Metabolic study of Angelica dahurica extracts using a reusable liver microsomal nanobioreactor by liquid chromatography-mass spectrometry

Abstract

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC-MS as trans-imperatorin hydroxylate (M1), cis-imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans-isoimperatorin hydroxylate (M1') and cis-isoimperatorin hydroxylate (speculated M2'). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1' and M3') were isolated by preparative HPLC for structure elucidation by (1) H-NMR and MS(2) analysis. M3' was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3' was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin.

Keywords: Angelica dahurica; HPLC-MS; furocoumarins; immobilized liver microsomes; magnetic nanoparticles; metabolites.

Fig 1

Chemical structures of imperatorin, isoimperatorin, and the proposed metabolites. M1, trans-imperatorin hydroxylate; M2, cis-imperatorin hydroxylate; M3, isoimperatorin epoxide; M1’, trans-isoimperatorin hydroxylate; M2’, cis-isoimperatorin hydroxylate; and M3’, isoimperatorin epoxide.

Fig 2

HPLC-UV chromatograms obtained from the metabolic solutions of imperatorin (A) and isoimperatorin (B) at incubation times of 0 and 30 min, respectively.

Fig 3

MS² properties of the imperatorin metabolites detected in the metabolic solution with LMMNPs for 30 min.

Fig 4

MS² spectrometric properties of the isoimperatorin metabolites detected in the metabolic solution with LMMNPs for 30 min.

Fig 5

The reusability of LMMNPs using isoimperatorin as the test compound.

Fig 6

Overlaid HPLC-UV chromatograms obtained from imperatorin-LMMNP (trace a), isoimperatorin-LMMNP (trace b), A. dahurica extract-LMMNP (trace c) incubation solutions, and A. dahurica extract (trace d).