Article pubs.acs.org/JAFC
Human Ether-à-go-go Related Gene (hERG) Channel Blocking Aporphine Alkaloids from Lotus Leaves and Their Quantitative Analysis in Dietary Weight Loss Supplements Ulrike Grienke,†,‡ Christina E. Mair,†,‡ Priyanka Saxena,§ Igor Baburin,§ Olaf Scheel,# Markus Ganzera,† Daniela Schuster,⊥ Steffen Hering,§ and Judith M. Rollinger*,†,‡ †
Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria ‡ Department of Pharmacognosy, and §Department of Pharmacology and Toxicology, Faculty of Life Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria # Cytocentrics Bioscience GmbH, Tannenweg 22k, 18059 Rostock, Germany ⊥ Institute of Pharmacy/Pharmaceutical Chemistry, Computer-Aided Molecular Design Group, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria S Supporting Information *
ABSTRACT: Blockage of the human ether-à-go-go related gene (hERG) channel can result in life-threatening ventricular tachyarrhythmia. In an in vitro screening of herbal materials for hERG blockers using an automated two-microelectrode voltage clamp assay on Xenopus oocytes, an alkaloid fraction of Nelumbo nucifera Gaertn. (lotus) leaves induced ∼50% of hERG current inhibition at 100 μg/mL. Chromatographic separation resulted in the isolation and identification of (−)-asimilobine, 1, nuciferine, 2, O-nornuciferine, 3, N-nornuciferine, 4, and liensinine, 5. In agreement with in silico predicted ligand−target interactions, 2, 3, and 4 revealed distinct in vitro hERG blockages measured in HEK293 cells with IC50 values of 2.89, 7.91, and 9.75 μM, respectively. Because lotus leaf dietary weight loss supplements are becoming increasingly popular, the identified hERGblocking alkaloids were quantitated in five commercially available products. Results showed pronounced differences in the content of hERG-blocking alkaloids ranging up to 992 μg (2) in the daily recommended dose. KEYWORDS: Nelumbo nucifera, lotus, dietary supplement, cardiotoxic risk, hERG, patch clamp assay, nuciferine, weight loss
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INTRODUCTION Blockage of the human ether-à-go-go-related gene (hERG) potassium channel leads to the prolongation of cardiac repolarization, manifested as a prolonged QT interval. This is a major risk factor for ventricular arrhythmias, so-called Torsades de Pointes (TdP), a potentially life-threatening cardiac disorder.1−3 Several drugs have been restricted in their use or have even been withdrawn from the market due to these severe hERG-related cardiac issues, for example, cisapride (gastroprokinetic), terfenadine (antihistaminic), astemizole (antihistaminic), and sertindole (antipsychotic).4 Accordingly, the hERG channel represents a primary antitarget in drug development. The importance of disclosing the hERG-blocking properties of drug candidates is reflected in the preclinical guideline to an integrated risk assessment to reveal the QT prolonging potential of human pharmaceuticals proposed by the International Conference on Harmonization (ICH) (ICH S7B Guideline).5 Moreover, in vitro assays can also be combined with computational approaches using, for example, pharmacophore models to predict a compounds’ probability to interact with the hERG channel.6−9 Because natural products, commonly consumed botanicals, and dietary supplements are, in contrast to drugs, not systematically screened for their hERG-blocking effects, there is an urgent need to critically assess their potential cardiotoxic © XXXX American Chemical Society
risks. Remarkably, to date, only a small number of natural compounds have been identified as hERG blockers,10 for example, iboga alkaloids such as ibogaine and voacangine7,11−14 or protoberberines such as dihydroberberine isolated from the methanolic extract of the traditional Chinese medicine (TCM) herbal drug Coptidis rhizoma.15 In the course of an ongoing European Union project (hERGscreen, FP7-People-IRSES-295174) with the aim of assessing the cardiotoxic risk of natural products, a library of ∼60 lead-like enhanced herbal extracts16 or fractions were screened for hERG channel blocking using an automated twomicroelectrode voltage clamp assay on Xenopus laevis oocytes.17 Among all samples tested, an enriched alkaloid fraction of the leaves of Nelumbo nucifera Gaertn. (Nelumbonaceae) induced 50.4 ± 10.3% (n = 3, data not shown) inhibition of the peak tail hERG current at 100 μg/mL. N. nucifera, commonly called lotus, is a well-known plant used as food and medicine. Beneficial effects have been reported for almost all organs including rhizomes, leaves, flowers, and seeds. Major secondary metabolites are alkaloids, flavonoids, and triterpenoids.18 Received: April 15, 2015 Revised: June 2, 2015 Accepted: June 2, 2015
