Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G

Published OnlineFirst October 4, 2018; DOI: 10.1158/1940-6207.CAPR-18-0178

Research Article Cancer Prevention Research Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inﬂata Extracts Modulate Estrogen Metabolism in ACI Rats Shuai Wang1, Tareisha L. Dunlap1, Lingyi Huang1, Yang Liu1, Charlotte Simmler1,2, Daniel D. Lantvit1, Jenna Crosby1, Caitlin E. Howell1, Huali Dong1, Shao-Nong Chen1,2, Guido F. Pauli1,2, Richard B. van Breemen1,3, Birgit M. Dietz1, and Judy L. Bolton1

Abstract

Women are increasingly using botanical dietary sup- tion, the effect of licorice extracts and compounds on plements (BDS) to reduce menopausal hot flashes. biomarkers of estrogen chemoprevention (CYP1A1)as Although licorice (Glycyrrhiza sp.) is one of the frequent- well as carcinogenesis (CYP1B1) was studied. LicA ly used ingredients in BDS, the exact plant species is was extensively glucuronidated and formed GSH often not identified. We previously showed that in adducts; however, free LicA as well as LigC were breast epithelial cells (MCF-10A), Glycyrrhiza glabra bioavailable in target tissues after oral intake of lic- (GG) and G. inflata (GI), and their compounds differ- orice extracts. GG, GI, and LicA caused induction of entially modulated P450 1A1 and P450 1B1 gene NQO1 activity in the liver. In mammary tissue, GI expression, which are responsible for estrogen detoxi- increased CYP1A1 and decreased CYP1B1,whereas fication and genotoxicity, respectively. GG and isoli- GG only increased CYP1A1. LigC may have contrib- quiritigenin (LigC) increased CYP1A1, whereas GI and uted to the upregulation of CYP1A1 after GG and GI its marker compound, licochalcone A (LicA), decreased administration. In contrast, LicA was responsible for CYP1A1 and CYP1B1. The objective of this study was to GI-mediated downregulation of CYP1B1. These studies determine the distribution of the bioactive licorice highlight the polypharmacologic nature of botanicals compounds, the metabolism of LicA, and whether GG, and the importance of standardization of licorice BDS to GI, and/or pure LicA modulate NAD(P)H quinone specific Glycyrrhiza species and to multiple constituents. oxidoreductase (NQO1) in an ACI rat model. In addi- Cancer Prev Res; 11(12); 819–30. 2018 AACR.

Introduction

1UIC/NIH Center for Botanical Dietary Supplements Research, College of Phar- In 2018, breast cancer will account for nearly one-third macy, University of Illinois at Chicago, Chicago, Illinois. 2Center for Natural of new cancer cases in women (1). A decline in hormone Product Technologies, Department of Medicinal Chemistry and Pharmacognosy, therapy (HT) usage was observed after the Women's College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois. 3Linus Pauling Institute, Oregon State University, Corvallis, Oregon. Health Initiative study in 2002 due to the increased breast cancer risk caused by the estrogen þ progestin Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). HT regimen (2). Besides the well-known hormonal S. Wang and T.L. Dunlap contributed equally to this article. estrogen carcinogenesis pathway, breast cancer risk is also inﬂuenced by changes in estrogen oxidative metab- Current address for Y. Liu: Research and Innovation, United States Pharmaco- peia, Rockville, MD 20852. olism (3). In the breast, P450 1A1 and P450 1B1 catalyze the metabolism of estrogens to 2- and 4-hydroxylated Corresponding Authors: Birgit M. Dietz, University of Illinois at Chicago, 833 S Wood St, M/C 781, Chicago, IL 60612. Phone: 312-996-2358; Fax: 312-996-7107; catechols, 2-OHE1/E2 and 4-OHE1/E2,respectively E-mail: [email protected]; and Judy L. Bolton, Phone: 312-996-5280; E-mail: (Fig. 1; ref. 4). The 2-hydroxylated metabolites are [email protected] strongly associated with reduced breast cancer risk (5) doi: 10.1158/1940-6207.CAPR-18-0178 because they inhibit E2-induced proliferation (6) and are 2018 American Association for Cancer Research. converted to nontoxic quinones (Fig. 1). On the other

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Figure 1. Biological targets and phase II metabolism of chemopreventive licorice compounds. A, The GI-speciﬁc compound, LicA, is a Michael acceptor that can covalently modify Keap1 to upregulate the detoxiﬁcation enzyme, NQO1, in MCF-10A and liver cells (21) as well as in liver tissue. LicA is also an AhR antagonist that can downregulate P450 1A1/1B1-mediated estrogen oxidative metabolism (11). In this animal model (ACI rats), GG and GI increased P450 1A1 gene expression (CYP1A1) in mammary tissue (Fig. 5B). In addition, GI also decreased P450 1B1 gene expression (CYP1B1)inthe mammary gland as indicated with arrows. B, LicA is mainly metabolized to various glucuronides by UDP- glucuronosyltransferases (UGT; Supplementary Fig. S2). As a Michael acceptor, LicA also forms GSH conjugates. In the liver and serum, LicA sulfate conjugates catalyzed by sulfotransferases (SULT) were detected as minor metabolites (Supplementary Fig. S2).

