Abstract!

Inhibition of the human ether-a-go-go-relatedgene channel is the single most important riskfactor leading to acquired long QT syndrome.Drug-induced QT prolongation can cause severecardiac complications, including arrhythmia, andis thus a liability in drug development. Consider-ing the importance of the human ether-a-go-go-related gene channel as an antitarget and the dai-ly intake of plant-derived foods and herbal prod-ucts, surprisingly few natural products have beentested for channel blocking properties. In an as-sessment of possible human ether-a-go-go-re-lated gene liabilities, a selection of widely usedherbal medicines and edible plants (vegetables,fruits, and spices) was screened by means of afunctional two-microelectrode voltage-clamp as-say with Xenopus oocytes. The human ether-a-go-go-related gene channel blocking activity of se-lected extracts was investigated with the aid of ahigh-performance liquid chromatography-based

profiling approach, and attributed to tannins andalkaloids. Major European medicinal plants andfrequently consumed food plants were found tohave a low risk for human ether-a-go-go-relatedgene toxicity.

Abbreviations!

hERG: human ether-a-go-go-related geneIhERG: hERG potassium currentLQTS: long QT syndromeOTC: over-the-counterPE: petroleum etherPLE: pressurized liquid extractionQTc: corrected QT intervalSPE: solid phase extractionTCM: traditional Chinese medicineWHO: World Health Organization

Supporting information available online athttp://www.thieme-connect.de/products

Natural Products as Potential Human Ether-a-Go-Go-Related Gene Channel Inhibitors –Outcomes from a Screening of Widely Used HerbalMedicines and Edible Plants

Authors Anja Schramm1, Evelyn A. Jähne1, Igor Baburin2, Steffen Hering2, Matthias Hamburger1

Affiliations 1 Division of Pharmaceutical Biology, University of Basel, Basel, Switzerland2 Institute of Pharmacology and Toxicology, University of Vienna, Vienna, Austria

Key wordsl" herbal drugsl" dietary plantsl" hERG channel inhibitionl" Xenopus oocyte assayl" HPLC‑based activity profilingl" alkaloids

received February 27, 2014revised June 25, 2014accepted June 30, 2014

BibliographyDOI http://dx.doi.org/10.1055/s-0034-1382907Published online August 4, 2014Planta Med 2014; 80:1045–1050 © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. Matthias HamburgerDepartment of PharmaceuticalSciencesDivision of PharmaceuticalBiologyUniversity of BaselKlingelbergstrasse 504056 BaselSwitzerlandPhone: + 41612671425Fax: + 41612671474Matthias.Hamburger@unibas.ch

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Introduction!

A growing number of non-antiarrhythmic drugshave been shown to exhibit side effects associatedwith QT prolongation [1]. Drug-induced LQTS cancause ventricular tachyarrhythmia (torsades depointes arrhythmia) and sudden cardiac death.Due to this potentially fatal side effect, drugs suchas terfenadine and cisapride had to be withdrawnfrom the market. The most important determi-nant of acquired LQTS is the inhibition of IKr(IhERG), the rapidly activating component of thedelayed rectifier potassium current that is medi-ated by the hERG channel. The reduction of IhERGcan delay the repolarization phase of the cardiacaction potential and, as a consequence, lead toprolongation of the QT interval [2]. Hence, hERGchannel blockage is nowadays considered a major

Schramm A e

safety liability in preclinical drug developmentand clinical practice.Medicinal plants and phytomedicines are usedworldwide, either as alternatives to conventionalpharmacotherapy or as complementary medi-cines. For example, preparations containing gink-go, garlic, echinacea, saw palmetto, or St. Johnʼswort belong to the top-selling herbal products inthe United States [3]. An increased understandingof the impact of nutrition on human health led tospecific dietary recommendations aimed at low-ering the incidence of certain diseases. Dietaryphytochemicals such as flavonoids and organo-sulfur compounds are believed to possess preven-tive effects in chronic diseases, e.g., cancer andcardiovascular diseases [4–6]. The WHO dietaryguideline recommends a daily intake of at least400 g of fruits and vegetables [7]. Consumption

