In Vitro and in Vivo Studies of the Metabolic Activation of 8-Epidiosbulbin E Acetate

Abstract

Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our preliminary studies demonstrated that administration of EEA caused acute hepatotoxicity in mice, and the observed toxicity required cytochromes P450-mediated metabolism. Metabolic activation studies of EEA were performed in vitro and in vivo. Microsomal incubations of EEA supplemented with N-acetyl lysine (NAL) and glutathione (GSH) generated six metabolites (M1-M6). M1-M4 were characterized as pyrrole derivatives, and M5 and M6 were pyrrolinones. M2-M6 were detected in bile and/or urine of rats given EEA. Dimethyldioxirane-mediated oxidation of EEA in the presence of NAL and GSH produced M1-M6, all of which were generated in microsomal incubations. The structures of M3 and M6 were confirmed by (1)H and (13)C NMR. These findings provide evidence for the metabolic activation of EEA to the corresponding cis-enedial intermediate both in vitro and in vivo. Ketoconazole inhibited the microsomal production of the cis-enedial, and P450 3A4 was found to be the primary enzyme involved in the bioactivation of EEA.

Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds

“…8-Epidiosbulbin E acetate (EEA) is another diterpenoid lactone isolated from DBL in 1984 (Murray et al, 1984). Unexpectedly, EEA is the most abundant diterpenoid in some DBL and its contents depend on the commercial source (Lin et al, 2015). Our previous studies found that the mice administered with EEA-containing DBL extracts showed 7.4-and 10.7-fold higher serum ALT and AST activities (ALT: 9128 ± 3487 U/L; AST: 7189 ± 4560 U/L) than those of the mice treated with EEA-free DBL extracts (ALT: 1093 ± 322 U/L; AST: 613 ± 194 U/L) (Lin et al, 2015).…”

“…The furan moiety was proven to be metabolized to EEA-derived cis-enedial intermediate (EDE) in vitro and in vivo, which was subsequently trapped by GSH and/or NAL to offer six cyclic GSH/NAL conjugates (Scheme 10), and CYP3A4 was the dominant enzyme to catalyze the process (Lin et al, 2015). Serum ALT and AST levels of EEA-treated mice presented time-and dosedependent elevations.…”

“…Scheme 10. Proposed metabolic activation of EEA mediated by P450 enzymes and the formation of GSH and/or NAL conjugates (Lin et al, 2015).…”

Metabolic Activation of the Toxic Natural Products From Herbal and Dietary Supplements Leading to Toxicities

“…Animal studies also revealed that oral administration of ethanol extracts of D. bulbifera could cause the significant liver injury, together with increased lipid peroxide levels in liver ( Wang et al, 2010 ). Belonging to clane-type diterpene lactone with furane ring, diosbulbin B, diosbulbin D, and 8-epidiosbulbin E Acetate are principal constituents of D. bulbifera , which were verified to cause obvious liver toxicity towards on rat or mouse, respectively ( Lin et al, 2015 ; Lin et al, 2016a ; Lin et al, 2016b ). Although there is no furan in the structure of pulegone, it can be initially biotransformed to menthofuran after metabolism ( Thomassen et al, 1990 ), and menthofuran can further generate toxic metabolites, after metabolic activation ( Ravindranath et al, 1984 ; Ravindranath et al, 1986 ).…”

Metabolism of five diterpenoid lactones from Dioscorea bulbifera tubers in zebrafish

“… 18 The metabolic generation of six cyclic GSH/ N -acetyl lysine conjugates from EEA were detected both in vitro and in vivo . 19 To fully understand the hepatotoxicity effects and mechanism of the DLs, it is essential to obtain early information regarding its metabolism. However, compared with extensive researches on DIOB and EEA, the knowledge of the metabolites and metabolic pathways focusing on other DLs are limited.…”