hand, P450 1B1 is linked to carcinogenesis because antioxidant, and induce detoxification enzymes (10). it is overexpressed in malignant tissues (7). P450 1B1 However, the effects of most BDS on estrogen oxidative produces 4-OHE1/E2 which are oxidized by peroxidases/ metabolism are unknown. Although licorice belongs P450s to genotoxic quinones (4-OHE1/E2-Q) that alkyl- to one of the most popular botanicals contained in BDS ate DNA and generate depurinating adducts (estrogen used for women's health issues, several licorice species chemicalcarcinogenesis,Fig.1;refs.4,8).Hence,upre- (Glycyrrhiza sp., Fabaceae) are used to source these BDS gulation of the 2-hydroxylation (P450 1A1) and down- without discrimination (11). Also, clinical evidence for regulation of the 4-hydroxylation pathway (P450 1B1) efficacy is generally lacking (10). may significantly inhibit estrogen chemical carcinogen- Botanically, over 30 species of licorice exist (12, 13). esis in the breast. In addition, NAD(P)H quinone oxi- From a pharmacopoeial perspective, three species are cur- doreductase 1 (NQO1) decreases depurinating estrogen- rently used in BDS interchangeably: Glycyrrhiza glabra DNA adducts (4) due to reduction of the reactive 4- (GG), G. inflata (GI), and G. uralensis. Notably, these three OHE1/E2-Q to its catechol. Catechol-O-methyltransfer- species have distinctive and very different chemical pro- ase (COMT) also prevents quinone formation through files, as already demonstrated in previous studies (Table 1; methylation of estrogen catechols to produce stable refs. 12, 14). Cultivated in China, GI is naturally more metabolites, 2-MeOE1/E2 and 4-MeOE1/E2 (Fig. 1; popular in Asia. The roots from this species contain an ref. 9). abundant species-specific chemopreventive Michael accep- Due to fear of increased breast cancer risk with HT, many tor, licochalcone A (LicA; ref. Fig. 2). GG is the most women have turned to botanical dietary supplements popular licorice species in the United States and Europe, (BDS) as a complementary approach therapy for meno- and its species-specific compound is glabridin (Table 1; pausal symptom relief. Female consumers generally per- Fig. 2; refs. 15, 16). Both licorice species contain isoliquir- ceive BDS as safer modalities because BDS often contain itigenin (LigC, C for chalcone), also a chemopreventive constituents that are reported to be anti-inflammatory and Michael acceptor, and liquiritigenin (LigF, F for flavanone),

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Table 1. Concentration of bioactive compounds in licorice extracts determined by UHPLC-UV Compounds (% w/w crude extract) Species Glabridin LicA LigCa LigC equivalentsb LigF LigF equivalents GG 1.34 0.02 ––3.61 0.07 0.19 0.01 8.55 0.06 GI – 7.07 0.61 0.10 0.07 2.32 0.04 0.24 0.05 3.67 0.31 aLigC was below the limit of detection in GG. bThe term equivalents is used to represent the total amount of aglycone plus glycosides of LigC (i.e., isoliquiritigenin, isoliquiritin, isoliquiritin apioside, and licuraside) or LigF (liquiritigenin, liquiritin, liquiritin apioside, and liquiritigenin-7-O-apiosylglucoside) in each crude extract. The values are expressed as mean SD of independent measures.

a phytoestrogen with estrogen receptor (ER) b preferential carcinogenesis studies (19). LicA was administered in properties (refs. 13, 17; Fig. 2). Both LigC and LigF are parallel to determine its role in GI's bioactivity and its spontaneously interconvertible isomers (Fig. 2; ref. 18), metabolic profile. LicA's distribution in liver and mam- found mainly as glycosides (e.g., liquiritin and isoliquir- mary tissues was determined and compared with LigC and itin) in the roots (Table 1). Due to the species-specific LigF. Levels of 2-MeOE1 in serum were quantified by compound profile, each licorice species has a unique LC-MS/MS, as a biomarker for overall estrogen oxidative bioactivity that could lead to differential clinical activity. metabolism, because rat P450 1B1 predominantly per- Previously, we showed that GG and LigC increased forms estrogen 2-hydroxylation (20). CYP1A1 and CYP1B1 estrogen oxidative metabolism (2- and 4-hydroxylation), expressions were determined in mammary tissues, and whereas GI and LicA decreased metabolism in MCF-10A NQO1 activity was measured in liver and mammary tis- "normal" breast epithelial cells (11). The purpose of the sues. Studies in MCF-10A cells with GI revealed different current study was to determine the chemopreventive effects results between in vitro and in vivo studies and identified of GI and GG on estrogen oxidative metabolism in the ACI potential bioactive compounds. The current experiments rat model, which is frequently used for in vivo estrogen carried out in ACI rats provide crucial information to

Figure 2. Key compounds of GG and GI including LigF and LigC equivalents.