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of plant-derived foods (vegetables, fruits, and spices) and the useof medicinal herbs/phytomedicines result in a remarkable intakeof total plant secondary metabolites. It has been estimated thatthe human dietary intake of phytochemicals may reach up to sev-eral grams per day [8,9]. Plant-derived compounds possess aspectrum of beneficial properties, but they may also show ad-verse effects and/or interactions with prescription and OTC drugs[10,11].Considering the importance of hERG as an antitarget, surpris-ingly few natural products have been tested for hERG channelblocking properties. A number of structurally diverse naturalproducts have been shown to diminish hERG channel activity invitro, e.g., naringenin, trimethyl-apigenin, curcumin, lobeline,chelerythrine, papaverine, and capsaicin [12–18]. It has beenshown that the consumption of 1 L of freshly squeezed pinkgrapefruit juice (containing the hERG channel blocking flavanonenaringenin) leads to a mild prolongation of the QTc interval inboth young healthy volunteers and patients suffering fromcardiomyopathy [12,19]. The fact that not only synthetic drugsubstances but also widely occurring natural products, such asflavonoids and alkaloids, block IhERG warrants an assessment ofwidely usedmedicinal and dietary plants for their potential to in-hibit IhERG. Also, the concomitant use of QT prolonging medica-tions along with hERG channel blocking natural products maylead to clinically relevant drug interactions and may be a concernwith respect to consumer safety.In the present study, we wanted to address the following ques-tions: (i) Do herbal medicines and plant-derived foods containhERG channel blocking constituents and, if so, (ii) does their in vi-tro activity, daily intake in diet and/or herbal medicines point to-wards possible risks that would warrant an investigation in ani-mal models for cardiac arrhythmia? For this purpose, we pre-pared a focused library of plant extracts from herbal drugs widelyused in Europe, the US, and China, and from frequently consumedspices, vegetables, and fruits. Food plants were selected accordingto consumption patterns in Central Europe, taxonomic consider-ations (in order to cover a broad range of chemically diverse sec-ondary metabolites), and seasonal availability. hERG channel in-hibitionwas assessed by means of a two-microelectrode voltage-clamp assay with transfected Xenopus laevis oocytes.

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Results and Discussion!

A total of 79 plant samples were successively extracted with sol-vents of increasing polarities to afford a library of 187 extracts.This library was tested in a functional assay with transfectedX. laevis oocytes expressing hERG channels (Tables 1S–3S, Sup-porting Information). An initial test concentration of 100 µg/mLwas used, and extracts inhibiting IhERG by ≥ 30% were selectedfor further investigation. Of the extracts tested, six were foundto be active, including methanolic extracts from cinnamon (Cin-namomum zeylanicum Nees, Lauraceae), guarana (Paullinia cupa-na Kunth, Sapindaceae), nutmeg (Myristica fragrans L., Myristica-ceae), and Coptidis rhizoma (Coptis chinensis Franch., Ranuncu-laceae), and ethyl acetate and methanolic extracts from the fruitsof black pepper (Piper nigrum L., Piperaceae).The active extracts were submitted to high-performance liquidchromatography (HPLC)-based activity profiling in order to iden-tify and characterize hERG channel blocking constituents [20].Prior to microfractionation, they were analyzed by LC‑PDA‑ESI‑MS to obtain a qualitative phytochemical fingerprint, and

