Characterization of stable and reactive metabolites of piperine formed on incubation with human liver microsomes

Abstract

Black pepper, though commonly employed as a spice, has many medicinal properties.It consists of volatile oils, alkaloids, pungent resins, etc., of which piperine is a major constituent. Though safe at low doses, piperine causes alteration in the activity of drug metabolising enzymes and transporters at high dose and is known to precipitate liver toxicity. It has a potential to form reactive metabolite(s) (RM) owing to the presence of structural alerts, such as methylenedioxyphenyl (MDP), α, β-unsaturated carbonyl group (Michael acceptor), and piperidine. The present study was designed to detect and characterize stable and RM(s) of piperine formed on in vitro incubation with human liver microsomes. The investigation of RMs was done with the aid of trapping agents, viz, glutathione (GSH) and N-acetylcysteine (NAC). The samples were analysed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) using Thermo Scientific Q Exactive Plus Orbitrap. Full scan MS followed by data-dependent MS 2 (Full MS-ddMS 2 ) mode was used to establish mass spectrometric fragmentation pathways of protonated piperine and its metabolites. In total, four stable metabolites and their isomers (M1a-c, M2a-b, M3a-c, and M4a-b) were detected. Their formation involved removal of carbon (3, M1a-c), hydroxylation (2, M2a-b), hydroxylation with hydrogenation (3, M3a-c), and dehydrogenation (2, M4a-b). Out of these metabolites, M1, M2, and M3 are reported earlier in the literature, but their isomers and two M4 variants are novel. In addition, six novel conjugates of RMs, including three GSH conjugates of m/z 579 and three NAC conjugates of m/z 435, were also observed.

Identification of the metabolites of piperine via hepatocyte incubation and liquid chromatography combined with diode‐array detection and high‐resolution mass spectrometry

“…Its MS/MS spectrum (Figure 7B) showed three indicative product ions at m/z 201.0544, 135.0440, and 110.0602, which indicated that dehydrogenation had occurred on the piperidine ring. This metabolite was similar to one reported previously 19 …”

“…The MS/MS spectrum of their [M + H] + ions (Figure 5C) showed two indicative product ions at m/z 201.0545 and 102.0911, which indicated that hydroxylation occurred on the piperidine ring. These compounds were similar to previously reported metabolites 19 …”

“…Its MS/MS spectrum (Figure 5D) showed two indicative product ions at m/z 189.0545 and 123.0441, which provided support for ring opening of the 1,3‐benzodioxole group. This metabolite was also reported in previous studies 18,19 …”

“…The MS/MS spectrum of the [M + H] + ion (Figure 4A) showed two characteristic product ions at m/z 504.1798 and 450.1689, which were formed through the loss of a glutamyl radical (129 Da) and glycine (75 Da), respectively. Both metabolites were identical to those of previously reported metabolites, and it is proposed that they were formed through the ortho ‐quinone intermediate 19 …”

“…Gao et al investigated the in vivo metabolism of piperine in the rat and identified 12 metabolites in rat urine 18 . Praneetha and coworkers characterized the stable and reactive metabolites of piperine in human liver microsomes 19 . These studies did provide some useful information on the metabolism of piperine.…”