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continue studying these licorice species and their effects on vent Systems; ref. 24), these data suggested that the corre- estrogen carcinogenesis in long-term studies with ACI rats sponding solvent system may elute the target analyte into a and eventually estrogen metabolism in women. The data high-resolution separation range (the sweet spot, partition herein explore additional chemopreventive modes to help coefficient, K value from 0.25 to 4 of a countercurrent develop more beneficial and safer standardized licorice separation method; ref. 25). Regarding verification of the dietary supplements for women's health. selected solvent system, an analytical scale of high-speed countercurrent chromatography (HSCCC; 16 mL, Tauto Materials and Methods Biotech) was applied, which achieved a higher purity of Materials, chemicals, and reagents LicA than that from the literature reported solvent system All chemicals and reagents were purchased from Fisher on the same instrument (Supplementary Fig. S1; ref. 26). Scientific or Sigma except for the following: 2-methoxyes- The scaled-up separation is described in the Supplemen- tary Information. The purity of LicA used in this animal trone (MeOE1)-1,4,16,16-d4 was obtained from CDN study was determined as 95% (w/w) by qHNMR. isotope, E2 and 2-/4-MeOE1/E2 reference compounds were acquired from Steraloids Inc., LigF and LigC were obtained from ChromaDex, and rat serum for initial standardization Animal experiment studies was purchased from BioreclamationIVT. Female August-Copenhagen Irish (ACI) rats were purchased from Harlan Laboratories at 5 weeks of age, accli- Plant material, extraction, and characterization mated for 1 week, fed a phytoestrogen-free diet (AIN-76A), GI was provided as a gift to SNC by Dr. Liang Zhao and randomly divided into five groups with 6 rats each: (Lanzhou Institute of Chemical Physics CAS), and GG was vehicle, estradiol benzoate (EB, 1 mg/kg/day), LicA purchased from Mountain Rose Herbs. Raw materials were (80 mg/kg/day) þ EB, GG (2 g/kg/day, gavage) þ EB, and identified by macroscopic/microscopic analyses and DNA GI (2 g/kg/day, containing 141 mg LicA) þ EB. At 6 weeks barcoding, as previously described (14). Ground roots of of age, one vehicle was applied subcutaneously (s.c., GG and GI were extracted by maceration and percolation at sesame oil) and one by gavage (50% corn oil with 50% room temperature with a solvent mixture [ethanol (200 PEG/H2O), EB and LicA were given s.c., and the licorice USP proof), isopropanol, and water (90:5:5, v/v)] at the extracts were administered by gavage for 4 days. The rats fi ratio of botanical to solvent as 1:15 (g/mL). The extracts were sacri ced by CO2 asphyxiation on day 5. Blood was were concentrated and freeze dried to yield 12% w/w of the collected, and serum prepared immediately after collec- initial ground roots. Extracts were analyzed by UHPLC-UV tion; mammary tissues, uterus, and liver were collected to quantify major chalcone and flavanone constituents, as and snap frozen in liquid nitrogen and stored at 80 C previously reported (21, 22). Briefly, a standard curve until analysis. The animal protocol complied with the containing the following 11 reference standards was used Guide for the Care and Use of Laboratory Animals, and for their quantitation in both licorice extracts. The AUC was all procedures were approved by UIC's Institutional Ani- taken at 360 nm for all chalcones (isoliquiritin, isoliquir- mal Care and Use Committee (Protocol No. 16-033). itin apioside, licuraside, LigC, and LicA), and at 275 nm for all flavanones (liquiritin, liquiritin apioside, liquiritigenin Preparation of serum and tissue samples for LicA 7-O-apiosylglucoside, and LigF) and for glabridin. Quan- metabolism profile analysis and for LicA titative results obtained for each LigF glycoside or LigC quantitation in vivo glycoside were converted by their molecular weight, there- LicA was quantified in serum, liver, and mammary gland by leading to their concentration as LigF or LigC equiva- samples from the LicA þ EB and GI þ EB treatment groups lents, respectively (Table 1). (Fig. 3C; Supplementary Table S2). After thawing at room temperature, serum (50 mL) was transferred to a 1.5 mL Preparation and characterization of LicA Eppendorf tube and mixed with 10 mL of ACN containing The crude LicA sample, enriched from GI (eLicA, purity naringenin (500 nmol/L) as the internal standard (IS). of LicA, 50%), was a gift to SNC from Qinghai Lake Liver and mammary tissues (500–800 mg for liver and 50– Medicinal CO., Ltd. A loss-free countercurrent separation 100 mg for mammary gland) were weighed and homog- was implemented for the purification of LicA from the enized in 70% aqueous methanol (containing 0.1% formic eLicA, as follows: TLC-based solvent system strategy (23) acid) at 5 mL for liver and 1 mL for mammary tissues. The was performed for screening a proper solvent system. homogenate (200 mL) was taken and spiked with 20 mL Among screened solvent systems (Supplementary Table naringenin (500 nmol/L). Ice-cold ACN (600 mL) was S1), LicA was eluted by the organic phase of n-hexane-ethyl added, and the mixture was centrifuged for 15 minutes at acetate-methanol-water (HEMWat, 4:6:5:5, v/v) to Rf ¼ 13,000 x g at 4 C for protein precipitation. The supernatant 0.43 on a precoating normal-phase Si TLC plate (400 mL) was transferred to a new Eppendorf tube and (MACHEREY-NAGEL). Considering the main principles evaporated to dryness under a stream of nitrogen. The of the GUESS method (Generally Useful Estimate of Sol- residue was reconstituted in 100 mL of 20% ACN, and 5 mL

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Figure 3. UHPLC-MS/MS analysis of key licorice compounds. Licorice compounds, LicA, LigC, LigF, and glabridin (GB), were detected by UHPLC-MS/MS in the crude extracts and serum, liver, and mammary gland after administration of (A) GG (2 g/kg/day) and EB and (B) GI (2 g/kg/day) and EB to ACI rats for 4 days. C, Free LicA was quantiﬁed by UHPLC-MS/MS in rat serum, liver, and mammary gland after GI and LicA administration.

was injected into LC-MS/MS for analysis. The same rat temperature: 250C; heating block temperature: 400C; samples were analyzed for LicA metabolites. drying gas flow: 10 L/min. The data were acquired using selected reaction monitoring (SRM) with positive ion þ UHPLC-MS/MS analyses electrospray as follows: naringenin ([MþH] m/z 273 to þ UHPLC-MS/MS analysis was performed as described 153 and m/z 273 to 147, IS); and LicA ([MþH] m/z 339 previously (27, 28). Briefly, a Shimadzu LCMS-8060 to 121 and m/z 339 to 297). triple quadrupole mass spectrometer equipped with a For determination of LicA metabolites, the positive ion Shimadzu Nexera UHPLC system was used for analysis. electrospray SRM transitions for each analyte were estab- For quantitative analysis, analytes were separated on a lished as m/z 515 to 339 for LicA monoglucuronides, m/z Waters Acquity UPLC BEH C18 2.1 50 mm column 646 to 339 for the glutathione conjugate of LicA, m/z 419 to (1.7 mm particle size). Mass-spectrometer parameters 339 for LicA sulfate, m/z 531 to 339 for monooxygenated were as follows: nebulizing gas flow: 2.5 L/min; heating LicA glucuronides, and m/z 545 to 369 for catechol-O- gas flow:10L/min;interfacetemperature:300C; DL methylated LicA glucuronides.