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DOI: 10.1021/acs.jafc.5b01901 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
period of 10 min. All chemicals and solvents used were of analytical grade. Plant Material. The leaves of N. nucifera Gaertn. (Nymphaeaceae) were obtained from Plantasia (MMag. E.A. Stöger, Oberndorf/ Salzburg, Austria). A voucher specimen (JR-20100922_A1) is deposited in the Herbarium of the Department of Pharmacognosy, University of Vienna, Austria. Extraction, Isolation, and Identification of Pure Compounds. The dried ground leaves of N. nucifera (712 g) were macerated with 6 L of CH2Cl2/MeOH (1:1) (at 22 °C, three times for 72 h each). After removal of the solvent under vacuum, 56 g of extract was obtained. This extract was then redissolved in CH2Cl2 and aqueous MeOH (1:1) and separated via liquid−liquid partition using petroleum ether followed by CH2Cl2. After removal of MeOH from the aqueous phase under vacuum, it was alkalized to a pH of 10 to extract alkaloids with CH2Cl2 to obtain 885 mg of an enriched alkaloid fraction (NN-A). For the isolation of the alkaloids, NN-A was subjected in pilot scale (50 mg) to fast centrifugal partition chromatography (FCPC) using a biphasic solvent system of petroleum ether/ethyl acetate/methanol/ water (1:5:1:5, v/v/v/v). The FCPC instrument was operated in descending mode (lower phase as mobile phase). The sample was dissolved in a mixture of mobile and stationary phase (1:1, v/v). After filling the stationary phase into the system, the mobile phase was pumped in while equilibrating at 800 rpm (column volume = 200 mL). Finally, the sample solution was injected, and separation was carried out at a flow rate of 1.8 mL/min and a rotation speed of 1200 rpm in a counterclockwise direction. Resulting fractions were monitored and combined according to their TLC pattern to give nine fractions (A1− A9). A7 was identified as pure compound 3 (9.96 mg). In a large-scale approach, another 882 mg of NN-A was fractionated by FCPC applying the same parameters and conditions as described above for the small-scale separation (column volume = 200 mL). In sum, 18 fractions were obtained (B1−B18). Fractions B9 (14.35 mg) and B10 (42.24 mg) were purified via silica gel column chromatography (Merck silica gel 60 PF254, 43 cm × 2 cm) by applying an isocratic system of acetone/CH 2 Cl 2 /EtOH/NH 3 (4:3:1:0.1, v/v/v/v) to yield compound 4 (5.41 mg) and compound 5 (8.75 mg). B7 (18.35 mg) and B17 (28.56 mg) were purified via Sephadex LH-20 column chromatography (mobile phase, MeOH), yielding compounds 1 (4.10 mg) and 2 (7.03 mg). All isolated compounds (1−5) could be identified by using HPLC, MS analysis, optical rotation, and 1D- and 2D-NMR experiments. The physical and spectroscopic data of compounds 1−5 agreed with those published previously for (−)-asimilobine, 1, nuciferine, 2, Onornuciferine, 3, N-nornuciferine, 4, and liensinine, 5.21−24 Their purity was checked using TLC and LC-MS and revealed to be >98% in all cases. Expression of hERG Channels in Xenopus Oocytes and Voltage Clamp Studies. Preparation of stage V−VI oocytes from X. laevis (NASCO), synthesis of capped runoff complementary RNA (cRNA) transcripts from linearized complementary DNA (cDNA) templates, and injection of cRNA were performed as described previously.25 cDNA of hERG (accession no. NP000229) was kindly provided by Dr. Sanguinetti (University of Utah). Currents through hERG channels were studied 1−4 days after microinjection of the cRNA by the two-microelectrode voltage clamp technique making use of a TURBO TEC-03X amplifier (npi electronic GmbH, Tamm, Germany). The bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2·6H2O, 1.8 mM CaCl2·2H2O, and 5 mM HEPES (all Sigma, pH 7.4). Voltage-recording and current-injecting microelectrodes (Harvard Apparatus) were filled with 3 M KCl and pulled to have resistances between 0.5 and 2 MΩ. Oocytes with maximal current amplitudes >3 μA were discarded to avoid voltage clamp errors. The pClamp software package v.10.0 (Molecular Devices, Inc.) was used for data acquisition. Unless otherwise stated, the following voltage protocol was used: from a holding potential of −80 mV, the cell membrane was initially depolarized to +20 mV (300 ms) to achieve channel activation and subsequent rapid inactivation. During the following repolarization to −50 mV (300 ms) channels recover from inactivation and elicit the
Neferine, 6, a benzylisoquinoline alkaloid (Figure 1), isolated from the seed embryo of N. nucifera has been reported to block
Figure 1. Chemical structures of lotus alkaloids: (−)-asimilobine, 1, nuciferine, 2, O-nornuciferine, 3, N-nornuciferine, 4, liensinine, 5, and neferine, 6.
