findings suggest that the metabolism-based herb–drug interactions mediated by Danshen were most likely caused by the lipophilic tanshinones, rather than hydrophilic components. In summary, this study investigated the in vitro inhibition of CYP2C19 activity by five tanshinones isolated from Danshen and compared the inhibitory effects of both ethanol and water extracts of the herb on this metabolic enzyme. In order to confirm these herb–drug interactions and fully understand the different interaction types, further studies should include in vivo studies on CYP enzyme activity as well as the actions of tanshinones on drug transporters. Nevertheless, the current findings demonstrate the potential herb–drug interactions between tanshinones and drugs metabolized by CYP2C19. The interactions should be considered more carefully for patients using Danshen, especially the formulations containing more lipophilic tanshinones as an alternative medicine for different diseases.
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Conflict of Interest
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The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Acknowledgements
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Tao Hu is a recipient of postgraduate student scholarship from The Chinese University of Hong Kong. The paper is also dedicated to Prof. John H.K. Yeung for his life-long contributions in drug metabolism in particular the research on CYP enzymes.
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Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies
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Tao Hu ⇑, Xuelin Zhou, Lin Wang, Penelope M.Y. Or, John H.K. Yeung, Yiu Wa Kwan, Chi Hin Cho
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School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
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a r t i c l e
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Article history: Received 23 November 2014 Received in revised form 20 January 2015 Accepted 6 February 2015 Available online xxxx Keywords: Tanshinones CYP2C19 Omeprazole Human liver microsomes Molecular docking
a b s t r a c t Objectives: This study aimed to investigate the effects of five tanshinones, the lipophilic components from Danshen (Salvia miltiorrhiza), on CYP2C19 activity in pooled human liver microsomes (HLMs). Methods: The effects of tanshinones on CYP2C19 activity were compared by enzyme inhibition study using omeprazole 5-hydroxylation in pooled HLMs. The inhibition constant (Ki) values and inhibition modes of effective tanshinones were evaluated by enzyme kinetic study. Molecular docking analysis was used to simulate the binding conformations of tanshinones to the active cavity of human CYP2C19. Results: Dihydrotanshinone and miltirone showed potent inhibitory effects on CYP2C19 activity in a concentration-dependent manner. Tanshinone I showed weaker inhibitory effect, whereas tanshinone IIA and cryptotanshinone had no inhibitory effect. Further enzyme kinetic study showed that the inhibition by dihydrotanshinone and miltirone was a mixed type. The effects of tanshinones were also confirmed by a molecular docking study. Besides, the ethanol extract of Danshen also showed a mixed type of inhibition, whereas the water extract had no inhibitory effect. Conclusions: The current findings demonstrate the inhibition of CYP2C19 activity by the ethanol extract of Danshen and its components tanshinones, implicating the potential herb–drug interactions between Danshen and therapeutic agents metabolized by CYP2C19 in clinical practice. Ó 2015 Published by Elsevier Ireland Ltd.
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1. Introduction
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Danshen, the dried root of Salvia miltiorrhiza Bunge, is widely used in Asian countries especially in China for the prevention and treatment of cardiovascular, cerebrovascular and hepatic diseases [1]. Danshen is often co-administrated with other drugs in order to improve therapeutic efficacies, thus, precaution must be taken for the possible herb–drug interactions. For instance, combination treatment of Danshen with warfarin, an anticoagulant, can give rise to gross anticoagulation and bleeding complications [2–4]. Tanshinones, the lipophilic components isolated from Danshen, are a series of abietane diterpenes [5]. Numerous studies have demonstrated the potential applications of tanshinones in a broad spectrum of diseases such as atherosclerosis, cardiac arrhythmias, hypertension, obesity, metabolic syndrome and cancer [6]. They are promising drug candidates for these diseases.
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⇑ Corresponding author at: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Fax: +86 852 2603 5139. E-mail address: [email protected] (T. Hu).