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For qualitative determination of LigC, LigF, LicA, and 1-Step RT-PCR Master Mix, and CYP1A1 and CYP1B1 glabridin in the GI and GG extracts, serum, and tissues, the primer with FAM-MGB probe, and GAPDH primer with licorice extracts (GG and GI) were dissolved in 50% aque- VIC-MGB probe. Data were analyzed with the compar- ous methanol at 10 mg/mL. Rat serum and tissue samples ative CT (DDCT) method and expressed as fold induction were prepared as described above. Samples were analyzed relative to the vehicle control group. using UHPLC-MS/MS with negative ion electrospray and SRM (28) as follows: liquiritin and isoliquiritin, m/z 417 to Statistical analysis 255; liquiritin apioside, isoliquiritin apioside, and licura- The data were expressed as mean SEM from 6 animals side, m/z 549 to 255; LigF and LigC, m/z 255 to 119; per group or SEM for three independent experiments in glabridin, m/z 323 to 201; and glycyrrhetinic acid, m/z MCF-10A cells. Significance was determined using the 469 to 425. Student t test to compare two samples or one-way ANOVA with Dunnett posttest to compare multiple samples with < Analysis of estrogen oxidative metabolism (2-MeOE1) the control ( , P 0.05). and E1/E2 in serum LC-MS/MS analysis was performed as previously Results fi described with minor modi cations (29). Serum samples Measurement of bioactive compounds in Glycyrrhiza m (150 L) were incubated at 37 C for 4 hours after adding species m glucuronidase and sulfatase hydrolysis buffer (300 L) and Extracts were analyzed by UHPLC-UV, and all com- the IS, 2-methoxyestrone-d4. After the incubation, sample pounds were expressed as % w/w of each extract preparation and analysis were conducted as previously (Table 1). Glabridin (Fig. 2) was present at 1.34% of described (29). The SRM transitions were as follows: m/z total GG extract, whereas LicA represents one of the 534.3 to 171.2, m/z 504.3 to 171.0, and m/z 506.3 to 171.0, major compounds of GI crude extract (7.07%). Glabri- for 2-MeOE1,E1, and E2, respectively. Results are expressed din and LicA exist as aglycones, but LigC and LigF occur as fold change from average amount of analytes of rats primarily as glycosylated compounds, which exceeded treated with EB alone. the corresponding aglycones by 15-fold. LigC glycosides (isoliquiritin, isoliquiritin apioside, and licuraside) and Analysis of NQO1 activity in liver tissue and LigF glycosides (liquiritin, liquiritin apioside, and liquir- mammary gland itigenin-7-O-apiosylglucoside; Fig. 2) together with the The NQO1 activity in frozen liver and mammary tissue corresponding aglycones are represented as total LigC was determined as described previously (30). The NQO1 and LigF equivalents, as the glycosides are deglycosylated activity was determined in a clear supernatant solution of in vivo (Fig. 3A and B). GG extract contained 1.5-fold the tissue homogenate (5 mg of liver protein and 30 mgof more LigC equivalents and over 2-fold more LigF equiva- mammary gland protein) as previously described (31). The lents than GI. The most abundant LigF glycoside (>70%) absorbance was measured at 610 nm, and the results were in GG extract was liquiritin apioside (Table 1). expressed as fold induction of NQO1 activity relative to the control group. Distribution of LicA, LigF, LigC, and glabridin in serum, liver, and mammary tissue after GG and GI Quantification of P450 1A1/1B1 mRNA expression administration via RT-qPCR LigC/LigF, LigC/LigF glycosides, glabridin, and LicA were Mammary tissues (100 mg) were homogenized in qualitatively determined in crude extracts, serum, liver, and TRIzol reagent. The total RNA was extracted using the mammary tissues by UHPLC-MS/MS analysis. The con- RNeasy lipid tissue Kit (Qiagen), and RNA (5 mg) was centration of glabridin was low in the serum, and it was not reverse transcribed with Invitrogen's SuperScript III First- detected in liver and mammary tissues (Fig. 3A). The three Strand Synthesis System. RT-qPCR was performed using other licorice compounds, LicA, LigF, and LigC, were all TaqMan rat CYP1A1, CYP1B1, and ACTB primers with available in the liver and mammary gland. The most FAM/MGB probe (Applied Biosystems) as described striking observation was that most of the LigC/LigF glyco- previously (29). MCF-10A cells were obtained from the sides were hydrolyzed, which increased LigC/LigF aglycone ATCC and authenticated via short tandem repeat pro- concentrations in the serum and both tissue samples. LigC filing (Promega). MCF-10A cells were cultured as and LigF concentrations were similar in mammary tissues, described previously (29). For the in vitro CYP1A1/ although LigC was notably higher in serum but signifi- CYP1B1 induction experiments, cells having approxi- cantly lower than LigF in liver tissues (Fig. 3A and B). In the mately 15 through 20 passages were plated in 96-well GI crude extract, LicA levels were much higher than free plates and treated with vehicle (DMSO), GG, GI, and LigC; however, because LigC equivalents were hydrolyzed licorice compounds for 24 hours. RT-qPCR was per- in vivo, the concentration of free LigC exceeded LicA con- formed as previously described (29) using TaqMan centrations in serum and mammary tissues (Fig. 3B). After

