Hypericum perforatum: Which Constituents may Induce Intestinal MDR1 and CYP3A4 mRNA Expression?

• Abstract
• Introduction
• Materials and Methods
• Chemicals and extracts
• HPLC analysis
• Cell cultures
• Real-time PCR
• Cytotoxicity assay
• Statistics
• Results
• Discussion
• References

Abstract

In vitro and in vivo studies suggest that extracts of St John’s wort (Hypericum perforatum, L.; SJWE) interact with various drugs, by enhancing their elimination, due to induction of intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp), the gene product of multidrug resistance gene 1 (MDR1/ABCB1). The aim of our study was to identify the major constituents responsible for this induction and their relative importance. Therefore, plant extracts were investigated that vary in these constituents with respect to their effect on mRNA expression of MDR1/CYP3A4. First, different pure constituents of Hypericum perforatum L. were investigated. Secondly, diverse SJWE with different concentrations of hyperforin, quercitrin and hypericin were investigated. The concentrations of hyperforin, hypericin, and quercitrin in the plant extracts were determined by HPLC, and an ”artificial extract” containing the same mixture of these constituents was investigated. Different plant extracts, pure constituents or ”artificial extracts” were applied to the human colon carcinoma-derived cell line (LS180) and the induction of MDR1 and CYP3A4 expression was analyzed by quantitative RT-PCR. MDR1 and CYP3A4 mRNA expression were both induced by single constituents of SJW such as hypericin and hyperforin in a concentration of 10 μM. Additionally, CYP3A4 mRNA expression was induced by quercitrin. SJW extracts containing hyperforin induced significantly MDR1 mRNA expression, whereas no CYP3A4 induction was observed after treatment with any of the investigated SJWE. These effects could be mimicked by ”artificial extracts” containing the same compositions of hyperforin, hypericin and quercitrin as the plant extracts.

Introduction

Extracts of Hypericum perforatum L. (St John’s wort; SJWE) are used in the treatment of mild to moderate depression [1]. A number of pharmacokinetic interactions have been identified between SJWE and other drugs, such as cyclosporine [2], [3], [4], digoxin [5], indinavir [6], amitriptyline [7] and oral contraceptives [8]. Previous studies have shown that SJWE induces the expression of the drug-metabolizing cytochrome P450 isoform 3A4 (CYP3A4) and the drug efflux pump P-glycoprotein (Pgp) in vitro [9] and in vivo [10]. This induction and the subsequent enhanced elimination of the affected drugs are thought to be the basis for many of these herb-drug interactions.

The aim of this study was to find out which of the major constituents of SJWE are able to regulate MDR1/ABCB1 and CYP3A4 mRNA expression. First, single constituents were investigated. Secondly, different variants of SJWE were investigated which were derived by different extraction methods from the same plant variety and contained different concentrations of hyperforin, quercitrin and hypericin. As a control, Ze117 a widely used SJW extract with low hyperforin content was used. Thirdly, ”artificial” SJW extracts were made by mixing the pure constituents hyperforin, hypericin and quercitrin in concentrations as measured in the investigated plant extracts. This was done to test the hypothesis that these mixtures could completely explain the effects of the respective whole SJWE on MDR1 and CYP3A4 mRNA expression. In the case of the same effects of SJW extracts and their specific mixtures, a major contribution of other relevant yet non-identified constituents could be excluded.

Materials and Methods

Chemicals and extracts

The SJWE Ze117 was extracted from Hyperici herba with 50 % (m/m) ethanol and provided by Zeller AG, Romanshorn (CH). From the flowering part of one Hypericum perforatum accession (genotype HP52, Vitaplant AG; Witterswil, Switzerland) an 80 % (v/v) methanolic extract as well as different extract fractions were obtained by subsequent extraction of the powdered plant material with ethanol 60 % (v/v), hexane, ethyl acetate, methanol 80 % (v/v) and ethanol 20 % (v/v). The following extracts were obtained with the respective extracting agent: EtOH60: ethanol 60 %; Hex: hexane; Ethylac: ethyl acetate; MeOH80: methanol 80 %; EtOH20: ethanol 20 %; and Ze117: ethanol 50 %.

Quercitrin, isoquercitrin, hyperoside and rutin were purchased from Extrasynthèse (Genay, France), hypericin and quercetin from Sigma (Buchs, Switzerland), and hyperforin from Phytoplan (Heidelberg, Germany). Additional hyperforin was a kind gift from Zeller AG (Romanshorn, Switzerland). The purity of the compounds is for hypericin 95 %, quercetin ≥ 98 %, hyperforin > 90 % and for all other compounds > 98 %. Rifampicin was purchased from Sigma (Buchs, Switzerland). All other chemicals were purchased from commercial sources of best quality available.