Thus, the potential herb–drug interactions between tanshinones with the current therapeutic agents should be evaluated at the early stage of drug development. Herb–drug interactions include pharmacodynamic and pharmacokinetic interactions. Pharmacokinetic interactions can change the absorption, distribution, metabolism and excretion of drugs, thereby affecting drug concentrations, therapeutic effects and toxicities in the body. Most of the pharmacokinetic interactions are mediated by the drug metabolizing enzymes in particular the cytochrome P450 (CYP) as well as membrane transporters [7]. Cytochrome P450 enzymes located in the liver and intestine are responsible for the biotransformation of drugs and other xenobiotics. Modulation of these enzymes can change the metabolism of drugs and their metabolites, leading to drug–drug interactions. Previous studies in our laboratory have demonstrated different inhibitory effects of Danshen extracts and several tanshinones on the activities of CYP1A2, 2C9, 2D6, 2E1 and 3A4, indicating their potential roles in metabolism-based herb–drug interactions [8–10]. However, the effects of these tanshinones on CYP2C19, another important CYP isoform, are still unknown.
http://dx.doi.org/10.1016/j.cbi.2015.02.006 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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Fig. 1. Chemical structures of tanshinones present in this study.
2.2. Preparation of Danshen extracts
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For the preparation of ethanol extract, 200 g Danshen root was minced and boiled in 250 mL 95% ethanol twice under reflex condition. Filtrate was collected and dried using a rotary evaporator with warming no more than 50 °C. The brown residue was redissolved in ethyl acetate and the ethyl acetate layer was collected and dried by a rotary evaporator. The reddish brown crystals obtained finally represented the ethanol fraction in which the percentage yield was about 1%. For water extract, the crude root of Danshen (200 g) was cut into small pieces and boiled in 250 mL distilled water in reflux for 1 h. Subsequently, the filtrate was collected and the residue was mixed with additional 250 mL distilled water and boiled for another one hour. The two filtrates were combined and allowed to cool at room temperature. Water in the filtrate was removed by freeze-drying and about 35 g (17.5% of yield) of the water extract was obtained. The content of individual tanshinones and phenolic acid in ethanol and water extracts was analyzed by HPLC-UV and listed in Table 1 as described in our previous study [12]. Miltirone was not detected in water and ethanol extracts used in this study as compared with other studies, probably due to the different batches of the herb.
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As the third member of human CYP2C subfamily, CYP2C19 is involved in the metabolism of various clinically important therapeutic agents, including proton pump inhibitors (e.g., omeprazole, lanzoprazole, pantoprazole and rabeprazole), tricyclic antidepressants (e.g., amitryptiline, imipramine and lomipramine), antiepileptics (e.g., diazepam, phenytoin, nordazepam and phenobarbital), warfarin, clopidogrel, propranolol and cyclophosphamide. Modulation of CYP2C19 activity can change the pharmacokinetics, safety and efficacy profiles of these drugs. Indeed, it has been reported that the herb–drug interactions between Danshen and warfarin were partly due to the effect of Danshen on the CYP2C19 activity [11]. As the bioactive components of Danshen, it is speculated that tanshinones could contribute to the Danshen-mediated herb–drug interactions. Therefore, in this study we focused on the inhibitory effects of five tanshinones, namely tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone (Fig. 1) on CYP2C19 activity in human liver microsomes [6]. Besides, we also compared the effects of ethanol and water extracts of Danshen, the most commonly used formulations in clinical practice. This study is pivotal to assess the potential herb–drug interactions between Danshen and some commonly used therapeutic agents in humans.
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2. Materials and methods
2.3. Omeprazole hydroxylation assay
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2.1. Materials
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Pooled human liver microsomes (Catalog Number: 452161) were obtained from GenTest Corporation (Woburn, MA, USA) and stored at 80 °C. All unspecified compounds were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA). Tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone were purchased from Chengdu Cogon Bio-tech Co., Ltd. (Sichuan, People’s Republic of China); Miltirone was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, People’s Republic of China). The purities of tanshinones were >98% as determined by HPLC-UV. The dried herb of Danshen (Batch No. 050822; from Sichuan Province, China) was supplied by Winsor Health Products Ltd. (Hong Kong, People’s Republic of China). After extraction, the extracts were validated with the authentic standards from the Hong Kong Jockey Club Institute of Chinese Medicine (Hong Kong, People’s Republic of China).
