**5. Conclusions**

In sum, the results point out that uridine 5- -diphospho-glucuronosyltransferasecatalyzed conjugation reactions play a crucial role in phase II metabolism of asarone derivatives. This study confirms *O*-demethylation as an important metabolism pathway in humans for the first time. A fast renal excretion within 24 h is observed after calamus tea consumption. The recovery rate of only 42% emphasize that further work is still needed to characterize yet unknown human bA-related metabolites. A metabolization of bA via the epoxide-diol-pathway is suggested and should be considered for human risk assessment.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/foods10092032/s1, Figure S1: HPLC-qTOF-MS chromatogram of a selected urine sample after intake of fresh prepared bA-containing calamus tea, Table S1: Substances and concentrations used for phase II sulfonation experiments, Table S2: HPLC-MS/MS setup for the quantitation of *threo*- and *erythro*-asarone diols in urine samples, Table S3: HPLC-qTOF-MS setup for the screening of liver microsome samples for phase-II-metabolites originating from beta-asarone epoxide (bAE) and 3- hydroxyasarone (3- OH), Table S4: Characterization of unknown metabolites: Differences to HPLC-qTOF-MS setup presented in Table S2.

**Author Contributions:** Conceptualization, M.E.; methodology, L.H. and B.C.; investigation, L.H. and J.R.; data curation, L.H.; writing—original draft preparation, L.H.; writing—review and editing, B.C. and M.E.; visualization, B.C. and M.E.; supervision, M.E.; funding acquisition, M.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by German Federal Ministry of Education and Research, grant number 01FP13061F.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of University Hospital Münster, Germany (protocol code 2020-002-f-S, date of approval: 17 February 2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained within the article or Supplementary Materials.

**Acknowledgments:** We would like to thank Alexander Cartus and Simone Stegmüller for isolating and providing beta-asarone and beat-asarone epoxide.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