HPLC analysis

An aliquot of the extracts was filtered through an RC membrane (0.45 μm) and analyzed in a Jasco HPLC system with UV-VIS detection (200 - 600 nm) using a Hypersil 120 - 5 ODS (250 × 4.6 mm) column with a precolumn of the same material (both Macherey Nagel; Oensingen, Switzerland) as stationary phase. The mobile phase consisted of two solvent systems [A: 19 % acetonitrile, 80 % water, 1 % H₃PO₄; and B: 59 % acetonitrile, 40 % methanol, 1 % H₃PO₄ (85 %) in a linear gradient (0 - 8 min 100 % A, 8 - 30 min 50 % A, 30 - 75 min 0 % A)]. Quantification of a part of the flavonoids was performed by the external standard method. External standards of rutin, hyperoside, isoquercitrin, quercitrin, quercetin, kaempferol, biapigenine, amentoflavone, hypericin, and hyperforin were used. Precision and selectivity of the method were determined by the comparison of the R_F values and UV spectra of the single compounds and a mixture of these. In the absence of commercially available reference substances, not all of the detected flavonoid-like peaks (e. g., quercetin glycosides) could be identified, and quantified. Intra- and interassay variability of the assay was below 10 % (CV).

Cell cultures

The LS180 cell line (used between passage 36 and 40) was purchased from ATCC (Manassas, VI, USA). It was cultured as previously described [11]. After the cells had reached confluence they were treated with the compound of interest for 72 h. Medium containing the compound of interest was freshly prepared and changed every 24 hours and experiments were performed in the dark. The single compounds were dissolved in dimethyl sulfoxide (DMSO). Rifampicin 10 μM was used as control for CYP3A4 and MDR1/ABCB1 mRNA induction [12]. The final DMSO concentration did not exceed 0.5 %. Up to this concentration, the solvent did not change significantly the expression level of any gene of interest compared to control without DMSO. SJWE were dissolved in DMSO and a final concentration of 0.01 mg/mL extract was used in all induction experiments.

Real-time PCR

Quantitative real-time PCR was performed as described before (Pfrunder et al. [11]). For MDR1/ABCB1 detection the following primers and probe were used: 5′-AAGCTGTCAAGGAAGCC-AATGCCTATGACTT-3′ (probe), 5′-CTGTATTGTTTGCC ACCACGA-3′ (forward), and 5′-AGGGTGTCAAATTTATGAGGCAGT-3′ (reverse). For CYP3A4 detection the following primers and probes were used: 5′-TTCTCCTGGCTGTCAGCCTGGTGC-3′ (probe), 5′-TCTCATCCCAGACTTGGCCA-3′ (forward), and 5′-CATGTGAATGAGTTCCATATAGATAGA-3′ (reverse). As internal standard ribosomal 18 s gene was used. The expression level of the endogenous control gene 18 s did not alter under any of the different treatments.

Cytotoxicity assay

The cytotoxicity of SJWE was assessed by sulforhodamine B (SRB) cytotoxicity assay previously described by Skehan et al. [13].

Statistics

For statistical comparison, data of groups were compared by analysis of variance (ANOVA). The level of significance was P = 0.05. If this analysis revealed significant differences, pair-wise comparison between groups was performed by two-sided unpaired t-test. P values were adjusted by Bonferroni’s correction for multiple comparisons. All comparisons were performed using SPSS for Windows software (version 12.0).