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HPLC-based activity profiling was then carried out after optimi-zation of separation. Aliquots of each extract (5mg) were sepa-rated by semipreparative RP-HPLC, and microfractions of 90 seach were collected and tested in the oocyte assay.The methanolic extracts of C. zeylanicum, P. cupana, and M. fra-grans were among the most active extracts in our screening (in-hibition of IhERG by 64.5 ± 5.7%, 45.3 ± 10.2%, and 42.3 ± 1.0%, re-spectively). However, the chromatograms all showed broadhumps in the region of activity (l" Fig. 1, left column), indicativeof a possible presence of tannins in the extract. Therefore, tanninswere selectively removed from the extracts by SPE on polyamidecolumns [21]. Comparison of HPLC profiles before and after poly-amide filtration confirmed the effectiveness of tannin removal,without significant alterations of the HPLC chromatograms(l" Fig. 1, right column). The hERG inhibitory activity of tannin-depleted extracts was significantly lower (see insets in l" Fig. 1),and we concluded that tannins were responsible for the in vitrohERG channel blocking properties of these extracts. Given thenegligible oral bioavailability of tannins [22,23], one can reason-ably assume that no in vivo effects are to be expected. Moreover,gallic acid (1), ellagic acid (2), and (+)-catechin (3) (l" Fig. 3),which can be formed by the intestinal microflora as breakdownproducts of tannins and are known to be resorbed [22,23], weredevoid of hERG inhibitory activity when tested at 100 µM in theoocyte assay. We conclude that the activity of tannins in our invitro assay was likely due to nonspecific interactions [20,21].High levels of tannins are present in nuts, fruits, and somewidelyused spices (e.g., bay leaves, cinnamon, star anise, nutmeg, all-spice, and juniper), whereas most vegetables lack them com-pletely [24–26].Ethyl acetate and methanolic extracts from the fruits of blackpepper (P. nigrum) reduced IhERG by 32.4 ± 0.5% and 36.9 ± 9.1%,respectively. For both extracts, the activity profiles showed thatthe activity peak correlated with the major peak in the HPLCchromatogram (l" Fig. 2A–B). By analysis of LC‑PDA‑ESI‑MS dataand comparison with literature data, this peak was readily iden-tified as piperine (4) (l" Fig. 3) [27]. The hERG inhibitory activityof the alkaloid was determined in the oocyte assay. Piperine (4)inhibited IhERG by 15.3 ± 1.1% and 35.7 ± 2.5% when tested at100 µM and 300 µM concentrations, respectively (l" Fig. 2C–D).With an IC50 value > 300 µM, piperine (4) was a weak hERG chan-nel blocker. The pronounced activity peak observed in the EtOAcextract (microfraction ten inhibited IhERG by 64.3 ± 7.3%) was dueto the high piperine content in the extract [28]. Piperine (4) hasbeen previously identified as a promising scaffold for novel GA-BAA receptor modulators with anticonvulsant and anxiolytic ac-tivity [27,29].The methanolic extract of the TCM herbal drug Coptidis rhizoma(C. chinensis) reduced IhERG by 31.7 ± 2.0%. By means of HPLC-based activity profiling of the crude extract, the hERG channelblocking constituents were tracked to known protoberberine al-kaloids. Dihydroberberinewas themost active, as it reduced IhERGat 100 µM by 30.1 ± 10.1%, while berberine at the same concen-tration was less active (inhibition of IhERG by 16.3 ± 2.0%) [30].While these values do not point towards a high-affinity block,possible effects on ventricular repolarization cannot be ruledout. Even relatively weak hERG in vitro inhibitors can produceclinically relevant QT prolongation if plasma levels are suffi-ciently high [31]. Moreover, if the metabolites are more lipophilicthan the administered compound, they could accumulate in thesystemic compartment more efficiently. This has been recentlydemonstrated for berberine and its main metabolite berberru-

Fig. 1 HPLC‑PDA analysis of (A) cinnamon (C. zey-lanicum) MeOH extract, (B) guarana (P. cupana)MeOH extract, and (C) nutmeg (M. fragrans) MeOHextract. Activity profiling of crude extracts (left col-umn) with HPLC chromatograms and correspond-ing activity profiles (% inhibition of IhERG) of time-based microfractions. HPLC fingerprints of tannin-depleted extracts (right column) are shown forcomparison purposes. Inhibition of IhERG by crudeextracts and tannin-depleted extracts (100 µg/mL)is indicated on the upper right side of each chro-matogram. UV traces were recorded at 254 nm. Forreference compound cisapride, the IC50 for hERGcurrent inhibition at a pulse frequency of 0.3 Hz was1.6 ± 0.4 µM (n = 3, Fig. 1S of the Supporting Infor-mation, see also [36] for comparison).

