**1. Introduction**

Alpha-asarone (aA) and beta-asarone (bA) are phenylpropanoids, whose chemical structures only differ in the conformation of the double bound of the respective C3 side chain. These compounds mainly occur in essential oils of *Acorus* species, which are widely disseminated in Europe, North America and East Asia [1,2]. The predominant class is *Acorus (A.) calamus* L. Nevertheless, asarone contents strongly vary depending on several factors such as country of origin, polyploidy and plant materials e.g., roots or leaves [3]. In Indian tetraploid varieties, bA is present at high amounts of up to 95%, whereas aA amounts are on average about 15% [4].

Besides flavoring properties and bioactivity, essential oils as well as dried rhizomes or leaves of calamus are mainly used in herbal teas, frozen desserts, yoghurts, alcoholic and non-alcoholic beverages or food supplements [5]. Traditional phytomedicine and cosmetics also represent significant human exposure routes [2,4,5]. Moreover, asarone isomers and calamus-derived preparations are considered to show positive effects on human health such as antioxidant, anti-inflammatory, antidepressant, anti-microbial, neuro-, chemo- and radioprotective properties that facilitate their use as phytopharmaceutical summarized in Das et al. [6] and Chellian et al. [7]. Besides all positive effects, these substances are also of toxicological concern. Among acute and chronic toxicity, hepatotoxic properties in rodents as well as cytotoxic, genotoxic and mutagenic effects in vitro are reported for

**Citation:** Hermes, L.; Römermann, J.; Cramer, B.; Esselen, M. Phase II Metabolism of Asarone Isomers In Vitro and in Humans Using HPLC-MS/MS and HPLC-qToF/MS. *Foods* **2021**, *10*, 2032. https://doi.org/ 10.3390/foods10092032

Academic Editors: Andreas Eisenreich and Bernd Schaefer

Received: 14 July 2021 Accepted: 26 August 2021 Published: 29 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

parent compounds and several oxidative phase I metabolites [7–11]. Furthermore, DNA repair mechanisms are also activated in response to asarone-mediated genotoxic effects in cells [10].

Phase I metabolism of aA and bA is elucidated in liver microsomes of different species. The main metabolite of aA is reported as 3- -oxoasarone, which arises out of (*E*)-3- -hydroxyasarone (3- OH) via further oxidation steps, whereas bA is mainly metabolized via an epoxide intermediate to *erythro*- and *threo*-1- ,2- -dihydroxyasarone (*erythro*- and *threo*-asarone diols) and 2,4,5 trimethoxy-phenylacetone (asarone ketone) [12–14]. Even though in vitro phase I metabolism has been deeply characterised in the literature, little is known so far about potential phase II conjugation of these compounds. Nonetheless, the intake of *A. calamus* oil preparations in a human intervention trial proposes a renal hydroxylated bA metabolite after glucuronidase treatment, emphasizing glucuronic acid conjugation as possible phase II pathway [15]. This metabolite is also observed in urine of rats after an intraperitoneal application of aA [16].

Currently, there is no human data available regarding toxicokinetic parameters. In studies with rodents, for aA, bioavailability of 34% up to 78% is reported, which is further enhanced by inhalation [7]. Plasma half-life times of 29 min for aA and 13 min for bA are found after intravenous treatment of rats [17]. Depending on application form, longer half-life periods for oral and inhalative aA application, with values of 65 min up to 95 min, are reported [17,18]. In blood serum of rats, the half-life value of approximately 54 min indicate that bA is rapidly excreted [19]. Furthermore, marginal oral bioavailability has been considered, due the hydrophilic character of the asarone isomers, summarized in Chellian et al. [7]. Nevertheless, both isomers are shown to cross the blood brain barrier [18,20].

So far, in the European Union a maximum level of 1 mg/kg for bA is defined for alcoholic beverages, and for food a recommended maximum of 0.1 mg/kg is given [5,21]. For calamus preparations, it is recommended to use diploid varieties, which contain almost no or little bA amounts. For herbal medicine products, an intake of 2 μg/kg body weight/day is temporarily acceptable [5,22]. aA is not further regulated by law in the European Union, while bA is classified as a genotoxic carcinogen, thus the margin of exposure approach is used for risk assessment and a value below 10,000 is considered a high priority [23]. Toxicokinetic and toxicodynamic data of the genotoxin bA have to be urgently improved for adequate risk evaluation.

In consideration of the limited metabolism data of asarone isomers, in this study microsomal phase II metabolites were characterized using the respective phase I metabolites. Moreover, a human study was performed to identify main renal phase II metabolites. Ten participants consumed a commercially available calamus-containing infusion and gave urine samples over a period of 48 h. Furthermore, the study provided new insights into the excretion kinetic over the observation period and the overall rate of excretion, which was calculated considering the ingested asarone amounts. A high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-qTOF-MS) approach was used to identify new metabolites in microsome samples and in human urine. Kinetic and excretion data were generated using validated high-pressure liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) detection for quantitation.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

All solvents used for sample dilution or chromatography were of LC-MS-grade and purchased from Carl Roth (Karlsruhe, Germany), Fisher Scientific (Schwerte, Germany) or Sigma-Aldrich (Steinheim, Germany), if not stated otherwise. Water was purified using a Purelab Flex 2 system (Veolia Water Technologies, Celle, Germany). Formic acid (FA), and magnesium chloride (MgCl2) were ordered from Merck (Darmstadt, Germany), tris(hydroxymethyl)aminomethane (TRIS) from VWR (Darmstadt, Germany) and ammonium hydrogen carbonate from Carl Roth (Karlsruhe, Germany). Uridine-5- -diphosphate glucuronic acid (UDPGA), glucose-6-phosphate-dehydrogenase (G6P-DH) and NADP+ were purchased from Sigma-Aldrich and *E*. *coli* beta-glucuronidase from Romer Labs GmbH (Butzbach, Germany). G6P was from AppliChem (Darmstadt, Germany).

Microsomes from the species pig and horse were isolated according to the protocol of LAKE 1987 and stored at −<sup>80</sup> ◦C in storage buffer [24]. Human microsomes (Corning® UltraPool™, Corning, NY, USA) were purchased from Corning Inc. (Durham, NC, USA).

bA, 3- OH and (Z)-asarone-1- ,2- -epoxide (bAE) were kindly provided by our project partner from the University of Kaiserslautern (Germany). *Threo*- and *erythro*-asarone diols were isolated from a decomposed epoxide solution. Further information can be found in the literature [10]. Additionally, 7- -hydroxycoumarin and 4-methylumbelliferyl-β-D-glucuronide were purchased from Sigma-Aldrich (Steinheim, Germany). All analyte solutions were prepared in acetonitrile and stored at −20 ◦C.