CYP2C19 activity was assessed by the formation of 5-hydroxyomeprazole from omeprazole, a probe substrate of CYP2C19, in pooled human liver microsomes (HLMs) [13]. Briefly, HLMs (0.8 mg/mL) were incubated with the NADPH-regenerating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, 0.4 U/mL glucose 6phosphate dehydrogenase and 3.3 mM MgCl2) and tanshinones in Tris/KCl buffer (pH 7.4) with a total volume of 250 lL. The incubation mixture was pre-incubated at 900 rpm and 37 °C for 5 min without HLMs, then the incubation was initiated by adding icecold HLMs into the mixture. After incubation for 1 h, the reaction was terminated by adding 250 lL ice-cold acetonitrile (ACN). Subsequently, all the incubation tubes were centrifuged at 10,000 rpm for 10 min to precipitate protein. The supernatant was collected and extracted with 250 lL ethyl acetate at 1400 rpm and 25 °C for 30 min. 10 lL propranolol (50 lg/mL in methanol) was added as the internal standard. Afterwards, the tubes were centrifuged at 10,000 rpm for 5 min and the organic layer was transferred into
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Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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T. Hu et al. / Chemico-Biological Interactions xxx (2015) xxx–xxx Table 1 Major components in water extract and ethanol extract of Danshen, as determined by HPLC-UV. (Data were present as mean ± SD, n = 3) [12]. Compounds
Tanshinone I Tanshinone IIA Cryptotanshinone Dihydrotanshinone Danshensu Salvianolic acid B
Water extract
Ethanol extract
Absolute value (lg/g)
Percentage (%)
10.4 ± 0.40 22.6 ± 0.51 34.6 ± 0.86 12.7 ± 0.46 3.1 ± 0.03 37.3 ± 0.50
(1.04 ± 0.04) 10 (2.26 ± 0.05) 10 (3.46 ± 0.09) 10 (1.27 ± 0.05) 10 0.31 ± 0.003 3.73 ± 0.05
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a glass tube for evaporation under a gentle stream of nitrogen. Eventually, the residue was re-suspended in 60 lL solvent (ACN:H2O = 1:2.4; pH 2.7, adjusted by H3PO4) for high-performance liquid chromatography (HPLC) analysis.
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2.4. HPLC analysis
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HPLC analysis of omeprazole and 5-hydroxyomeprazole was performed as previously described with minor modifications [14]. In brief, the Hewlett–Packard (HP) 1050 series pumping system with a multiple wavelength detector was used for the determination. Omeprazole, 5-hydroxyomeprazole and propranolol were separated using a reversed-phase Alltech Alltima C18 column (250 mm 4.6 mm, 5 lm), equipped with an Eclipse XDB-C18 guard column. The mobile phase consisted of 10 mM ammonium formate (pH 4), methanol, and ACN (60:25:15, v/v/v) at a flow rate of 1 mL/min. The wavelength of UV detection was 302 nm. The detection method was validated over the concentration range of 0.05–1 lg/mL (5-hydroxyomeprazole) and 5–100 lg/mL (omeprazole) with good intra-day and inter-day precision (relative standard deviation 100 lM >100 lM >100 lM 0.6 lM 26.9 lM 30.9 lg/mL >1000 lg/mL 0.2 lM
NDa NDa NDa 3.4 lM 7.0 lM 8.7 lg/mL NDa 0.9 lM
NDa NDa NDa Mixed type Mixed type Mixed type NDa Competitive
Not determined. Positive control.
Fig. 2. Inhibition of CYP2C19 activity in pooled human live microsomes by tanshinone I (A), tanshinone IIA (B), cryptotanshinone (C), dihydrotanshinone (D), miltirone (E), nootkatone (F), ethanol extract (G) and water extract (H) of Danshen. Data are expressed as mean ± SEM. (n = 3) ⁄p < 0.05 and ⁄⁄⁄p < 0.001 indicate significant difference as compared with the control group.