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bine formed by O-demethylation [32]. Also, biodistribution, me-tabolism, and accumulation in target organs may differ betweensingle-dose and chronic administration.To our knowledge, this is the first screening study evaluating thepotential of herbal drugs and plant-derived foods for in vitrohERG channel inhibition. Our data suggest that widely used Euro-pean medicinal plants, frequently consumed spices, vegetables,and fruits are associated with a low risk for hERG toxicity. How-ever, the case of Coptidis rhizoma emphasizes the need for amore extensive assessment of herbal remedies from other tradi-tional health systems (e.g., TCM, Ayurveda, Kampo, and Unani)that are increasingly used worldwide for therapeutic purposesand/or as nutritional supplements.

Materials and Methods!

General experimental proceduresAnalytical and semipreparative HPLC separations were carriedout with SunFire C18 columns (3 × 150mm i.d., 3.5 µm; 10 ×150mm i.d., 5 µm). ESI‑MS data were recorded on an Esquire3000 plus ion trap mass spectrometer (Bruker Daltonics) coupledvia a T-splitter (split ratio 1:5) to an Agilent 1100 system consist-ing of an autosampler, degasser, binary pump, column oven, andPDA detector. Semipreparative HPLC separations were performedon an Agilent 1100 system consisting of an autosampler, quater-nary pump with degasser module, column thermostat, and PDAdetector. Unless otherwise stated, H2O (solvent A) and MeCN(solvent B) were used as the mobile phase. The flow rate was0.5mL/min for LC‑PDA‑ESI‑MS, and 4mL/min for semiprepara-tive HPLC. Detection was at 254 nm, while PDA spectra weremeasured from 210 to 400 nm. Data acquisition and analysiswere performed using HyStar 3.2 software.

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Fig. 2 HPLC-based activity profiling of black pep-per (P. nigrum) fruit extracts. A EtOAc extract,B MeOH extract. HPLC chromatograms (254 nm)and corresponding activity profiles (% inhibition ofIhERG) of time-based microfractions are shown. C,D Concentration-dependent inhibition of IhERG bypiperine (4). The control current is superimposedwith the current traces recorded during a pulse trainof 1 Hz (15 pulses) in the presence of piperine(100 µM and 300 µM).

Fig. 3 Structures of compounds 1–4 tested for hERG channel inhibitoryactivity.

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Solvents and chemicalsSolvents used for extraction and polyamide filtration were of an-alytical grade, whereas HPLC-grade solvents were used for HPLCseparations. HPLC-grade water was obtained by an EASY-pure IIwater purification system. Formic acid (98.0–100.0%) and HPLC-grade MeCN were from Scharlau. Ellagic acid dihydrate (≥ 97% byHPLC), (+)-catechin (≥ 98% by HPLC), gallic acid (≥ 98.5% by GC),and piperine (≥ 97% by TLC) were purchased from Sigma-Aldrich.

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Plant materialThe origin of the plant species used in this study is listed in Tables1S–3S of the Supporting Information. Fresh plant material andfreshly squeezed grapefruit juice were shock frozen and lyophi-lized. Voucher specimens are preserved at the Division of Phar-maceutical Biology, University of Basel, Switzerland.

