bChinese Materia Medica (CMM) is a general termcovering medicines, mostly of botanical origin,prescribed in accordance with the principles andtheories of traditional Chinese medicine (TCM)[1,2]. In recent years, the use of CMM has enor-mously increased worldwide, and so has the in-ternational attention regarding its safe and effec-tive use [2–4]. Similarities in morphology and no-menclature can lead tomisidentification and con-fusion [2,5], which may result in poisoning inci-dents. A case in point has been the aristolochic ac-id (AA) nephropathy caused by a slimming teawhich contained Aristolochia due to mistakenidentity of CMM, and consequent misuse of Aris-tolochia species in some herbal preparations [2,5–8]. Aristolochia species (Aristolochiaceae) havebeen used in TCM as anti-inflammatory herbs.However, their use is no longer permitted inmany countries due to their content of AA shownto be nephrotoxic, carcinogenic, and mutagenic[2,6].In recent years we have been successfully identi-fying new structural scaffolds for GABAA receptormodulators [9–11] by an extract library-based ap-proach and HPLC-based activity profiling [12,13],

using an automated functional two–microelec-trode voltage clamp assay with Xenopus laevis oo-cytes that transiently express the GABAA receptorsubtype !1"2#2s [14]. One of the active samples inthe library was a petroleum ether extract of acommercial sample labelled as “Chaihu” (poten-tiation of IGABA by 156% ± 22% when tested at100 µg/mL). The Chinese herbal drug Chaihu (Bu-pleuri Radix) refers to the dried roots of Bupleu-rum chinense DC. (Bei-chaihu) or B. scorzonerifo-lium Willd. (Nan-chaihu) (Apiaceae) [15,16]. It isawell-knownTCM drug claimed to posses, amongothers, sedative properties [17]. It is widely usedin TCM as the principal ingredient of many multi-herb remedies due to its broad spectrum of tradi-tional uses [16,18].By means of HPLC-based activity profiling [13], amajor peak of activity could be located in fraction11 (potentiation of IGABA by 212% ± 14%)(l" Fig. 1). We isolated the peak contained in thisfraction by semipreparative HPLC. A molecularformula C15H20O2 of compound 1 was derivedfrom the accurate mass obtained by LC-TOFMSanalysis. A database search with this molecularformula gave no entries for B. chinense [19]. For

Abstract!

In a two–microelectrode voltage clamp assay us-ing Xenopus laevis oocytes, a petroleum ether ex-tract prepared from a commercial sample of thetraditional Chinese herbal drug labelled as “Chai-hu” (Bupleurum chinense DC. roots) enhanced theIGABA by 156% ± 22%when tested at 100 µg/mL. Bymeans of HPLC-based activity profiling combinedwith high-resolution LC‑MS and microprobeNMR, the germacranolide aristolactone (1) wasidentified as one of the main active compounds(EC50 56.02 µM± 5.09 µM). However, aristolac-tone has been previously reported only from thegenus Aristolochia (Aristolochiaceae), suggesting

a possible adulteration.With the aid of a validatedHPTLC protocol for detection of aristolochic acidsand with reference samples, the commercial sam-ple was confirmed to be a mixture of Aristolochiamanshuriensis root and Bupleurum chinense root.This finding was corroborated by macroscopic in-spection of the drug. This case of adulterationwith a highly nephrotoxic drug raises concernsabout adequate quality control of TCM drugscommercialized in Europe.

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Discovery of GABAA Receptor Modulator Aristolactonein a Commercial Sample of the Chinese HerbalDrug “Chaihu” (Bupleurum chinense Roots) UnravelsAdulteration by Nephrotoxic Aristolochia manshuriensisRoots

Authors Diana C. Rueda1, Janine Zaugg1, Melanie Quitschau1, Eike Reich2, Steffen Hering3, Matthias Hamburger1

Affiliations 1 Division of Pharmaceutical Biology, University of Basel, Basel, Switzerland2 CAMAG Laboratory, Muttenz, Switzerland3 Institute of Pharmacology and Toxicology, University of Vienna, Vienna, Austria

Key wordsl" Bupleurum chinensel" Apiaceael" Aristolochia sp.l" Aristolochiaceael" aristolactonel" aristolochic acidsl" traditional Chinese medicinel" adulteration

received August 24, 2011revised Dec. 5, 2011accepted Dec. 18, 2011

BibliographyDOI http://dx.doi.org/10.1055/s-0031-1298171Published online January 23,2012Planta Med 2012; 78: 207–210© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

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

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bthe entire genus Bupleurum, there was only one entry with thismolecular formula, corresponding to saikodiyne A. However, the1H NMR and 2DNMR spectra did not match with this polyacety-lenic compound. Detailed analysis of the spectra finally led toidentification of 1 as aristolactone (l" Fig. 2A; spectroscopic datain Supporting Information). Concentration dependent GABAA re-ceptor modulation of 1was tested in the oocyte assay, at concen-trations between 0.1 and 1000 µM. Aristolactone (1) potentiatedIGABA in a concentration-dependent manner, with an EC50 of56.0 µM± 5.1 µM and a maximum potentiation of 70.7% ± 2.6%(l" Fig. 2B). No significant direct activation of GABAA receptorswas observed (l" Fig. 2C), but concentrations lower than 100 µMinduced inhibition of IGABA. This may indicate a high potency neg-ative modulation binding site interfering with a low potency pos-itive modulation, since concentrations in the range of 100 µM to1mM induced moderate enhancement of IGABA (l" Fig. 2B and2C).Aristolactone is a characteristic marker for the genus Aristolochia.Due to its unexpected presence in the extract, we suspected adul-teration of B. chinense roots with an Aristolochia species. There-fore, we subjected the commercial herb sample to HPTLC analysisusing a validated protocol for detection of AA [20]. The presenceof AAwas confirmedwith the aid of reference solutions, whereasan authentic sample of Bupleurum chinense roots was devoid ofAA (l" Fig. 3). Further HPTLC fingerprint analyses and macro-scopic inspection revealed that the sample was a mixture (ap-

