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

(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.

Quantitative Analysis of ß-Asarone Derivatives in Acorus calamus and Herbal Food Products by HPLC-MS/MS.

α-Asarone and ß-asarone are reported as bioactive constituents of Acorus calamus. Phase I metabolism of asarone isomers results in a multiple spectrum of genotoxic metabolites. Thus, the question arises whether structural analogues of the known phase I metabolites also naturally occur in A. calamus-based food products. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for three product classes, herbal infusions, alcoholic beverages, and food supplements. High asarone contents were detected in herbal infusions (total mean 9.13 mg/kg, n = 8) and food supplements (total mean 14.52 mg/kg, n = 6); hence, these food products can highly contribute to human exposure to genotoxic asarone derivatives. Also, the occurrence of asarone oxidation products found in food and food supplements has to be taken under consideration because data on toxicity is limited so far.

α-Asarone, ß-asarone, and γ-asarone: Current status of toxicological evaluation.

Asarone isomers are naturally occurring in Acorus calamus Linné, Guatteria gaumeri Greenman, and Aniba hostmanniana Nees. These secondary plant metabolites belong to the class of phenylpropenes (phenylpropanoids or alkenylbenzenes). They are further chemically classified into the propenylic trans- and cis-isomers α-asarone and ß-asarone and the allylic γ-asarone. Flavoring, as well as potentially pharmacologically useful properties, enables the application of asarone isomers in fragrances, food, and traditional phytomedicine not only since their isolation in the 1950s. However, efficacy and safety in humans are still not known. Preclinical evidence has not been systematically studied, and several pharmacological effects have been reported for extracts of Acorus calamus and propenylic asarone isomers. Toxicological data are rare and not critically evaluated altogether in the 21st century yet. Therefore, within this review, available toxicological data of asarone isomers were assessed in detail. This assessment revealed that cardiotoxicity, hepatotoxicity, reproductive toxicity, and mutagenicity as well as carcinogenicity were described for propenylic asarone isomers with varying levels of reliability. The toxicodynamic profile of γ-asarone is unknown except for mutagenicity. Based on the estimated daily exposure and reported adverse effects, officials restricted or published recommendations for the use of ß-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.

DNA double strand break repair as cellular response to genotoxic asarone isomers considering phase I metabolism.

The phenylpropenes α-asarone and ß-asarone are widely spread in the marsh plant Acorus calamus. Both isomers are classified as carcinogenic in rodents. However, the respective genotoxic mechanisms are not elucidated so far. The present study gives deeper insights into the genotoxic effects of asarone isomers as well as their known oxidative phase I metabolites, (E)-3'-oxoasarone and asarone epoxide. We show that asarone metabolites highly increase DNA strand breaks after 1 h of incubation, markedly metabolic activation contributes to their carcinogenic mode of action. All test compounds act as aneugens and potently enhance the amounts of micronuclei in binuclear cells. However, a prolonged incubation time of 24 h results in a decrease of DNA damage. This work suggests that asarone metabolites also induce DNA double strand breaks , why we put a strong focus on homologous recombination and non-homologous end joining. The obtained results herein indicate that asarone epoxide-induced DNA strand breaks are repaired via a homologous repair pathway.

Alternaria toxins in South African sunflower seeds: cooperative study.

Sunflower seed samples (N = 80) from different sunflower cultivars originating from different localities in South Africa were analyzed for 15 toxins produced by fungi of the genus Alternaria by means of a simple one-step extraction dilute-and-shoot HPLC-MS/MS approach. References for valine-tenuazonic acid (Val-TeA), altenusin (ALTS), and altenuisol (ALTSOH) were isolated from fungal culture extracts and spectroscopically characterized. Additionally, valine-tenuazonic acid was tested regarding its cytotoxicity in comparison with tenuazonic acid (TeA) and showed less activity on HT-29 cells. Furthermore, alternariol monomethyl ether-3-O-ß-D-glucoside (AME-3G) was produced by fermentation of alternariol monomethyl ether (AME) with the fungus Rhizopus oryzae. The seed samples were analyzed both with and without hulls. The method covers the AAL toxins TA1 and TA2, altenuene (ALT) and iso-altenuene (iso-ALT), altenuisol, altenusin, altertoxin I (ATX-I) and altertoxin II (ATX-II), alternariol (AOH) and alternariol monomethyl ether, alternariol monomethyl ether-3-O-ß-D-glucoside, tenuazonic acid, allo-tenuazonic acid (allo-TeA) and valine-tenuazonic acid, and tentoxin (TEN). More than 80% of the samples were positive for one or more analytes above the respective limit of detection (0.2-23 µg/kg). Alternariol, its monomethyl ether, tentoxin, tenuazonic acid, altenuisol, and valine-tenuazonic acid were found in quantifiable amounts. The highest prevalences were found for tentoxin (73% positive, mean content 13.2 µg/kg, maximum level 130 ± 0.9 µg/kg) followed by tenuazonic acid (51% positive, mean content 630 µg/kg, maximum level 6300 ± 560 µg/kg). The obtained data were further analyzed statistically to identify quantitative or qualitative relationships between the levels of Alternaria toxin in the samples.