Food Chemistry 134 (2012) 1096–1105 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Metabolic pathways of the psychotropic-carboline alkaloids, harmaline and harmine, by liquid chromatography/mass spectrometry and NMR spectroscopy Ting Zhao a, Shan-Song Zheng a, Bin-Feng Zhang a,b,c, Yuan-Yuan Li a, S.W. Annie Bligh d, ⇑ ⇑ Chang-Hong Wang a,b,c, , Zheng-Tao Wang a,b,c, a Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201210, China b The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, 1200 Cailun Road, Shanghai 201210, China c Shanghai R&D Center for Standardization of Chinese Medicines, 199 Guoshoujing Road, Shanghai 201210, China d Institute for Health Research and Policy, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK article info abstract Article history: The b-carboline alkaloids, harmaline and harmine, are present in hallucinogenic plants Ayahuasca and Received 3 June 2011 Peganum harmala, and in a variety of foods. In order to establish the metabolic pathway and bioactivities Received in revised form 25 January 2012 of endogenous and xenobiotic bioactive b-carbolines, high-performance liquid chromatography, coupled Accepted 6 March 2012 with mass spectrometry, was used to identify these metabolites in human liver microsomes (HLMs) Available online 16 March 2012 in vitro and in rat urine and bile samples after oral administration of the alkaloids. Three metabolites of harmaline and two of harmine were found in the HLMs. Nine metabolites for harmaline and seven Keywords: metabolites for harmine, from the rat urine and bile samples, were identified. Among them, four Harmaline in vivo metabolites were isolated and fully characterised by NMR analysis. For the first time, harmaline Harmine Metabolites is shown transforming to harmine by oxidative dehydrogenation in rat. Five metabolic pathways were b-Carboline alkaloids therefore proposed, namely, oxidative dehydrogenation, 7-O-demethylation, hydroxylation, O-glucuro- nide conjugation and O-sulphate conjugation. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction such as harmane, harmine, harmaline and ibogaine can also be found in common plant-derived foodstuffs (e.g. wheat, rice, corn, The b-carboline alkaloids, harmaline and harmine, are the main barely, soy, beans, rye, grapes, mushrooms and vinegar), well- active components of the hallucinogenic plant, Ayahuasca, which is cooked meat, plant-derived beverages (e.g. wine, beer, whisky, used as an ingredient of the popular sacred and psychoactive brandy and sake), and plant-derived inhaled substances (e.g. to- drinks in South American Indian cultures. It is also known as Caapi, bacco) (Agüía, Peña-Farfala, Yáñez-Sedeño, & Pingarróna, 2007; Pinde, Natema or Yaje, which is widely used for prophecy, divina- Alves, Mendes, Oliveira, & Casal, 2010; Conning, 1989; Crotti, tion, and as a sacrament in the northern part of South America Gates, Lopes, & Lopes, 2010; Derakhshanfar, Oloumi, & Mirzaie, (Samoylenko et al., 2010). The b-carboline alkaloids also present 2010; Guan, Louis, & Zheng, 2001; Herraiz, Guillén, & Arán, in Peganum harmala, which has traditionally been used for ritual 2008). Because of their natural presence in the food chain and envi- and medicinal preparations in the Middle East, central Asia, and ronment, there appears to be a risk associated with extra exposure South America. According to previous reports, the entire plant to these b-carbolines from dietary sources, smoking, and consump- and seeds of P. harmala have been separately used to treat diseases tion of alcoholic beverages. In fact, the occurrence of b-carbolines such as cough, asthma, rheumatoid arthritis and swelling pain in human blood and excreta, beef and sardines under normal phys- (Cheng et al., 2010; Hemmateenejad, Abbaspour, Maghamia, Miri, iological conditions has been reported in the literature, which & Panjehshahin, 2006; Kartal, Altun, & Kurucu, 2003; Samoylenko implies that b-carboline alkaloids may be present in biological sys- et al., 2010; Sourkes, 1999; Zheng et al., 2009). The b-carbolines, tems (Guan et al., 2001; Herath, Mikell, Ferreira, & Khan, 2003; Riba et al., 2003). The b-carboline alkaloids have been of interest due to their ⇑ Corresponding authors. Address: The MOE Key Laboratory for Standardization psychotropic properties. These compounds affect the content of of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, 1200 neurotransmitters by strong reversible inhibition of monoamine Cailun Road, Zhangjiang Hi-Tech Park, Shanghai 201210, China. Tel.: +86 21 oxidase (Kim, Sablin, & Ramsay, 1997; Schwarz, Houghton, Rose, 51322511; fax: +86 21 51322519. E-mail addresses: [email protected] (C.