Induction of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) by Glycyrrhiza Species Used for Women's Health: Differential Effects of the Michael Acceptors Isoliquiritigenin and Licochalcone A

Abstract

For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme,

Nad(p)h: quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human hepatoma (HepG2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in nontumorigenic breast epithelial MCF-10A cells. In female Sprague-Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 in vivo most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability in vivo. These data show the chemopreventive potential of licorice species in vitro could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the in vivo effects in the rat model after four-day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible.

Figure 1

Electrophilic compounds, A) XH from hops, curcumin from turmeric, and sulforaphane from broccoli, B) bioactive compounds from licorice species.

Figure 2

NQO1 activity in murine hepatoma hepa1c1c7 cells by A) licorice extracts from the three major species, GG, GU, and GI in comparison to hops extract. B) LigC/LigF, and LicA from licorice in comparison to XH from hops. C) Licorice bioactive constituents relative to XH, curcumin from turmeric, and sulforaphane from broccoli sprouts. D) Chemopreventive index was calculated as the ratio of a compound's LC₅₀ over its CD value in the NQO1 activity assay. Results are shown as fold induction and are the means of three independent determinations.

Figure 2

NQO1 activity in murine hepatoma hepa1c1c7 cells by A) licorice extracts from the three major species, GG, GU, and GI in comparison to hops extract. B) LigC/LigF, and LicA from licorice in comparison to XH from hops. C) Licorice bioactive constituents relative to XH, curcumin from turmeric, and sulforaphane from broccoli sprouts. D) Chemopreventive index was calculated as the ratio of a compound's LC₅₀ over its CD value in the NQO1 activity assay. Results are shown as fold induction and are the means of three independent determinations.

Figure 3

Induction of ARE-luciferase in stably transfected human hepatoma Hep-G2-ARE-C8 cells by A) licorice extracts from the three major species, GG, GU, and GI in comparison to hops extract, B) by LigC/LigF, and LicA from licorice, XH from hops, curcumin from turmeric, sulforaphane from broccoli. Results are normalized to the corresponding protein concentrations and the DMSO control. Results are shown as fold induction and are the means of three independent determinations.

Figure 4

Induction of NQO1 in non-tumorigenic ER (−) human mammary epithelial cells (MCF-10A). Quantitation of western blot analysis of the Induction of NQO1 in MCF-10A cells by A) crude extracts of G. glabra; GG (10 μg/mL), G. uralensis; GU (10 μg/mL, open bar), G. Inflata; GI (10 μg/mL) in comparison to hops extract (5 μg/mL) and B) characteristic compounds LigC (5 μM), LigF (10 μM), and LicA (5 μM) in comparison to XH (5 μM). Results are shown as fold induction and are the means of three independent determinations.

Figure 5

LC-MS/MS SRM chromatograms of LigC/LigF tissue distribution A) in the GG extract and the GG treated animal tissues B) in the LigC treated animal tissues.

Figure 6

The influence of licorice extracts and characteristic LigF and LigC on NQO1 induction in the A) liver and B) mammary glands. Animals were treated orally for 4 days with GG and GU at 1.3 g/kg BW per day, LigF at 80 mg/kg BW per day, and LigC at 40 mg/kg BW per day. 4’-Bromoflavone, the positive control was mixed in their diet at 150 mg/kg BW per day and induced NQO1 4.47 ± 0.13 folds in the liver and 2.15 ± 0.14 folds in the mammary tissue.

Scheme 1

Michael acceptors LigC and LigF modulate NQO1 through different mechanisms based on the in vitro observations. The left side clear arrow with black borders shows the inhibitory effect of LicA on AhR pathway. The blue arrow shows the effect of LicA on Keap1-Nrf2 pathway and the induction of ARE and NQO1 by this compound. Pink arrow shows the effect of LigC on Keap1-Nrf2 pathway and the induction of ARE and NQO1 by this compound. LigC interacts with Keap1 and the consequent translocation of Nrf2 to the nucleus and its interaction with ARE might result in NQO1 induction. LicA could influence the induction of NQO1 through two parallel, yet opposing molecular interactions at the promoter of NQO1. It can increase ARE induction through interacting with Keap1 and decrease XRE induction through inhibiting AhR, which might result in a lower NQO1 induction compared to that of LigC.