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LicA s.c. and GI gavage administration for 4 days, LicA was also quantiﬁed by UHPLC-MS/MS in serum, liver tissue, and mammary gland. Interestingly, after both applications, free LicA was available in serum, liver, and mammary gland 24 hours after the last dose (day 5; Fig. 3C). Although LicA is highly glucuronidated in vivo (Fig. 1B; Supplementary Fig. S2), considerable amounts of free LicA were observed in the target tissues after oral administration of the extract (Supplementary Table S2; Fig. 3C).

Metabolism of LicA in serum, liver tissue, and mammary gland LicA metabolites were analyzed in serum, liver, and mammary gland samples by UHPLC-MS/MS after s.c.

administration of LicA. In serum, liver, and mammary Figure 4. tissues, glucuronidation reactions dominated LicA metab- NQO1 induction by GG, GI, and LicA in the liver. NQO1 activity was olism and resulted in two major (MG1 and MG2) and one measured in the liver after administration of vehicle, EB (1 mg/kg/day), minor (MG3) LicA glucuronide metabolites (Fig. 1B; Sup- and EB (1 mg/kg/day) with GG (2 g/kg/day), GI (2 g/kg/day), or LicA (80 mg/kg/day) to ACI rats for 4 days. Results are normalized to EB control. plementary Fig. S2A–S2D). LicA also formed a GSH conjugate, which was a major metabolite in the liver (Supple- mentary Fig. S2; Fig. 1B) and a minor metabolite in the Supplementary Fig. S4) of the EB treatment group, serum and mammary tissues (Supplementary Fig. S2A and respectively. To analyze the influence of the botanicals S2C). Sulfation was minor in both serum and liver (Sup- on the overall amount of E2/E1 levels, the E2/E1 con- plementary Fig. S2A and S2B; Fig. 1B) and not detectable in centrations were also determined in serum. GG and GI mammary tissue (Supplementary Fig. S2C). LicA also slightly reduced E2/E1 levels; however, the difference did formed phase I metabolites (M1, M2, and M3); however, not reach significance (Supplementary Fig. S5). these metabolites were only detectable in vivo after their glucuronides were hydrolyzed with b-glucuronidase/ CYP1A1 and CYP1B1 expression in mammary tissues sulfatase (Supplementary Fig. S2D). These LicA phase I To assess the effect on P450 1A1 and 1B1 expression in and II metabolites have been previously determined from the mammary gland, CYP1A1 and CYP1B1 mRNA expres- incubations of LicA in liver microsomes (27). sion levels were measured. Treatment with EB significantly suppressed the expression of both, CYP1A1 and CYP1B1,to Licorice species and LicA increase NQO1 activity in the 3- and 5-fold lower than the vehicle control group, respec- liver tively (Fig. 5B). GG, GI, and LicA were administered with NQO1 activity was measured in liver and mammary EB; therefore, their effects were compared with the EB tissues. EB treatment alone did not affect the NQO1 activity treatment group to determine statistical significance. Inter- significantly in liver tissue (Fig. 4). EB treatment slightly estingly, both GG, but also GI, upregulated CYP1A1 to 7- reduced NQO1 activity in the mammary gland which is and 2-fold, respectively. Only GI caused significant down- consistent with previous reports (Supplementary Fig. S3; regulation of CYP1B1 expression, reducing it 3-fold (Fig. ref. 32). 5B); however, LicA-dosed rats showed no statistical differ- Upon cotreatment of EB with botanicals, GG and GI ence from the EB-dosed rats. significantly induced NQO1 activity to 2-fold of the EB fi control groups in liver tissue. LicA signi cantly increased CYP1A1 and CYP1B1 expression after treatment with GG, NQO1 activity to 1.5-fold (Fig. 4). No induction in NQO1 GI, and licorice compounds in MCF-10A cells activity by the licorice extracts or LicA was observed in To identify potential compounds that might be respon- mammary tissue (Supplementary Fig. S3). sible for the observed upregulation of CYP1A1 in mammary tissue by GG and especially GI, CYP1A1 and CYP1B1 GG, GI, and LicA significantly downregulate overall expressions were determined in MCF-10A cells after treat- estrogen oxidative metabolism in serum ment with GI, GG, LicA, LigC, and LigF. As expected from As rat P450 1B1 preferentially catalyzes estrogen previous results (11), GG caused an increase in both genes, 2-hydroxylation (20), the levels of 2-MeOE1 were mea- CYP1A1 (3-fold) and CYP1B1 (1.5-fold); however, after GI suredinserumsamplesasabiomarkerforoverallestro- treatment, CYP1A1 and CYP1B1 expressions were reduced gen oxidative metabolism. The results showed that all to nearly 13-fold and 6.5-fold less than basal levels, respec- three groups, GG, GI, and LicA, significantly reduced tively (Fig. 6A). LicA (20 mmol/L) was responsible for 2-MeOE1 by almost 60%, 70%, and 70% (Fig. 5A; decreases in both CYP1A1 and CYP1B1 expressions seen

