Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug 29;10(9):2032.
doi: 10.3390/foods10092032.

Phase II Metabolism of Asarone Isomers In Vitro and in Humans Using HPLC-MS/MS and HPLC-qToF/MS

Affiliations

Phase II Metabolism of Asarone Isomers In Vitro and in Humans Using HPLC-MS/MS and HPLC-qToF/MS

Lena Hermes et al. Foods. .

Abstract

(1) Background: Metabolism data of asarone isomers, in particular phase II, in vitro and in humans is limited so far. For the first time, phase II metabolites of asarone isomers were characterized and human kinetic as well as excretion data after oral intake of asarone-containing tea infusion was determined. (2) Methods: A high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-qTOF-MS) approach was used to identify phase II metabolites using liver microsomes of different species and in human urine samples. For quantitation of the respective glucuronides, a beta-glucuronidase treatment was performed prior to analysis via high pressure liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). (3) Results: Ingested beta-asarone and erythro and threo-asarone diols were excreted as diols and respective diol glucuronide conjugates within 24 h. An excretion rate about 42% was estimated. O-Demethylation of beta-asarone was also indicated as a human metabolic pathway because a corresponding glucuronic acid conjugate was suggested. (4) Conclusions: Already reported O-demethylation and epoxide-derived diols formation in phase I metabolism of beta-asarone in vitro was verified in humans and glucuronidation was characterized as main conjugation reaction. The excretion rate of 42% as erythro and threo-asarone diols and respective asarone diol glucuronides suggests that epoxide formation is a key step in beta-asarone metabolism, but further, as yet unknown metabolites should also be taken into consideration.

Keywords: asarone glucuronides; asarone isomers; human study; metabolites.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Study design including wash-out phase, diary keeping and urine collection after intake of 300 mL calamus tea infusion. Urine samples were collected for 48 h and total urine volume was determined. The participants collected a spot urine sample every time they urinated.
Figure 2
Figure 2
HPLC-qTOF-MS chromatograms after incubation of (a) 3′OH and (b) bAE in pig liver microsomes. Presented are the extracted-ion chromatogram (XICs) with the calculated mass of (a) m/z 399.1305 ± 0.01 for the 3′OH glucuronide and (b) m/z 417.1397 ± 0.01 for erythro- and threo-asarone diols-derived glucuronic acid conjugates. (c) HPLC-qTOF-MS spectrum of 3′OH glucuronide (m/z 399.1305 ± 0.01) with the respective structural formula and the suggested cleavage of the glucuronic acid majority to m/z 223.0984.
Figure 3
Figure 3
(a) Structural illustration of erythro- and threo-asarone diols and their stereochemistry. (b) HPLC-MS/MS chromatogram of a 1:10 diluted urine sample spiked with 5 ng/mL of erythro- and threo-asarone diols. Presented are the quantifier (m/z 225193) and qualifier (m/z 225167) SRM transition.
Figure 4
Figure 4
HPLC-MS/MS chromatogram of a randomly selected urine sample, which was given after consumption of a calamus tea infusion, (a) before; (b) after treatment with beta-glucuronidase.
Figure 5
Figure 5
Exemplary HPLC-qTOF-MS chromatograms of a randomly selected urine sample before enzyme treatment. Presented are the extracted-ion chromatogram (XICs) with the calculated mass for (a) erythro- and threo-asarone diol glucuronides (diol-glucuronides, m/z 417.1404 ± 0.02) and (b) demethylated erythro- and threo-asarone diol glucuronides (demethylated diol-glucuronides, m/z 403.1247 ± 0.01). (c) HPLC-qTOF-MS spectrum of the O-demethylated erythro- and threo-asarone diol glucuronides (m/z 403.1247 ± 0.01) with the respective structural formula. The fragment m/z 227.0923 ± 0.02 corresponds to the O-demethylated metabolites after glucuronic acid cleavage. The loss of a further methyl group is shown by the exact mass of m/z 212.0685 ± 0.01.
Figure 6
Figure 6
Erythro- and threo-asarone diols (diols) excretion kinetic [µg] of ten participants over a period of 48 h. Excretion is classified in two-hour blocks, except for the night hours (14–20 h) and the last 24 h, because the concentrations of the metabolites were mostly under the Limit of Quantification (LOQ).

References

    1. Zuo H.L., Yang F.Q., Zhang X.M., Xia Z.N. Separation of cis- and trans-asarone from Acorus tatarinowii by preparative gas chromatography. J. Anal. Methods Chem. 2012;2012:402081. doi: 10.1155/2012/402081. - DOI - PMC - PubMed
    1. Rajput S.B., Tonge M.B., Karuppayil S.M. An overview on traditional uses and pharmacological profile of Acorus calamus Linn. (Sweet flag) and other Acorus species. Phytomedicine. 2014;21:268–276. doi: 10.1016/j.phymed.2013.09.020. - DOI - PubMed
    1. Varshney V.K., Song B.H., Ginwal H.S., Mittal N. High Levels of diversity in the phytochemistry, ploidy and genetics of the medicinal plant Acorus calamus L. J. Med. Aromat. Plants. 2015;2015:1–9. doi: 10.4172/2167-0412.S1-002. - DOI
    1. Rana T.S., Mahar K.S., Pandey M.M., Srivastava S.K., Rawat A.K.S. Molecular and chemical profiling of ‘sweet flag’ (Acorus calamus L.) germplasm from India. Physiol. Mol. Biol. Plants. 2013;19:231–237. doi: 10.1007/s12298-013-0164-8. - DOI - PMC - PubMed
    1. Scientific Committee on Food. European Commission Opinion of the Scientific Committee on Food on the Presence of Beta-asarone in Flavourings and Other Food Ingredients with Flavouring Properties (SCF/CS/FLAV/FLAVOUR/9 ADD1 Final) 2002. [(accessed on 27 August 2021)]. Available online: https://ec.europa.eu/food/system/files/2016-10/fs_food-improvement-agent....

LinkOut - more resources