Results

One aim of our study was to find out which of the major constituents of SJWE are able to regulate MDR1/ABCB1 and CYP3A4 mRNA expression. First, single constituents in a concentration of 10 μM were investigated. As illustrated in Fig. [1] A, MDR1/ABCB1 mRNA expression was significantly up-regulated by rifampicin (positive control), hypericin (∼4.8-fold) and hyperforin (∼5-fold). Quercetin, hyperoside, rutin, isoquercitrin and quercitrin had no effect on the mRNA expression level of MDR1/ABCB1. CYP3A4 mRNA expression was significantly up-regulated by rifampicin, hypericin (˜11-fold) and quercitrin (˜7-fold). Hyperforin induced CYP3A4 mRNA expression (7-fold) (Fig. [1] B). Quercetin, hyperoside, rutin and isoquercitrin had no effect on the CYP3A4 mRNA expression in LS180 cells. Then the potential of different variants of SJW extracts to induce MDR1 and CYP3A4 mRNA expression was investigated. The constitutions of six different extracts of SJW (Ze117; extracts EtOH60, Hex, Ethylac, MeOH80, and EtOH20) were first analyzed by HPLC. As shown in Table [1], these extracts differed mainly with respect to their composition of hyperforin, hypericin and quercitrin. The different extracts investigated had similar contents of rutin, hyperoside, isoquercitrin and quercetin (data not shown).The cytotoxicity of SJW extracts is displayed in Fig. [2]. All SJW extracts showed a concentration-dependent toxicity. Extract Hex showed the lowest IC₅₀ value (IC₅₀ : 0.017 mg/mL), indicating the highest toxicity on LS180 cells. Extract Ethylac exhibited also a moderately high toxicity (IC₅₀ : 0.024 mg/mL), whereas extract ETOH60 exhibited intermediate toxicity (IC₅₀ : 0.075 mg/mL). Ze117 (IC₅₀ : 0.569 mg/mL), MeOH80 (IC₅₀ : 0.381 mg/mL), and EtOH20 (IC₅₀ : 0.455 mg/mL) exhibited the highest IC₅₀ values, indicating lowest toxicity. In the induction experiments all extracts were investigated at a concentration of 0.01 mg/mL where no apparent toxicity was observed. Solely for extract Hex, a small toxic effect was seen. This is possibly caused by its high hyperforin concentration. Treatment with extracts EtOH60, Hex, and Ethylac led to a significant increase in MDR1 mRNA expression (Fig. [3] A), which was in the same range as with the positive control rifampicin. Cells incubated with extract EtOH60, Hex and Ethylac as well as LS180 cells incubated with rifampicin showed a 4- to 5.5-fold higher MDR1/ACB1 mRNA expression than the untreated controls. Treatment with extracts MeOH80, EtOH20 and Ze117 had no significant effect on MDR1/ABCB1 mRNA expression.

CYP3A4 transcriptional expression was only induced after rifampicin treatment. Neither Ze117 nor treatment with different extracts of the plant variety HP52 (EtOH60, Hex, Ethylac, MeOH80, and EtOH20) resulted in a significant increase in CYP3A4 mRNA expression (Fig. [3] B).

Finally, the hypothesis was tested that hyperforin, hypericin and quercitrin are the main constituents involved in MDR1/ABCB1 and CYP3A4 induction. Therefore ”artificial plant extracts” were generated that mimicked the concentration of hyperforin, hypericin and quercitrin of the different SJWE. Table [2] shows the concentration of hyperforin, quercitrin and hypericin of the artificial plant extracts. Since total hypericin content consists additionally of pseudohypericin we tried to mimic the amount of pseudohypericin with a corresponding additional amount of hypericin (denoted with an additional ”+”). By this approach a similar amount of total hypericin in the ”artificial extracts” as in the whole plant extract (extracts AEtOH60+, AEthylac+, AMeOH80+, and AZe117+) was obtained. In Fig. [4], the results of MDR1 and CYP3A4 mRNA expression are illustrated. In general, the effect of the whole plant extracts on MDR1/ABCB1 and CYP3A4 mRNA expression could be mimicked by the ”artificial plant extracts”. Incubation with the ”artificial extracts” led to a significantly elevated MDR1/ABCB1 mRNA expression (Fig. [4] A). The artificial extract AEtOH60, AHex, and AEthylac showed a 3- to 5-fold higher MDR1 mRNA expression whereas a 4- to 5.5-fold higher MDR1 mRNA expression was observed with the whole plant extracts. The ”artificial extracts” AMeOH80, AEtOH20, and AZe117 like the respective whole plant extracts had no effect on MDR1/ABCB1 mRNA expression. The level of CYP3A4 mRNA expression was not significantly altered by incubation with the ”artificial extracts” AHex, AEthylac, AMeOH80, AEtOH20 and AZe117 which is comparable to the effect of the corresponding whole plant extracts (Fig. [4] B). Addition of hypericin in order to mimic additional pseudohypericin (extracts AEtOH60+, AEthylac+, AMeOH80+, and AZe117+) showed no additional effect, either on MDR1/ABCB1 induction (Fig. [4] A) or on CYP3A4 induction (Fig. [4] B).

Discussion

Dürr and co-workers showed that SJWE LI160 induces intestinal MDR1/ABCB1 and CYP3A4 functional expression [9]. As SJW extract is a composition of many structurally diverse chemical constituents, we tested the ability of several major constituents to induce MDR1/ABCB1 and CYP3A4 transcriptional expression in the intestinal cell line LS180.