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Figure 5. Inﬂuence of GG, GI, and LicA on estrogen oxidative metabolism in serum and modulation of CYP1A1 and CYP1B1 expression in mammary tissue. A, Serum

analyzed for 2-MeOE1 after ACI rats were dosed for 4 days with vehicle, EB (1 mg/kg/day), and EB (1 mg/kg/day) coadministered with GG (2 g/kg/day), GI (2 g/kg/day), or LicA (80 mg/kg/day). The vehicle control group did not contain quantifiable amounts of 2-MeOE1 (Supplementary Fig. S4). Significance was calculated by comparing EB treatment with the treatment groups (EB plus GG, or GI, or LicA). B, Mammary tissues were collected from these ACI rats, and CYP1A1/CYP1B1 expression was analyzed with RT-qPCR. Significance was determined by comparing EB treatment with vehicle control and by comparing the treatment groups (EB plus GG, or GI, or LicA) with EB treatment.

by GI (Fig. 6B), which were 34-fold and 10-fold lower than model, we explored GI's effect on detoxification basal levels, respectively. In contrast, LigC (20 mmol/L) (CYP1A1) and genotoxic (CYP1B1) pathways involved caused an increase in CYP1A1 and CYP1B1 expression. in estrogen chemical carcinogenesis and compared it Interestingly, LigC preferentially increased CYP1A1 to 4- with GG's effect. In addition, the contribution of LicA fold compared with a 2.5-fold induction of CYP1B1, and to GI's bioactivity and LicA's metabolism and distribu- LigF significantly increased CYP1B1 to 2-fold of control tion were analyzed. Although LicA was significantly (Fig. 6B). conjugated with glucuronic acid (Fig. 1B; Supplementary Fig. S2A–S2D), free LicA was still detected in serum, liver, and mammary tissues 24 hours after the last LicA injec- Discussion tion and oral administration of the GI extract (Supple- Licorice is contained in very popular BDS utilized for mentary Table S2; Supplementary Fig. S2; Fig. 3). This women's health and regarded as a potential chemo- demonstrated that LicA was bioavailable in its free, preventive agent (10, 33). It is also one of the prevalent bioactive form in target tissues. The present study cor- plants in Traditional Chinese Medicine (TCM) as about roborated previous reports (30, 35) that LigC and LigF 1/3 of TCM formulae contain licorice (Gan Cao; ref. 34). were bioavailable (Fig. 3A and B). In the United States, GG is the most frequently utilized GG, GI, and LicA induced NQO1 activity in the liver, but species. Besides GG, GI and also G. uralensis can be found not in mammary tissue (Fig. 4; Supplementary Fig. S3). The in European dietary supplements. In China, all three lack of NQO1 activity in the mammary gland is consistent species are cultivated and utilized without discrimina- with lower LicA levels in mammary tissue compared with tion as Gan Cao. GG, GI, and their compounds dem- the liver (Supplementary Table S2; Fig. 3C). In addition, onstrated differential effects on estrogen oxidative the inducibility of NQO1 in mammary epithelial cells has metabolism in MCF-10A cells due to their distinct chem- been demonstrated to be much lower than in liver cells ical profiles (11). In our previous studies, LigF had no (36). In support of this, other known NQO1 inducers, effect, and GG and LigC treatments led to an increase in 40bromoflavone, hops, and xanthohumol, caused only low estrogen oxidative metabolism, whereas GI and LicA (40bromoflavone) or no NQO1 induction in the mammary inhibited estrogen metabolism (11). In the ACI rat gland compared with significant NQO1 induction in liver

Figure 6. Modulation of CYP1A1 and CYP1B1 expression by GG, GI, and licorice compounds in MCF-10A cells. MCF-10A cells were treated with (A) licorice extracts, GG and GI (5 mg/mL), or (B) licorice compounds, LicA, LigC, and LigF (20 mmol/L), for 24 hours before analysis of CYP1A1/CYP1B1 expression by qPCR.