-H. Wang), [email protected] Jenner, & Lees, 2003) and the inhibition of acetylcholinesterase (Z.-T. Wang). (Zheng et al., 2009, 2011). They also exhibit some pharmacological 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.03.024 T. Zhao et al. / Food Chemistry 134 (2012) 1096–1105 1097 actions, such as anti-tumour and analgesic effects (Farouk, Laroubi, donors (Mongolian). Reverse-phase silica gel (C18) and MCI Gel Aboufatima, Benharref, & Chait, 2008; Jahaniani, Ebrahimi, CHP20P(75–150 lm) for column chromatography were purchased Rahbar-Roshandel, & Mahmoudian, 2005; Wang, Liu, & Zheng, from Mitsubishi (Tokyo, Japan). The MG II C18 column (75 mm  2002), vasorelaxant activities (Astulla et al., 2008), and antimicro- 2.0 mm, i.d. 3 lm) was purchased from Shiseido (Tokyo, Japan). bial properties (Arshad, Neubauer, Hasnain, & Hess, 2008). Hence, DMSO-d6 was supplied by Armar Chemicals (Switzerland). b-carboline alkaloids have potential in the treatment of psychiatric A surveyor HPLC system (Thermo-Finnigan, San Jose, CA, USA) disorders and diseases. However, these compounds have some was used for sample separation. A Finnigan LCQ DECA XP, plus toxic effects especially in the central nervous system. For instance, ion-trap mass spectrometer equipped with an electrospray the structures of the N-methylated b-carboline alkaloids resemble ionisation (ESI) source, was used for mass analysis and detection N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and partic- (Thermo-Finnigan, San Jose, CA, USA). High resolution mass spec- ularly its neurotoxic metabolite, N-methyl-4-phenylpyridinium trometry (HRMS) was recorded using an ion mobility spectrometry (MPP+), may act as an endogenous or environmental neurotoxic (IMS)-mass spectrometry (MS) instrument (SYNAPT G2, Waters agent (Cobuzzi, Neafsey, & Collins, 1994). It was also found that Technologies, Milford, USA). A Bruker AV 400 MHz NMR spectrom- b-carboline alkaloids were co-mutagens in the presence of aro- eter (Faellanden, Switzerland) was used to record 1HNMR matic amines such as aniline, toluidine and inhibitors of mutagens (400 MHz), 13C NMR (100 MHz), HMBC and HSQC spectra. and carcinogens in mutagenic assays (Herraiz et al., 2008). The metabolic outcome and potential bio-activation of endoge- 2.2. Microsomal incubation with NADPH nous and xenobiotic bioactive b-carbolines are matters of research interest. However, not much is currently known about their Each incubation was performed in a 100 mM phosphate buffer metabolism. A previous study found that the main metabolic path- at pH 7.4 containing HLMs (final protein concentrations were ways of harmaline and harmine were O-demethylation mediated 1 mg/ml), the NADPH-generating system contained 10 mM glucose by CYP 2D6, CYP1A2 and CYP2C9, and hydroxylation mediated 6-phosphate, 1 mM NADP+, 4 mM magnesium chloride, and 1 unit/ by CYP1A2 and CYP1A1 (Yu, Idle, Krausz, Küpfer, & Gonzalez, ml of glucose 6-phosphate dehydrogenase, and various alkaloids 2003). Both in vivo and in vitro studies have revealed that the major (harmaline, harmine, harmalol, harmol) which were previously fate of harmaline and harmine was demethylation to form harma- dissolved in methanol (final methanol concentration was 1%, v/v) lol and harmol, respectively, which were subsequently excreted as with concentration 200 lM in a total volume of 100 ll. The reac- the glucuronide conjugate and sulphate conjugate (Ho, Estevez, tion system was incubated for 3 min at 37 °C before the reaction Fritchie, & Tansey, 1971; Mulder & Hagedoorn, 1974; Slotkin & was initiated by the addition of NADP+, and the incubation contin- DiStefano, 1970a, 1970b; Slotkin, DiStefano, & Au, 1970). Two ued at 37 °C for 60 min. The reaction was quenched by adding the hydroxylated metabolites were also found in human or mouse liver same volume of ice-cold acetonitrile and the reaction system was microsomes (Tweedie & Burke, 1987; Yu et al., 2003), but the posi- placed in an ice bath for 30 min. The incubation mixtures were tion of the hydroxyl group could not be definitively assigned. These then centrifuged for 10 min at 20,000g and an aliquot of the super- results are of further interest because of the biological features of natant was injected for LC-MSn analysis. these xenobiotic and endogenous bioactive alkaloids and their potential interactions with mutagens and carcinogens. Unambiguous identification of metabolites can usually only be 2.3. Microsomal incubation with UDPGA accomplished by spectroscopic characterisation, especially by MS and