ExtractionPrior to extraction, the dried plant material was ground in a ZM 1ultra centrifugal mill (sieve size 2.0mm; Retsch). Unless other-wise stated, plant extracts were prepared by PLE using an ASE200 instrument connected to a solvent controller (all Dionex).Ground plant material was packed into steel cartridges (11mL;Dionex) and consecutively extracted with solvents of increasingpolarity (PE, EtOAc, and MeOH). The extraction temperature wasat 70°C, and the pressure was set at 120 bar. Duration of a staticextraction cyclewas 5min, and three extraction cycles were usedfor each solvent to obtain exhaustive extraction [33,34]. Plant-derived foods containing high amounts of sugars, proteins, or tri-glycerides were extracted by percolation at room temperature,using the same solvents as for PLE. The following edible plantswere extracted by percolation: Brassica nigra, Citrus paradisi,Coffea arabica, Euterpe oleracea, Ficus carica, Glycine max, Lyciumbarbarum, Malus domestica, Morinda citrifolia, M. fragrans, Opun-tia ficus-indica, Phaseolus vulgaris, Rheum rhabarbarum, Sinapisalba, Theobroma cacao, and Vaccinium macrocarpon. Extractswere dried under reduced pressure, and stored at 4°C until use.

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High-performance liquid chromatography-basedactivity profilingA validated HPLC-based profiling protocol for the identificationof hERG channel inhibitors in herbal extracts was used [30].Briefly, an aliquot (5mg) of crude extract (100 µL of 50mg/mL inDMSO) was separated by semipreparative RP-HPLC using H2O(solvent A) andMeCN (solvent B) as themobile phase, unless oth-erwise stated. The gradient profiles were as follows: MeOH ex-tract of P. cupana: 10–70% B in 20min, hold 70% B for 5min(l" Fig. 1B); MeOH extract of M. fragrans: 20–100% B in 30min,hold 100% B for 5min (l" Fig. 1C); EtOAc extract of P. nigrum:30–100% B in 30min, hold 100% B for 10min (l" Fig. 2A); MeOHextract of P. nigrum: 20–100% B in 30min, hold 100% B for 10min(l" Fig. 2B). The MeOH extract of C. zeylanicum was separatedwith 0.1% aqueous formic acid (solvent C) and MeCN containing0.1% formic acid (solvent D) using the following gradient: 5–70%D in 30min, hold 70% D for 5min (l" Fig. 1A). Time-based micro-fractions of 90 s each were manually collected into glass tubes.The evaporation of microfractions was achieved with an EZ-2Plus vacuum evaporator (Genevac). Prior to testing for hERGchannel inhibition, residues were re-dissolved in 30 µL of DMSOand diluted with 2.97mL of bath solution.

Polyamide solid phase extractionTo remove tannins frommethanolic extracts of cinnamon, guara-na, and nutmeg, the crude extracts were subjected to SPE onpolyamide. Polyamide (0.05–0.16mm, Carl Roth GmbH) was con-ditioned in MeOH for 24 h, and packed into glass columns of1.7 cm in diameter to give bed heights of approximately 6 cm. Aportion (100mg) of each extract was dissolved in MeOH (50mL),applied to the column, and eluted at a flow rate of 1mL/min. Col-umns were washed with 300mL of MeOH. For the crude cinna-mon extract, polyamide filtration was also performed usingMeOH/H2O 7:3 (v/v) as the eluent. The tannin-depleted effluentswere collected, evaporated to dryness, and analyzed along withthe untreated extracts.