826 Cancer Prev Res; 11(12) December 2018 Cancer Prevention Research

Chemopreventive Activities of Licorice In Vivo

tissues of Sprague–Dawley rats (37). In comparison with its drug interaction potential are warranted. In contrast to vehicle, EB moderately reduced NQO1 activity in the the 2-MeOE1 levels, E1/E2 levels were only slightly influ- mammary gland in this study (Supplementary Fig. S3). enced by GG, GI, and LicA, suggesting that the decrease in Estradiol has been shown before to reduce NQO1 in ER- estrogen oxidative metabolism did not lead to an increase positive cells and estrogen-sensitive tissue (32). However, in E1/E2 levels (Supplementary Fig. S5). EB did not change NQO1 activity in the liver in the present To gauge the effects of GG, GI, and LicA on estrogen and previous investigations (Fig. 4; ref. 32). metabolism in mammary tissues, CYP1A1 and CYP1B1 The increase in NQO1 activity observed in liver tissues expressions were analyzed. These data closely correlated from GG, GI, and LicA groups confirmed the observed with 2-MeOE1 and 4-MeOE1 levels in our previous estro- in vitro NQO1-inducing properties in liver cells (21). LicA gen metabolism studies in MCF-10A and MCF-7 cells (Fig. 2) contains a Michael acceptor group that reacts with (11, 29, 46). A significant reduction in CYP1A1 and sulfhydryl groups, such as in Keap1. LicA GSH conjugates CYP1B1 by EB was observed in mammary tissues were detected in liver tissues which show reaction with (Fig. 5B). This estrogen effect on CYP1A1 has previously sulfur nucleophiles (Fig. 1B; Supplementary Fig. S2B and been described in MCF-7 cells (46). As GG and GI caused S2D). Activation of the Keap1/Nrf2 and ARE pathway by upregulation of CYP1A1 and GI additionally caused LicA and also LigC has been demonstrated previously in CYP1B1 downregulation, GG and GI promote estrogen in vitro studies (21, 38) leading to reduction of oxidative detoxification and GI also reduces the estrogen genotoxic stress (39, 40). However, although LigC formed GSH pathway in mammary tissue (Fig. 5B). Our current study conjugates in vivo (Sprague–Dawley rats) similar to LicA, and literature reports indicated that LigC preferentially LigC did not induce NQO1 activity in in vivo models increased biomarkers of the 2-hydroxylation pathway, (21, 30). In previous in vitro studies, LigF had no effect on 2-MeOE1 and CYP1A1, in MCF-10A cells (Fig. 6B; ref. 11) NQO1 activity (21). In the case of GG, these data suggest and increased CYP1A1 levels (6.84-fold) in mammary that other phytoconstituents other than LigC or LigF must tissues from female Sprague–Dawley rats (30). Thus, LigC contribute to the observed NQO1-inducing activity is suggested to significantly contribute to the reversal of (Fig. 4). E2-mediated CYP1A1 downregulation as seen after GG and GG, GI, and LicA caused a significant decrease in 2- GI administration (Fig. 5B). Conversely, LicA, an AhR MeOE1 levels (Fig. 5A) in serum. P450 1B1 in rats primarily antagonist that inhibited CYP1A1/CYP1B1 expression in catalyzes estrogen 2-hydroxylation; therefore, 2-MeOE1 MCF-10A cells (Fig. 6B) and XRE-luciferase reporter activ- was used as a general biomarker for estrogen oxidative ity in HepG2 cells (11), primarily downregulated CYP1B1 metabolism (20). Estrogen hydroxylation in the liver is expression in vivo after GI administration (Fig. 5B). Sur- higher than in any other tissue and is predominantly prisingly, GI did not decrease CYP1A1 expression in ACI performed by P450 3A4 and P450 1A2 (41). P450 rats (Fig. 5B), although GI and LicA treatments caused a 1A1/2 and P450 1B1 are regulated by the aryl hydrocarbon significant reduction in CYP1A1 well below basal levels receptor (AhR) and the xenobiotic response element in MCF-10A cells (Fig. 6A). The GI crude extract used in (XRE; Fig. 1). LicA is an AhR antagonist (Fig. 1; ref. 11), MCF-10A cells and ACI rats (Table 1; Fig. 3B) contained and because P450 1A2 is regulated by AhR (42), it can be LigC glycosides that were hydrolyzed extensively in the downregulated by LicA similarly to P450 1A1 (Fig. 6B) gut/intestine, increasing the concentration of LigC agly- leading to reduced formation of 2-MeOE1 levels in serum. cone and consequently the LigC:LicA ratio and CYP1A1 It is interesting that GG, which led to an induction of induction in mammary tissues (Figs. 3B and 5B). LigF did 2-MeOE1 in MCF-10A cells (Fig. 6), downregulated not affect CYP1A1 expression in MCF-10A cells in these and 2-MeOE1 levels in serum in this study (Fig. 5A). Literature previous studies (Fig. 6B; ref. 11). Considering that data demonstrate that GG as well as GI, and licorice GG exhibits a very complex phytochemical profile, other compounds inhibit P450 3A4 and P450 1A2 activity constituents of GG may add to the observed CYP1A1 leading to reduction of estrogen oxidative metabolism induction (16). as demonstrated by reduced 2-MeOE1 levels (Fig. 5A; In summary, this study investigated the effects of two refs. 43, 44). Specifically, P450 1A2 and P450 3A4 activity popular licorice species on biomarkers of detoxification was moderately inhibited by GI and LicA and only weakly and genotoxic estrogen metabolism pathways in ACI rats by GG, glabridin, and LigC (43, 44). LicA inhibited P450 and breast epithelial cells. GG and GI increased NQO1 3A4 irreversibly as a mechanism-based inhibitor (44) and activity in the liver (Fig. 4) and the detoxification estrogen decreased P450 3A4 gene expression in HepG2 cells (45). 2-hydroxyation pathway in the mammary tissues of ACI These results suggest that GG, GI, and LicA might interfere rats (Fig. 5B). In addition, GI also decreased CYP1B1 with general P450 metabolism (44); however, it is unclear expression (genotoxic pathway), because it contains at this point if these two licorice species lead to clinically the chemopreventive AhR antagonist, LicA (Fig. 5B). relevant drug–botanical interactions. Clinical studies to Other studies suggest that GI has superior chemopreven- analyze the influence of GG and GI on P450 enzymes and tive properties to GG (11, 47). GI had the greatest

www.aacrjournals.org Cancer Prev Res; 11(12) December 2018 827

Wang et al.

