Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract

Abstract

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC₅₀) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC₄₀ value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC₅₀ value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.

Keywords: CYP inhibition; antimutagens; chemoprotection; heterocyclic amines; medicinal plant; xenobiotics.

Figure 1

Inhibition of ethoxyresorufin O-deethylation activity (EROD) by Asclepias subulata extract (non-heated and heated) on rat liver microsomes. Results are expressed as averages ± standard deviation (n = 3), ASE: Asclepias subulata extract. The dotted line represents 50% inhibition. A total of 100% activity of control (incubation mixture without ASE): 217.24 pmol/mg protein/min. ASE was evaluated at 2.5–880 µg/mL (non-heated) and 0.02–960 µg/mL (heated).

Figure 2

Inhibition of methoxyresorufin O-demethylation activity (MROD) by Asclepias subulata extracts, non-heated and heated, on rat liver microsomes. Results are expressed as averages ± standard deviation (n = 3), ASE: Asclepias subulata extract. A total of 100% activity of control (incubation mixture without ASE): 9.11 pmol/mg protein/min. ASE was evaluated at 160–880 µg/mL (non-heated) and 0.02–880 µg/mL (heated).

Figure 3

Structures of (A) calotropin and (B) corotoxigenin-3-O-glucopyranoside and similar compounds (C) oleanolic and (D) ursolic acids.

Figure 4

Validation modelling between α-naphthoflavone (light green) and CYP1A1. −14.5 kcal/mol. Center: (−19.6144, 36.1934, −37.9863), size: (44, 46, 49).

Figure 5

Validation modelling between α-naphthoflavone (light green) and CYP1A2. −14.4 kcal/mol. Center: (3.5573, 20.9096, 30.4746), size: (49.1593, 46.1562, 48.2646).

Figure 6

Molecular docking of corotoxigenin-3-O-glucopyranoside (yellow) with CYP1A1 (A) and CYP1A2 (B).

Figure 7

Molecular docking of (A) oleanolic and (B) ursolic acids (yellow) with CYP1A1.

Figure 8

Molecular docking of (A) oleanolic and (B) ursolic acids with CYP1A2.