Evaluation of Herb-Drug Interactions of Hovenia dulcis Fruit Extracts.

BACKGROUND: Hovenia dulcis (Rhamnaceae) fruits are popularly used as herbal medicines or dietary supplements in Asian countries due to functions such as liver protection and detoxification from alcohol poisoning. Accordingly, it is very likely for dietary supplemental products, including H. dulcis fruit extracts, to be taken with prescription drugs.
OBJECTIVE: In this study, possible food-drug interactions involving H. dulcis fruit extracts were evaluated based on the inhibition of cytochrome P450 (CYP) enzyme activity.
MATERIAL AND METHODS: The water extract of H. dulcis fruit extracts was incubated in human liver microsomes with CYP-specific substrates. The formation of the CYP-specific metabolites was measured using liquid chromatography-tandem mass spectrometry.
RESULTS: H. dulcis fruit extracts showed negligible effects on seven CYP isozyme activities at all concentrations tested.
CONCLUSION: This result suggests that H. dulcis fruit extracts may have minimal pharmacokinetic interactions with coadministered drugs through the modulation of CYP enzymes.
SUMMARY: Food-drug interactions involving H. dulcis fruit extracts were evaluated.The inhibition of CYPs by H. dulcis extracts was tested.H. dulcis extracts showed negligible effects on CYP activities.H. dulcis extracts may have minimal pharmacokinetic interactions with co-administered drugs. Abbreviations Used: CYP: cytochrome P450 enzymes, HPLC: High performance liquid chromatography, LC-MS/MS : liquid chromatography-tandem mass spectrometry, MRM: multiple-reaction monitoring.

INTRODUCTION

Herbal dietary supplements are receiving remarkable attention worldwide. Such products have traditionally been used as folk remedies and are liable to be regarded as safe.[1] Herbal products are widely used to treat diseases and to improve individuals’ health in combination with prescription medicines.[2] However, they are unregulated and many patients do not tell their physicians that they are taking herbal products. Therefore, there is growing concern about unexpected adverse effects induced by herb–drug interactions.[34] One of the representative mechanisms of herb–drug interactions are alterations in the absorption and disposition of drugs via the modification of pharmacokinetic regulators, such as cytochrome P450 (CYP) enzymes.[5] CYP enzymes are the major enzymes in the liver, which is a crucial organ for the drug metabolism of many conventional medicines.[67] Many herbs could affect the enzyme activity of CYP, which may cause alterations in the metabolic clearance of the substrate drug by inhibiting or inducing a specific CYP enzyme; these modifications may lead to decreased therapeutic effects or increased toxicity of certain drugs.[891011] For these reasons, assessments of herb–drug interactions involving CYP inhibition based on in vitro data should be executed, in order to prevent adverse effects caused by taking prescription medicines in combination with herbal products.

Hovenia dulcis Thunberg belongs to a small genus of Rhamnaceae that occurs naturally in East Asia.[12] This tree has been widely used as a traditional herb in Oriental medicine for many years, and the fruit stalk of H. dulcis is the main part of the tree that is used for herbal materials.[1213] The fruit stalks of H. dulcis have shown a variety of benefits, including alcohol detoxification,[1415] hepatoprotective effects,[1617] antioxidant effects,[18] and antidiabetic effects.[1920] Therefore, the fruit stalk of H. dulcis has been popularly used as an herbal dietary supplement in Asian countries.[21222324] H. dulcis products are mainly consumed as drinks, so it is very likely that they may be taken along with prescription or over-the-counter drugs. However, to our knowledge, information on the herb–drug interactions of H. dulcis fruit extracts has not yet been reported. Therefore, this study investigated the effects of fruit stalk extracts of H. dulcis on CYP-mediated drug metabolism using human liver microsomes, in order to assess and predict herb–drug interactions.

MATERIALS AND METHODS

Materials

Aqueous extract samples of fruit stalks of H. dulcis were provided by Lifetree Biotech Co., Ltd. (Suwon, Korea). The extract was standardized to contain 0.88 ± 0.05 mg/g of ampelopsin and 0.86 ± 0.05 mg/g of taxifolin by HPLC analysis [Figure 1]. Pooled human liver microsomes were purchased from BD Gentest (Woburn, MA, USA). Glucose-6-phosphate, β-NADP+, glucose-6-phosphate dehydrogenase, phenacetin, coumarin, paclitaxel, diclofenac, mephenytoin, dextromethorphan, midazolam, furafylline, methoxsalen, sulfaphenazole, ticlopidine, quercetin, quinidine, ketoconazole, and terfenadine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Solvents for HPLC were purchased from J. T. Baker (Phillipsburg, NJ, USA). Distilled water was prepared using a MilliQ purification system (Millipore, Billerica, MA, USA).

Figure 1

Representative HPLC chromatogram of H. dulcis fruit extracts. An Xbridge™ Shield RP18 column (4.6 mm I.D. × 150 mm, 3.5 μm) (Waters, Milford, USA) was used for the chromatographic separation. Column oven temperature maintained at 30°C. The mobile phase was composed of 0.1% acetic acid (solvent A) and acetonitrile (solvent B). The flow rate was 1.0 mL/min. The composition of solvent B was maintained at 20% for 3 min, linearly increased to 38% for 24 min, further increased to 90% for 1 min and maintained at 90% for 5 min, which was followed by equilibration to the initial composition for 6 min. The injection volume was 10 μL and the UV detection wavelength was 290 nm.

