A novel concept for detoxification: Complexation between aconitine and liquiritin in a Chinese herbal formula (‘Sini Tang’) Karoline Peter a,1 , Johann Schinnerl b,1 , Susanne Felsinger c , Lothar Brecker c,n , Rudolf Bauer d , Heimo Breiteneder a , Riurong Xu e , Yan Ma a,nn a Molecular Research in Traditional Chinese Medicine Group, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria b Chemodiversity Research Group, Department of Systematic and Evolutionary Botany, Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria c Institute of Organic Chemistry, University of Vienna, Waehringerstrasse 38, A-1090 Vienna, Austria d Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 4/I, A-8010 Graz, Austria e Affiliated Hospital of Shandong University of Traditional Chinese Medicine, 42 Wenhuaxi Road, Jinan, Shandong, China article info Article history: Received 9 April 2013 Received in revised form 15 July 2013 Accepted 16 July 2013 Available online 24 July 2013 Keywords: Sini Tang Decoction Aconitine Liquiritin Herbal interaction Complex formation abstract Ethnopharmacological relevance: Sini Tang (SNT, ) is a traditional Chinese herbal formulation consisting of three different herbs: Aconitum carmichaelii (Fuzi, ), Zingiber officinale (Ganjiang, ), and Glycyrrhiza uralensis (Gancao, ). For this study, we modified this mixture by adding the bark of Cinnamomum cassia (Rougui, ). Aconitum carmichaelii contains aconitine and its derivatives, all of which are highly toxic alkaloids. These compounds are commonly detoxified with pyrolytic and hydrolytic pretreatments, such as Heishunpian ( ), which requires repeated soaking in salt water, boiling until the roots turn black, and drying in the oven. We now demonstrate that Glycyrrhiza uralensis, which is often used in Traditional Chinese Medicine for detoxification, reduces the concentration of free aconitine in decoctions by forming a complex between liquiritin and aconitine. Materials and methods: Aqueous extracts of SNT, each individual herb or herbal mixture, and methanolic extracts of individual herbs were tested for free aconitine by HPLC coupled with a diode array detector. A detected complex was investigated by NMR and UV/vis spectroscopy. The continuous variations method and 1 H-NMR titrations provided the complex stoichiometry and binding constant. A 2D-ROESY experiment was performed to obtain the structural details of the formed complex. Results: A fast and simple HPLC method was developed to determine the amounts of aconitine and its derivatives found in herbal extracts. The Heishunpian pretreatment led to nearly complete pyrolysis and hydrolysis of the toxic compounds. However, in some batches, considerable amounts of aconitine remained. The addition of Glycyrrhiza uralensis to Aconitum carmichaelii, or liquiritin to free aconitine, led to a complexation with aconitine. The complex possessed a 1:1 stoichiometry and a binding constant of ca. 3000 L/mol to 4000 L/mol in mixtures of aqueous methanol. Conclusions: A new HPLC based method allows the concentration of toxic aconitine and other diester diterpene alkaloids in herbal extracts to be rapidly determined. This method provides a starting point for the development of routine quality control procedures. The complexation of free aconitine by adding an excess of Glycyrrhiza uralensis or free liquiritin to SNT formulations will make these formulations safer. & 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Traditional Chinese medicine (TCM) is an empirical healthcare system based on several millennia of treatment. For well-specified diseases, single herbs are usually prescribed. In contrast, formulas containing several herbs are required when treating complicated or chronic diseases to obtain maximum benefits and minimise side effects (Chen et al., 2004). “Sini Tang” (SNT) is a widely used Chinese herbal formula that has traditional use dating back to 150–219 A.D.; this formulation is a treatment for cardiovascular diseases and is officially recorded in China Pharmacopoeia Committee (2010). The SNT decoction is an aqueous extract of the roots of Aconitum carmichaelii Debeaux (aconite), the rhizome of Zingiber officinale Roscoe (ginger), and the roots of Glycyrrhiza Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.07.022 n Corresponding author. Tel.: +43 1 4277 52131; fax: +43 1 4277 9521. nn Corresponding author. Tel.: +43 1 40400 5784; fax: +43 1 40400 5130. E-mail addresses: lothar.brecker@univie.ac.at (L. Brecker), yan.ma@meduniwien.ac.at (Y. Ma). 1 These two authors contributed equally to the work. Journal of Ethnopharmacology 149 (2013) 562–569