Electrophysiological bioassay: expression of humanether-a-go-go-related gene channels in Xenopus oocytesand voltage-clamp experimentsOocyte preparation: Oocytes from the South African clawed frogX. laevis, were prepared as follows: After 15min exposure of fe-male X. laevis to the anesthetic (0.2% solution of MS-222; themethane sulfonate salt of 3-aminobenzoic acid ethyl ester;Sigma-Aldrich), parts of the ovary tissue were surgically re-moved. Defolliculation was achieved by enzymatical treatmentwith 2mg/mL collagenase type 1A (Sigma-Aldrich). Stage V–VIoocytes were selected and injected with the hERG-encodingcRNA. Injected oocytes were stored at 18°C in ND96 bath solu-tion containing a 1% penicillin-streptomycin solution (Sigma-Al-drich). The ND96 bath solution contained 96mMNaCl, 2mM KCl,1mM MgCl2 × 6H2O, 1.8mM CaCl2 × 2H2O, and 5mM HEPES(pH 7.4).Automated two-microelectrode voltage-clamp studies: Currentsthrough hERG channels were studied with the two-microelec-trode voltage-clamp technique using a TURBO TEC-03X amplifier(npi electronic GmbH). Electrophysiological experiments wereperformed one to three days after cRNA injection. Voltage-re-cording and current-injecting microelectrodes (Harvard Appara-tus) were filledwith 3MKCl and had resistances between 0.5 and2MΩ. Oocytes with maximal current amplitudes > 3 µAwere dis-carded to avoid voltage-clamp errors. The following voltage pro-

tocol was used: from a holding potential of − 80mV, the cellmembrane was initially depolarized to + 20mV (300ms) in orderto achieve channel activation and subsequent rapid inactivation.During the following repolarization to − 50mV (300ms), thechannels recover from inactivation and elicit IhERG. A final step tothe holding potential ensured that the channels returned to theclosed state. The protocol was applied either in 3-s intervals (ex-tract screening) or in 1-s intervals (bioactivity studies on frac-tions and pure compounds). Measurements were started afterthe initial current ”run up“ (slow increase of hERG current ampli-tudes during repetitive pulsing) reached a steady baseline. Theautomated fast perfusion system ScreeningTool (npi electronicGmbH, see [35] for details) was used to apply the test solutionsto the oocyte. Sample stock solutions (prepared in DMSO) werefreshly diluted every day with ND96 bath solution. The steady-state block of IhERG was evaluated at an ambient temperature(20–24°C). Decreases in tail current amplitudes were taken as ameasure of block development during repetitive pulsing. The fi-nal maximum DMSO concentration (1%) in the test solutions didnot affect the hERG currents (data not shown). Cisapride (Sigma-Aldrich; purity ≥ 98%) was used as a positive control. The IC50 val-ue for the hERG current inhibition at a pulse frequency of 0.3 Hzwas 1.6 ± 0.4 µM (n = 3, Fig. 1S of the Supporting Information, seealso [36] for comparison). Data acquisition and processing wereperformed using pCLAMP 10.0 software and Clampfit 10.2 soft-ware, respectively.

Data analysisInhibition of IhERG was defined as [1 – I(hERG,drug)/I(hERG,ctrl)] × 100,where I(hERG,drug) is the current amplitude in the presence of theindicated test material (extract, fraction, or pure compound) andI(hERG,ctrl) is the control current amplitude. Data were analyzedusing Origin software 7.0 (OriginLab Corporation). Data pointsrepresent the mean ± SE from at least two oocytes and twooocyte batches.

Supporting informationData on the hERG channel inhibitory activity of 18 European me-dicinal plants (Table 1S), 5 traditional Chinese herbal drugs (Ta-ble 2S), and 56 food plants and spices (Table 3S) are available asSupporting Information.

Acknowledgments!

This project was conducted within the International ResearchStaff Exchange Scheme (IRSES), project “hERG Related Risk As-sessment of Botanicals” PIRSES‑GA-2011–295174 Marie CurieActions funded under the 7th Framework Programme of theEuropean Commission. Financial support by the Swiss NationalScience Foundation (Project 31600–113109) is gratefully ac-knowledged (M.H.). We thank the Freiwillige Akademische Ge-sellschaft Basel (A.S.) and StudEx (Leonardo da Vinci Programm)(E.A. J.) for research scholarships.

Conflict of Interest!

The authors declare no conflict of interest.

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