prox. 1 :1) of B. chinense roots and A. manshuriensis roots (Fig. 2S,Supporting Information).The case described here highlights an apparent gap in appropri-ate control of TCM herbal drugs imported in Europe. In principle,wholesale distributors importing bulk herbal drugs are legallybound to ensure proper quality control measures using the re-spective pharmacopoeia monographs. Massive adulteration of awidely used and safe drug, Bupleuri radix (Chaihu), with highlytoxic AA-containing Aristolochia species represents a serious riskfor consumer safety [5]. It is worrying to see that almost two dec-ades after the discovery of AA nephropathy [7,8], AA-containingdrugs and herbal products are still on the market across theworld [21,22], and that certain populations are at a measurablerisk of getting AA-related cancer [23]. At this point we can onlyspeculate about the reasons for this case of adulteration. Howev-er, root samples often possess few distinctive macroscopic fea-tures, especially in their commercial form of cut drugs, and thisfact lends to either accidental or intentional adulteration. Therather similar visual appearance of the two drugs in questionvisibly led to a situation which was only uncovered by chance.Thus, regulatory bodies should be aware of the specific issues re-lated to TCM herbal drugs, so that proper quality and safety canbe ensured. For authentication of TCM herbs, a broad range ofmethods is available, such as chromatographic and spectroscopictechniques [24,25], as well as DNA fingerprinting and genomicapproaches [26,27]. Yet, methods such as HPTLC fingerprintingand macroscopic and microscopic inspection [2,28] remain im-

Fig. 2 A Chemical structure of compound 1. B Concentration-responsecurve for 1 on GABAA receptors of the subunit composition !1"2#2S, using aGABA EC5–10. C Typical traces for modulation of IGABA for compound 1. Theflat segments in the currents indicate the absence of direct activation ofthe receptors.

Fig. 1 HPLC-based activity profiling of a petroleum ether extract of acommercial sample labelled as B. chinense DC. roots, for GABAA receptormodulatory activity. B HPLC chromatogram (210 nm) of a semipreparativeseparation of 10mg of extract. The 27 time-based fractions collected, 90 seach, are indicated with dashed lines. A Potentiation of the IGABA by eachfraction.

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bportant. The specific case discussed here highlights the high in-formation content of HPTLC fingerprints.

Materials and Methods!

Aristolochia reference samples were provided and identified bythe European Directorate for the Quality of Medicines & Health-Care (EDQM), and Bupleurum reference samples by the AmericanHerbal Pharmacopoeia. The herbal drug labelled as “Chaihu”(dried roots of Bupleurum chinense DC.) was purchased in 2002from the Institute of Chinese Medicine, PeterWeinfurth, Ennepe-tal, Germany. Prior to extraction for the extract library in 2002,the drug was submitted to macroscopic and microscopic analysisin comparison with the respective monograph of the ChinesePharmacopoeia [29]. A voucher specimen (00 363) is depositedat the herbarium of the Division of Pharmaceutical Biology, Uni-versity of Basel.Microfractionation of the extract for activity profiling was per-formed as previously described [11,13], with minor modifica-tions (see Supporting Information). A total of 27 time-based mi-crofractions of 90 s each were collected for assessment of GABAAreceptor modulatory activity, as previously described [10,11,14].Details of the bioassay are given in the Supporting Information.The peak in the active time window was isolated by means ofsemipreparative HPLC. LC‑PDA-ESITOFMS and microprobe NMRwere used for structure elucidation. For details, see SupportingInformation.Detection of AA in the commercial sample was performed byHPTLC, according to the PhEur Test for Aristolochic Acids in HerbalDrugs (2.8.21), method A [30]. HPTLC fingerprint analyses wereperformed as previously described [20], with minor modifica-tions specified in the Supporting Information. Macroscopic in-spection was carried to complement the information about thecomposition and purity of the sample obtained from HPTLC anal-

ysis. By visual comparison to an authentic reference drug, 60% ofthe sample was found to be different or questionable.

Supporting informationDetailed description of the experimental conditions, NMR spec-troscopic data of aristolactone (1), and HPTLC evidence of adul-teration of the commercial sample of B. chinense roots withA. manshuriensis roots are available as Supporting Information.

Acknowledgements!

Financial support was provided by the Swiss National ScienceFoundation through project 205320_126888/1 (MH). D.C. Ruedathanks the Swiss Federal Commission for Scholarships for ForeignStudents (FCS) for a fellowship. We are grateful to Eliezer Cenivi-va and Valeria Widmer, both from CAMAG Laboratory, for per-forming the macroscopic and HPTLC analysis.

Conflict of Interest!

The authors have declared no conflict of interest.

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