hERG channels stably expressed in human embryonic kidney (HEK293) cells. In whole-cell patch clamp studies, this compound induced a concentration-dependent decrease in hERG current amplitude with an IC50 of 7.42 μM.19 Moreover, Western blot analysis and immunofluorescence experiments gave insights into the effect of compound 6 on hERG protein levels in HEK293 cells. Liensinine, 5, a structurally similar benzylisoquinoline alkaloid (Figure 1) from N. nucifera seed embryos, was also described to be a hERG blocker, however, at concentrations below 10 μM in much lower potency than its more hydrophobic derivative, 6.20 The aim of the present study was to evaluate the hERGblocking activity of alkaloids present in N. nucifera leaves and to quantitatively analyze their content in commonly consumed herbal supplements for weight loss including a critical discussion of the potential cardiotoxic risk caused by these products.
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MATERIALS AND METHODS
General Experimental Procedures. Optical rotations were measured on a model 341 polarimeter (PerkinElmer, Waltham, MA, USA) at 25 °C. 1D- and 2D-NMR experiments were performed by using DRX300 and TXI600 NMR spectrometers (Bruker, Billerica, MA, USA) operating at 300 and 600 MHz, respectively. The samples were measured in CDCl3 (calibrated to the residual nondeuterated solvent signals). MS analysis was performed on an Esquire 3000 plus ion-trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with electrospray ionization (ESI) in the positive and negative modes: spray voltage, 4.5 kV; sheath gas, N2, 30 psi; dry gas, N2, 6 L/min, 350 °C; scanning range, m/z 50−1000. Column chromatography was performed using silica gel 60 (40−63 μm) from Merck (Darmstadt, Germany) and Sephadex LH-20 (20−100 μm) from Pharmacia (Stockholm, Sweden). Fast centrifugal partition chromatography was carried out on a FCPC A200 instrument from Kromaton (Annonay, France). Fractions obtained from all chromatographic steps were analyzed by TLC (mobile phase, acetone/CH3Cl/ EtOH/NH3 (4:3:1:0.1, v/v/v/v); stationary phase, Merck silica gel 60 PF254, sprayed with Dragendorff reagent at Vis, UV254, and UV366). HPLC was performed on an Agilent 1200 LC system (Santa Clara, CA, USA) with a photodiode array detector (DAD). LC parameters were as follows: stationary phase, 150 × 3.0 mm i.d., 5 μm, Phenomenex Gemini NX-C18; temperature, 35 °C; mobile phase, A = 95% ammonium acetate 50 mM/5% CH3CN (pH of mixture 6.8); B = 25% ammonium acetate 50 mM/75% CH3CN (pH of mixture 7.6); flow rate, 0.4 mL/min; DAD detection wavelength, 270 nm; injection volume, 10 μL. Separations were performed by gradient elution (80:20 A/B in 25 min to 30/70 A/B, then within 1 min to 2/98 A/B), followed by a 4 min column wash (2A:98B) and a re-equilibration B
DOI: 10.1021/acs.jafc.5b01901 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry hERG current (IhERG). A final step to the holding potential ensured that channels returned to the closed state. The protocol was applied in 1 s intervals (1 Hz pulse frequency). Drug Application and Data Analysis of Voltage Clamp Studies. Drugs were applied to Xenopus oocytes by means of a screening tool fast perfusion technique (npi electronic GmbH).26 Data were analyzed using Origin software 7.0 (OriginLab Corp., Northampton, MA, USA). Each data point represents the mean ± SE from at least three oocytes and two oocyte batches. Whole-Cell Patch Clamp Assay Using HEK293 Cells. Measurements were performed using the automated patch clamp device CytoPatch 2 Instrument.27 Whole-cell patch clamp recordings were applied to examine the effects of the selected lotus leaf alkaloids on the hERG ion channel. To elicit hERG-mediated currents, cells were clamped for 200 ms to −80 mV and then shifted for 200 ms to −50 mV. The voltage step to −50 mV was used to determine the leak current of the cell. Thereafter, the cell was depolarized for 2 s at +40 mV. By stepping back to −50 mV for 2 s, the hERG tail current was activated. At the end of the pulse protocol, the cells were clamped again to holding potential. This voltage protocol was repeated at a frequency of 0.1 Hz. The membrane resistance, Rm, was monitored during the complete recording time. Recordings during which Rm decreased to