In our studies, we found significantly elevated MDR1 and CYP3A4 mRNA expression in LS180 cells after exposure to hypericin and hyperforin. CYP3A4 mRNA expression was additionally significantly induced after quercitrin treatment. We cannot completely exclude that some of our observed effects of the pure constituents used in this experiments are partly due to toxicity. Hypericin exhibited low toxic effects already at a concentration of 5 μM (data not shown) and hyperforin has been found to be toxic in primary hepatocytes at concentrations of about 1 μM [14]. We detected no toxicity for quercitrin, rutin and hyperoside (data not shown). However, since some extracts (Hex) contained hyperforin in a concentration of 10.8 μM, we decided to test the single constituents at this high concentration.

In addition, we tested the potencies of several SJWE to induce MDR1/ABCB1 and CYP3A4 mRNA expression. Hyperforin seems to be the most important SJW constituent responsible for MDR1/ABCB1 mRNA induction. All extracts that induced MDR1 mRNA (EtOH60, Hex, and Ethylac) contained hyperforin (0.5 μM to 10.8 μM) whereas extracts that had no effect on MDR1 mRNA induction (MeOH80, EtOH20 and Ze117) contained no hyperforin. The finding that MDR1 mRNA expression is induced by hyperforin alone (Hex) is in line with clinical results that showed a lower incidence for drug interactions with a Hypericum variety (Ze117) [15] containing low amounts of hyperforin. Hyperforin was previously shown to bind with high affinity to PXR (EC₅₀ of 0.023 μM) [14]. PXR, a nuclear receptor, activated by a range of xenobiotics (for review see [16]) is regarded as a key transcriptional regulator of various genes such as CYP3A4 [17], CYP3A7, CYP2B6 [18] as well as MDR1/ABCB1 [19], bile salt export pump (BSEP) [20] and multidrug resistance related protein 2 (MRP2/ABCC2) [16]. Binding of hyperforin leads to activation of PXR that forms a heterodimer with retinoic acid receptor (RXR) and induces MDR1/ABCB1 and CYP3A4 gene transcription. Quercitrin leads most likely not to an increase in MDR1 mRNA expression since the extract containing the highest concentration of quercitrin (MeOH80) had no inductor effect on MDR1 expression. Extract MeOH80 contained also a significantly higher content of hypericin than extracts Hex and Ethylac, indicating that hypericin is not responsible for the inductor effect.

We observed no effect of the investigated plant extracts on the expression of CYP3A4 mRNA. These findings were confirmed several times, also with other plant varieties investigated (data not shown). This is in contrast to clinical trials where, after multiple administrations to human subjects, an increased CYP3A4 expression was observed in duodenal biopsies [9]. However, we have to consider the principle limitations of in vitro assays with multicomponent plant extracts, since it is difficult to estimate relevant concentrations and exposure times. This has been intensively discussed by Butterweck et al. [21]. Furthermore, isolated cells are devoid of compensatory mechanisms as present in living tissue. Therefore, confirmation of our results by in vivo experiments is mandatory.

In conclusion, our findings suggest that hyperforin is the most important constituent of SJWE that is involved in MDR1/ABCB1 mRNA induction. Since some extracts with low hyperforin content show also anti-depressive effects, it might be possible to separate the anti-depressive effect from the interacting potential in SJW extract by breeding plant varieties that contain only low amounts of hyperforin.

References

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  Thieme ConnectPubMedSearch in Google Scholar