CYP inhibition assay

The CYP inhibition assay was performed according to the previously established method.[25] Various concentrations of fruit stalk extracts from H. dulcis (1, 3, 10, 30, and 100 μg/mL in 70% methanol) were tested without and with a preincubation procedure, to test the possibility of mechanism-based inactivation. Well-known selective CYP inhibitors were used as positive controls. The inhibitors and their concentrations were as follows: 10 μM of furafylline for CYP1A2; 20 μM of methoxsalen for CYP2A6; 10 μM of quercetin for CYP2C8; 50 μM of sulfaphenazole for CYP2C9; 20 μM of ticlopidine for CYP2C19; 50 μM of quinidine for CYP2D6; and 5 μM of ketoconazole for CYP3A4. The resultant incubation samples were pretreated using solid-phase extraction, then analyzed based on multiple-reaction monitoring (MRM) detection using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CYP specific marker substrates used, and the metabolites monitored with their MRM precursor-product transition conditions, are tabulated in Table 1.

Table 1

CYP-specific marker substrates and their metabolites monitored

High performance liquid chromatography (HPLC) analysis

The content of the major bioactive constituents of H. dulcis, ampelopsin, and taxifolin was measured in the extract sample using HPLC. The HPLC analysis was performed based on the previously reported method.[26] The extract sample was dissolved in 80% methanol at a concentration of 250 mg/mL, filtered, and injected into the HPLC system. Ampelopsin and taxifolin were eluted at 6.2 and 10.8 min, respectively [Figure 1].

RESULTS AND DISCUSSION

The inhibitory effects of fruit stalk extract of H. dulcis were examined in human liver microsomes. The validity of the CYP assay system was assessed with well-known selective inhibitors of CYP isozymes: Furafylline (CYP1A2), methoxsalen (CYP2A6), quercetin (CYP2C8), sulfaphenazole (CYP2C9), ticlopidine (CYP2C19), quinidine (CYP2D6), and ketoconazole (CYP3A4). Each inhibitor selectively inhibited the corresponding CYP marker activity. The remaining activity of the CYP inhibitors was as follows: 2.79% for CYP1A2 (furafylline), 1.74% for CYP2A6 (methoxsalen), 6.64% for CYP2C8 (quercetin), 3.76% for CYP2C9 (sulfaphenazole), 16.57% for CYP2C19 (ticlopidine), 1.65% for CYP2D6 (quinidine), and 2.79% for CYP3A4 (ketoconazole) at designated concentrations. Tables 2 and 3 show the effect of fruit stalk extracts of H. dulcis on the drug-metabolizing activity of seven CYP isozymes. When fruit stalk extracts of H. dulcis were tested without preincubation, no significant inhibitory effects on CYP isozyme enzyme activities were observed at any of the concentrations tested [Table 2]. When the fruit stalk extracts of H. dulcis were preincubated and tested, the extent of inhibition on several CYP isozyme activities was slightly different from the results shown in [Table 3], but no inhibitory effects on CYP isozyme enzyme activities were observed, and the IC50 values were all >100 μg/mL [Table 3]. Therefore, fruit stalk extracts of H. dulcis are considered to have negligible effects on drug metabolism regulated by CYP isozymes as competitive or mechanism-based inhibitors.

Table 2

Remaining activities of CYP isozymes following treatment with H. dulcis extracts (without preincubation)

Table 3

Remaining activities of CYP isozymes following treatment with H. dulcis extracts (with preincubation)

There have been several published reports demonstrating the CYP 450 inhibitory effects caused by the major bioactive compounds of H. dulcis extracts, ampelopsin, and taxifolin. Huang et al.[27] reported that ampelopsin significantly inhibited CYP2C9 activity and induced CYP3A4 activity after oral administration in rats. Çelik et al.[28] showed that taxifolin acts as a weak inhibitor of cytochrome b5 reductase, which is involved in specific CYP450-mediated drug metabolism by donating a second electron to CYP450 cytochrome b5. These previous reports raised some concerns about the possibility of inhibited CYP enzyme activity by H. dulcis extracts. However, in this study, no considerable inhibitory effects of H. dulcis extracts on CYP 450 enzymes were observed. It is supposed that the chemical complexity of H. dulcis extracts may compensate for or dilute the effects of ampelopsin and taxifolin. The representative constituents of H. dulcis extracts are phenolic compounds and triterpene saponins.[26] As phenolic compounds, hovenodulinol, hovenitins I, II, and III, (+)-3,3’,5’,5,7-pentahydroflavanone, laricitrin, myricetin, (+)-gallocatechin, dihydrokaempferol, dihydromyricetin (ampelopsin), and quercetin have been reported. Saponin C2, β-daucosterol, hovenidulciosides A1, A2, B1, and B2, hodulosides I and III, and hovenidulcigenin have been reported as triterpene saponins. The property of each compound on CYP inhibition has not yet been fully elucidated. However, considering the content of ampelopsin and taxifolin in the extract (less than 0.1%), the effect on CYP activity by these compounds in the extract is expected to be negligible as observed in this study.

CONCLUSION

The potential effects of fruit stalk extracts of H. dulcis on human CYP enzyme activities were evaluated in vitro, and the results showed a possible negligible interaction with coadministered drugs by the modulation of CYP enzymes. However, differences may exist between in vitro and in vivo test results. In addition, there could be other herb–drug interaction mechanisms involved with H. dulcis extracts and continued and further studies of such interactions should be conducted.

Financial support and sponsorship

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2014 R1A1A1A05002840).

Conflicts of interest

There are no conflicts of interest.