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none, miltirone and ethanol extract (Fig. 3A–C), the plotted lines intersected at the point in the second quadrant, indicating that the inhibition was a mixed type. However, in the primary Lineweaver–Burk plot of nootkatone (Fig. 3D), the lines intersected on the y-axis, suggesting that the inhibition was competitive [19]. The inhibition by dihydrotanshinone and miltirone was a mixed type, in which the inhibitor binds to the enzyme regardless of whether the enzyme has already bound to the substrate. Whereas the inhibition of nootkatone was competitive type, in which both the inhibitor and the substrate compete for the same active site in the enzyme [20]. Thus, even though the dosage of the drug metabolized by CYP2C19 was increased, co-administrated tanshinones can still inhibit the enzyme activity due to their mixed inhibition types. This type of herb–drug interactions could be more clinically significant as one could not compensate the inhibitory action of the herbal medicine on the metabolic enzyme merely by increasing the dosages of therapeutic drugs in any human settings. Since the IC50 value depends on the experimental conditions, the value of a compound may vary between different reports [21]. In order to compare the potencies of tanshinones with other CYP2C19 inhibitors and predict the possible in vivo interactions, the inhibition constant (Ki) values of dihydrotanshinone and miltirone were determined by their secondary Lineweaver–Burk plots (Fig. 4), in which the x-intercept gave an estimate of the negative of Ki (–Ki) [22]. According to the results, the Ki value of nootkatone, a known CYP2C19 inhibitor, was 0.9 lM. This was consistent with the value (0.5 lM) indicated in the U.S. FDA guidance for drug interaction studies. The Ki values of dihydrotanshinone and miltirone were 3.4 lM and 7.0 lM, respectively.
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3.3. Molecular docking study
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The logarithm of binding free energies of all the test compounds was much lower than -7 (the threshold of positive binding), showing that they all can bind to the catalytic site of CYP2C19 (Table 3) [16]. The inhibition of tanshinones on the metabolism of omeprazole depended on whether their binding free energies were much lower than that of omeprazole or not. The structure of 0XV, an inhibitor existing in the crystal structure of CYP2C19, was
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re-generated by CORINA and re-docked into the crystal structure. As shown in Fig. 5A, the re-docked position of 0XV with binding free energy of 9.7 kcal/mol was comparable to the original one from the X-ray experiment, the related RMSD (root-mean-square deviation) value between docked pose and actual pose was 0.8 Å. It was also observed that the amino acid residues including Phe476, Thr-301, Ile-205, Ile-362, Leu-366, Ile-362 and Val-208 played key roles in this interaction as reported [23]. These results indicated that the simulation in this study was successful. Besides, the binding free energy ( 8.7 kcal/mol) of omeprazole also suggested its positive binding to the active cavity of CYP2C19 and the distances (>1.5 Å) of omeprazole and 0XV were not very close to Water601. The binding conformations of tanshinones were further analyzed. As shown in Fig. 5B–F, tanshinone I mainly interacted with Phe114, Val113, Ile205, Leu237, Gly296, Thr301, Ile362, Leu366 and Phe476 via van der Waals force, Asp293 and Ala297 via polarity, and Water601 via hydrogen interaction within a distance of 3 Å. Tanshinone IIA mainly interacted with Phe100, Leu102, Phe114, Ile205, Val208, Leu233, Ala297, Tr301 and Leu366 via van der Waals force, Gly296 via polarity, and Val208 via sigma-pi interaction within a distance of 3.8 Å, and Water601 via hydrogen interaction within a distance of 3.1 Å. Cryptotanshinone interacted withVal113, Phe114, Leu201, Asn204, Ile205, Leu237, Met240, Ala297, Leu366 and Phe476 via van der Waals force, and Asp293, Ala292, Gly296 via polarity. Dihydrotanshinone interacted with the amino acid residues including Val113, Ile205, Gly296, Leu366 and Phe476 via van der Waals force, as well as Ala292, Asp293 and Ala297 via polarity. Besides interaction with Asn107, Val208, Val113, Leu237, Ala297, Asp293, Thr301, Leu366 and Phe476 via van der Waals force, miltirone showed pi-pi interactions via Phe114 with a distance of 4 Å in the active cavity of CYP2C19. These van der Waals force and polarity mainly supported the interactions of tanshinones in the active cavity of CYP2C19.