Jürgen Drewe MD, MSc

Department of Clinical Pharmacology and Toxicology

University Clinic Basel

Petersgraben 4

4031 Basel

Switzerland

Phone: +41-61-265-3848

Fax: +41-61-265-8581

Email: juergen.drewe@unibas.ch

References

• 1 Linde K, Melchart D, Mulrow C D, Berner M. St John’s wort and depression.  JAMS. 2002;  288 447-8; discussion: 48 - 9
  PubMedSearch in Google Scholar
• 2 Ruschitzka F, Meier P J, Turina M, Luscher T F, Noll G. Acute heart transplant rejection due to Saint John’s wort.  Lancet. 2000;  355 548-9
  CrossrefPubMedSearch in Google Scholar
• 3 Mai I, Kruger H, Budde K, Johne A, Brockmoller J, Neumayer H H. et al . Hazardous pharmacokinetic interaction of Saint John’s wort (Hypericum perforatum) with the immunosuppressant cyclosporin.  Int J Clin Pharmacol Ther. 2000;  38 500-2
  PubMedSearch in Google Scholar
• 4 Barone G W, Gurley B J, Ketel B L, Lightfoot M L, Abul-Ezz S R. Drug interaction between St. John’s wort and cyclosporine.  Ann Pharmacother. 2000;  34 1013-6
  CrossrefPubMedSearch in Google Scholar
• 5 Johne A, Brockmoller J, Bauer S, Maurer A, Langheinrich M, Roots I. Pharmacokinetic interaction of digoxin with an herbal extract from St John’s wort (Hypericum perforatum).  Clin Pharmacol Ther. 1999;  66 338-45
  PubMedSearch in Google Scholar
• 6 Piscitelli S C, Burstein A H, Chaitt D, Alfaro R M, Falloon J. Indinavir concentrations and St John’s wort.  Lancet. 2000;  355 547-8
  CrossrefPubMedSearch in Google Scholar
• 7 Johne A, Schmider J, Brockmoller J, Stadelmann A, Stroemer E, Bauer S. et al . Decreased plasma levels of amitriptyline and its metabolites on comedication with an extract from St. John’s wort (Hypericum perforatum).  J Clin Psychopharmacol. 2002;  22 46-54
  CrossrefPubMedSearch in Google Scholar
• 8 Pfrunder A, Schiesser M, Gerber S, Haschke M, Bitzer J, Drewe J. Interaction of St John’s wort with low-dose oral contraceptive therapy: a randomized controlled trial.  Br J Clin Pharmacol. 2003;  56 683-90
  CrossrefPubMedSearch in Google Scholar
• 9 Dürr D, Stieger B, Kullak-Ublick G A, Rentsch K M, Steinert H C, Meier P J. et al . St John’s wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4.  Clin Pharmacol Ther. 2000;  68 598-604
  CrossrefPubMedSearch in Google Scholar
• 10 Hennessy M, Kelleher D, Spiers J P, Barry M, Kavanagh P, Back D. et al . St. John’s wort increases expression of P-glycoprotein: implications for drug interactions.  Br J Clin Pharmacol. 2002;  53 75-82
  CrossrefPubMedSearch in Google Scholar
• 11 Pfrunder A, Gutmann H, Beglinger C, Drewe J. Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines.  J Pharm Pharmacol. 2003;  55 59-66
  CrossrefPubMedSearch in Google Scholar
• 12 Schuetz E G, Schinkel A H, Relling M V, Schuetz J D. P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans.  Proc Natl Acad Sci USA. 1996;  93 4001-5
  CrossrefPubMedSearch in Google Scholar
• 13 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 14 Moore L B, Goodwin B, Jones S A, Wisely G B, Serabjit-Singh C J, Willson T M. et al . St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor.  Proc Natl Acad Sci USA. 2000;  97 7500-2
  CrossrefPubMedSearch in Google Scholar
• 15 Rätz A E, von Moos M, Drewe J. Johanniskraut: ein Phytopharmakon mit potenziell gefährlichen Interaktionen.  Schweiz Rundsch Med Prax. 2001;  90 843-9
  PubMedSearch in Google Scholar
• 16 Kast H R, Goodwin B, Tarr P T, Jones S A, Anisfeld A M, Stoltz C M. et al . Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor.  J Biol Chem. 2002;  277 2908-15
  CrossrefPubMedSearch in Google Scholar
• 17 Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M. et al . Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction.  Proc Natl Acad Sci USA. 1998;  95 12 208-13
  PubMedSearch in Google Scholar
• 18 Goodwin B, Moore L B, Stoltz C M, McKee D D, Kliewer S A. Regulation of the human CYP2B6 gene by the nuclear pregnane X receptor.  Mol Pharmacol. 2001;  60 427-31
  PubMedSearch in Google Scholar
• 19 Geick A, Eichelbaum M, Burk O. Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin.  J Biol Chem. 2001;  276 14 581-7
  PubMedSearch in Google Scholar
• 20 Schuetz E G, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C. et al . Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450.  J Biol Chem. 2001;  276 39 411-8
  PubMedSearch in Google Scholar
• 21 Butterweck V, Derendorf H, Gaus W, Nahrstedt A, Schulz V, Unger M. Pharmacokinetic herb-drug interactions: are preventive screenings necessary and appropriate?.  Planta Med. 2004;  70 784-91
  Thieme ConnectPubMedSearch in Google Scholar

Jürgen Drewe MD, MSc

Department of Clinical Pharmacology and Toxicology

University Clinic Basel

Petersgraben 4

4031 Basel

Switzerland

Phone: +41-61-265-3848

Fax: +41-61-265-8581

Email: juergen.drewe@unibas.ch