Hepatic Metabolism of Carcinogenic β-Asarone

Abstract

β-Asarone (1) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound 1 is found in several plants, e.g., Acorus calamus or Asarum europaeum. Compound 1-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although 1 has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic allylic phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. In vivo experiments suggested that this pathway does not play a major role in the carcinogenicity of the propenylic compound 1 as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rats, bovines, porcines, and humans using (1)H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of 1 presumably yielded (Z)-asarone-1',2'-epoxide (8a) which instantly was hydrolyzed to the corresponding erythro- and threo-configurated diols (9b, 9a) and the ketone 2,4,5-trimethoxyphenylacetone (13). This was the main metabolic pathway in the metabolism of 1 in all investigated liver microsomes. Hydroxylation of the side chain of 1 led to the formation of three alcohols at total yields of less than 30%: 1'-hydroxyasarone (2), (E)- and (Z)-3'-hydroxyasarone (4 and 6), with 6 being the mainly formed alcohol and 2 being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of 4 and 6 were further oxidized to the corresponding carbonyl compounds (E)- and (Z)-3'-oxoasarone (5, 7). 1'-Oxoasarone (3) was probably also formed in incubations with 1 but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-O-demethylated metabolites of 1 were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of 1. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71-75% of 1 will be metabolized via epoxidation, 21-15% via hydroxylation (and further oxidation), and 8-10% via demethylation at lower as well as higher concentrations of 1, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of 1 are the ultimate carcinogens formed from 1.

Engineering P450 Peroxygenase to Catalyze Highly Enantioselective Epoxidation of cis‐β‐Methylstyrenes

“…[17,18] trans-Anethole (1-propenyl-4-methoxy-benzene), which is found in the essential oils of severalp lants including fennel and Chinese star aniseed, has potent pharmacological effect by its metabolism to the epoxides. [18,19] Therefore, we studied the use of the T213M mutant and the T213Ym utant of P450 119p eroxygenase to catalyzet he asymmetric epoxidation of the cis-b-methylstyrenes with substituentso nt he benzene ring. Scheme 2s hows the synthesis of as electiono fcis-b-methyl styrene derivatives by hydrogenation of phenylpropynes in the presence of Lindlar catalyst.…”

“…For example, certain epoxidation products of cis-b-methylstyrenes such as b-asarone and cis-anethole are versatile precursors for the synthesis of pharmaceutically and biologically active molecules. [17][18][19] Although extensivee xperimental studies on the epoxidation of styrene and its derivatives by using the P450 monooxygenases have been reported, [1,3] and computational data is available on olefin epoxidation by using P450 enzymes, [2,20] highlye nantioselective epoxidationo fs ome specific substrates such as cis-b-methylstyrene remains as ignificant challengei nt he field of P450 catalysis. For example,O ritiz de Montellano reported that the wild-type P450cam monooxygenase catalyzest he epoxidationo fs tyrene and b-methylstyrene, but only 78 % ee was observed for the asymmetrice poxidation of cis-b-methylstyrene.…”

Cytotoxic effects of β‐asarone on Sf9 insect cells

“…β‐asarone has an oral LD 50 of β‐asarone 1,010 mg/kg in rats (JECFA, ) and 1,560 mg/kg in mice (Liu et al, ). More than 70% of a β‐asarone (500 mg/kg) is metabolized by human liver microsomes via a theoretical transient epoxidation step to β‐asarone‐1′,2′‐erythro‐diol, β‐asarone‐1′,2′‐threo‐diol and the ketone 2,4,5‐trimethoxyphenylacetone (Cartus et al, ), a derivative that is noncarcinogenic (Avadhani, Selvaraj, Rajasekharan, & Tharachand, ; Olas & Bryś, ) and the Committee on Herbal Medicinal Products (HMPC) of the European Medicines Agency (European Medicines Agency EMEA, ) concluded a limit of exposure from herbal medicinal products of approximately 115 mg/day (HMPC, 2005). Together, these results reinforce the understanding that phytobiologicals including β‐asarone are less harmful to humans than most conventional insecticides.…”

Safety aspects of herbal products containing compounds with a potential risk

“…U eksperimentima na životinjama, tokom dugotrajne primene velikih doza, pokazano je da je azaron genotoksičan i hepatokancerogen. Pretpostavlja se da se metaboliše u genotoksični karcinogen (E)-azaron-1',2'-epoksid (65,66), ali nije poznato da li se rezultati ispitivanja na životinjama odnose i na humani organizam. Metabolizam, a time i stvaranje genotoksičnih i karcinogenih metabolita, ne moraju biti identični kod ljudi i eksperimentalnih životinja (5).…”