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4. Discussion and conclusion
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CYP2C19 is responsible for the metabolism of various clinically important therapeutic agents, the modulation of CYP2C19 activity can change the pharmacokinetic properties of these drugs in clinic.
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Fig. 3. Primary Lineweaver–Burk plots for inhibitory effects of dihydrotanshinone (A), miltirone (B), ethanol extract of Danshen (C) and nootkatone (D) on omeprazole 5dihydroxylation mediated by CYP2C19 in pooled human liver microsomes. Data are expressed as mean (n = 3).
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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Fig. 4. Secondary Lineweaver–Burk plots for Ki values of dihydrotanshinone (A), miltirone (B), ethanol extract of Danshen (C) and nootkatone (D) on omeprazole 5-dihydroxylation mediated by CYP2C19 in pooled human liver microsomes. Data are expressed as mean (n = 3).
Table 3 Logarithm of binding free energies (kcal/mol) of tanshinones to the active cavity of human CYP2C19 (PDB code 4GQS) with or without considering the existence of conserved water molecule Water601. Compounds
Binding free energies (kcal/mol) Without Water601
Tanshinone I Tanshinone IIA Cryptotanshinone Dihydrotanshinone Miltirone 0XVa Omeprazole
9.4 9.2 9.1 9.6 9.3 9.7 8.7
With Water601 9.4 8.7 8.8 9.6 9.3 9.7 8.7
a
0XV: (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)-methanone, the positive control.
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For instance, it has been reported that CYP2C19 seems to play the most important role in the in vivo metabolic activation of clopidogrel, an antiplatelet agent [24]. A recent research work also demonstrated that grapefruit juice consumption could inhibit the metabolic activation of clopidogrel in healthy volunteers, mainly by inhibiting CYP2C19 in the liver and small intestine [25]. These results indicated the possible in vivo herb–drug interactions via inhibition of CYP2C19 in humans. The likelihood of an in vivo interactions is projected based on the [I]/Ki ratio where [I] represents the mean steady-state Cmax value for total drug following administration of the highest proposed clinical dose [26]. As reported, after a single-dose administration (1 g) of the Danshen ethanol extract in healthy volunteers, the Cmax values of dihydrotanshinone was 0.85 ng/mL [27]. The [I]/Ki value of dihydrotanshinone (Ki = 3.4 lM) was less than 0.1, thus, the in vivo inhibition on CYP2C19 by dihydrotanshinone alone seems to be remote. However, the same clinical study also revealed that treatment of the Danshen ethanol extract could
change the pharmacokinetic profiles of midazolam, a probe substrate of CYP3A4, indicating that the in vivo interactions between ethanol extract and CYP3A4 substrate drug did occur in humans [27]. As reported, tanshinone I, tanshinone IIA and cryptotanshinone also showed weak inhibitory effect on CYP3A4, dihydrotanshinone showed the most potent inhibitory effect with the Ki value of 2.1 lM in the in vitro incubation with human liver microsomes [18]. In this case, the [I]/Ki value of dihydrotanshinone was also less than 0.1. The discrepancy between this in vitro evaluation and the exact in vivo interactions presumably caused by the extensive tissue distribution of the tanshinones. Indeed, several in vivo studies in rats showed that after oral administration the concentrations of tanshinone IIA in the small intestine and liver were about 500 folds and 70 folds higher than its plasma concentration, respectively [28]. As the chemical structures of the tanshinones present in this study are very similar, they possibly show the similar tissue distribution in vivo. If this is the case, the concentration of dihydrotanshinone in the liver and intestine should be high enough to inhibit CYP2C19, leading to possible in vivo herb–drug interactions in humans. This is the first report on simulating drug binding in the catalytic site of CYP2C19 for CYP2C19-mediated drug–drug interactions by using a recently developed crystal structure of human CYP2C19. According to the molecular docking analysis, dihydrotanshinone was the strongest inhibitor with the lowest binding free energy ( 9.6 kcal/mol) among these tanshinones, its binding free energy was much lower than that of omeprazole ( 8.7 kcal/ mol). As compared with the in vitro CYP2C19 inhibition assay, dihydrotanshinone and miltirone showed the similar tendency in molecular docking study. However, when the existence of conserved water molecules in the active cavity of CYP2C19 was neglected, the binding free energies of tanshinone IIA and cryptotanshinone were not consistent with the in vitro inhibition assay. It was speculated that ligands should not be within the distance
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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Fig. 5. Molecular docking analysis illustrates the favorable binding positions of all tanshinones with lowest binding free energy in the active cavity of human CYP2C19 (PDB code 4GQS) when considering the existence of Water601. The three-dimensional diagram (A) shows that re-docked position of 0XV with labeled amino residues is comparable to the experimental one in the crystal structure, indicating the high accuracy of this simulation with low RMSD. The two-dimensional diagrams demonstrate the interactions of tanshinone I (B), tanshinone IIA (C), cryptotanshinone (D), dihydrotanshinone (E) and miltirone (F) with the amino acid residues in the active cavity of CYP2C19. The numerals labeled above the lines indicate the distances between major functional residues and chemical groups.
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of 1.5 Å to the conserved water molecule [29]. Thus, binding conformations of each ligand within the distance of 1.5 Å to the conserved water molecule were further dismissed. During this procedure, Water601 was found to be one of the water molecules close to most of the conformations of tanshinone IIA and cryptotanshinone within the distance of 1.5 Å. After considering the existence of Water601, the lowest binding free energies of tanshinone IIA and cryptotanshinone were 8.7 kcal/mol and 8.8 kcal/mol, respectively. This meant that tanshinone IIA and cryptotanshinone should not be the inhibitor of CYP2C19, consistent with the results in the in vitro assay. Besides, water molecules within the binding site typically act by forming a hydrogen-bonded network, mediating the interactions between the ligand and the protein. Hence the
network may stabilize the complex formed with one ligand but not another (as seen in different tanshinones in this case), thereby contributing to differential binding as observed [30]. Although the binding free energy of tanshinone I was much lower than that of omeprazole, its in vitro inhibition activity was not so strong (IC20 = 4.0 lM). More parameters should be considered in the future docking study with tanshinone I. However, it is difficult to simulate the non-competitive binding of tanshinones in the active cavity of human CYP2C19 because only one active site of CYP2C19 was currently found, and allosteric site in the crystal structure of CYP2C19 was still not investigated. Furthermore, we also compared the inhibition of CYP2C19 activity by ethanol extract and water extract of Danshen. The
Please cite this article in press as: T. Hu et al., Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: Enzyme kinetic and molecular docking studies, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.02.006
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ethanol extract contains much more lipophilic tanshinones than those in the water extract in which the major components are hydrophilic danshensu and salvianolic acid B [12]. IC50 values of dihydrotanshinone and ethanol extract were 0.6 lM (0.17 lg/ mL) and 30.9 lg/mL, respectively. This indicates that dihydrotanshinone mostly contributes the inhibitory effect of ethanol extract, although its content is very low (0.93%) in the ethanol extract. Other miltirone-like compounds in the ethanol extract should also be considered for their potent activities. However, the water extract, danshensu and salvianolic acid B showed no inhibitory effect. Previous studies also demonstrated that the water extract had no inhibitory effect on the activities of CYP1A2, 2C9, 2D6, 2E1 and 3A4 [8]. Therefore, these findings suggest that the metabolism-based herb–drug interactions mediated by Danshen were most likely caused by the lipophilic tanshinones, rather than hydrophilic components. In summary, this study investigated the in vitro inhibition of CYP2C19 activity by five tanshinones isolated from Danshen and compared the inhibitory effects of both ethanol and water extracts of the herb on this metabolic enzyme. In order to confirm these herb–drug interactions and fully understand the different interaction types, further studies should include in vivo studies on CYP enzyme activity as well as the actions of tanshinones on drug transporters. Nevertheless, the current findings demonstrate the potential herb–drug interactions between tanshinones and drugs metabolized by CYP2C19. The interactions should be considered more carefully for patients using Danshen, especially the formulations containing more lipophilic tanshinones as an alternative medicine for different diseases.
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The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Tao Hu is a recipient of postgraduate student scholarship from The Chinese University of Hong Kong. The paper is also dedicated to Prof. John H.K. Yeung for his life-long contributions in drug metabolism in particular the research on CYP enzymes.
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