anti-inflammatory activity among medicinal licorice spe- Analysis and interpretation of data (e.g., statistical analysis, bio- cies in macrophage cells because it contains two major anti- statistics, computational analysis): S. Wang, T.L. Dunlap, L. Huang, inflammatory compounds, LicA and LigC (11). Also, GI C.E. Howell, H. Dong, B.M. Dietz b Writing, review, and/or revision of the manuscript: S. Wang, contains the ER preferential agonist 8-prenylapigenin T.L. Dunlap, L. Huang, C. Simmler, C.E. Howell, S-N. Chen, fi (47), which may have a better safety pro le. Long-term G.F. Pauli, R.B. van Breemen, B.M. Dietz, J.L. Bolton, B.M. Dietz studies are planned to compare the effect of GG and GI on Administrative, technical, or material support (i.e., reporting or E2-induced mammary carcinogenesis in ACI rats and ulti- organizing data, constructing databases): S. Wang, T.L. Dunlap, mately estrogen metabolism in women. This study high- L. Huang, R.B. van Breemen, B.M. Dietz, J.L. Bolton lights the fact that the chemopreventive bioactivity of Study supervision: S. Wang, G.F. Pauli, B.M. Dietz, J.L. Bolton in vivo licorice species cannot be reduced to the activity of single Other (prepared LicoA for assays): Y. Liu Other (botanical integrity to ensure consistency and repro- bioactive compounds, but is rather a result of multiple ducibility): S.-N. Chen, C. Simmler, G.F. Pauli constituents leading to polypharmacologic actions. Fur- thermore, knowing that licorice species have very different Acknowledgments chemical profiles (14) that profoundly affect their bioac- This work was supported by NIH grant P50 AT000155 from the tivity in vitro as well as in vivo should help design safer and NIH Office of Dietary Supplements (ODS) and the National Center for more efficacious botanicals with the greatest chemo- Complementary and Integrative Health (NCCIH) to the UIC/NIH preventive potential in women. These data suggest that GI Center for Botanical Dietary Supplements Research and by NIH grant containing the chemopreventive compounds, LicA and U41 AT008706 from NCCIH/ODS to the Center for Natural Product Technologies. The construction of the UIC CSB NMR facility and LigC, might be the optimal licorice species used for instrumentation was funded by NIGMS grant P41 GM068944 to women's health. Dr. Peter Gettins. We thank Shimadzu for providing the LCMS-8060 mass spectrometer used in this investigation. We also thank Dr. Liang Disclosure of Potential Conflicts of Interest Zhao at Lanzhou Institute of Chemical Physics, CAS, and Qinghai Lake No potential conflicts of interest were disclosed. Medicinal CO., Ltd. for their generous gifts.

Authors' Contributions The costs of publication of this article were defrayed in part by the Conception and design: S. Wang, L. Huang, G.F. Pauli, B.M. Dietz, payment of page charges. This article must therefore be hereby marked J.L. Bolton advertisement in accordance with 18 U.S.C. Section 1734 solely to Development of methodology: S. Wang, L. Huang, Y. Liu, J.L. Bolton indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Wang, T.L. Dunlap, L. Huang, Received May 14, 2018; revised August 17, 2018; accepted October C. Simmler, D.D. Lantvit, J. Crosby, H. Dong, R.B. van Breemen 3, 2018; published ﬁrst October 4, 2018.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J 8. Bolton JL, Dunlap T. Formation and biological targets of qui- Clin 2018;68:7–30. nones: cytotoxic versus cytoprotective effects. Chem Res Toxicol 2. Manson JE, Chlebowski RT, Stefanick ML, Aragaki AK, Rossouw 2017;30:13–37. JE, Prentice RL, et al. Menopausal hormone therapy and health 9. Yager JD. Mechanisms of estrogen carcinogenesis: The role of E2/ outcomes during the intervention and extended poststopping E1–quinone metabolites suggests new approaches to preventive phases of the Women's Health Initiative randomized trials. JAMA intervention – A review. Steroids 2015;99:56–60. 2013;310:1353–68. 10. Dietz BM, Hajirahimkhan A, Dunlap TL, Bolton JL. Botanicals 3. Russo J, Russo IH. The role of estrogen in the initiation of breast and their bioactive phytochemicals for women's health. Pharma- cancer. J Steroid Biochem Mol Biol 2006;102:89–96. col Rev 2016;68:1026–73. 4. Yang L, Zahid M, Liao Y, Rogan EG, Cavalieri EL, Davidson 11. Dunlap TL, Wang S, Simmler C, Chen S-N, Pauli GF, Dietz BM, NE, et al. Reduced formation of depurinating estrogen-DNA et al. Differential effects of Glycyrrhiza species on genotoxic adducts by sulforaphane or KEAP1 disruption in human estrogen metabolism: licochalcone A downregulates P450 1B1, mammary epithelial MCF-10A cells. Carcinogenesis 2013; whereas isoliquiritigenin stimulates it. Chem Res Toxicol 2015; 34:2587–92. 28:1584–94. 5. Sampson JN, Falk RT, Schairer C, Moore SC, Fuhrman BJ, Dallal 12. Kondo K, Shiba M, Yamaji H, Morota T, Zhengmin C, Huixia P, CM, et al. Association of estrogen metabolism with breast cancer et al. Species identification of licorice using nrDNA and cpDNA risk in different cohorts of postmenopausal women. Cancer Res genetic markers. Biol Pharm Bull 2007;30:1497–502. 2017;77:918–25. 13. Hajirahimkhan A, Simmler C, Yuan Y, Anderson JR, Chen S-N, 6. Gupta M, McDougal A, Safe S. Estrogenic and antiestrogenic Nikolic D, et al. Evaluation of estrogenic activity of licorice species activities of 16alpha- and 2-hydroxy metabolites of 17beta- in comparison with hops used in botanicals for menopausal estradiol in MCF-7 and T47D human breast cancer cells. J Steroid symptoms. PLoS One 2013;8:e67947. Biochem Mol Biol 1998;67:413–9. 14. Simmler C, Anderson JR, Gauthier L, Lankin DC, McAlpine JB, 7. Wen W, Ren Z, Shu XO, Cai Q, Ye C, Gao Y-T, et al. Expression of Chen SN, et al. Metabolite profiling and classification of DNA- cytochrome P450 1B1 and catechol-O-methyltransferase in authenticated licorice botanicals. J Nat Prod 2015;78:2007–22. breast tissue and their associations with breast cancer risk. Cancer 15. Tao W, Duan J, Zhao R, Li X, Yan H, Li J, et al. Comparison of three Epidemiol Biomarkers Prev 2007;16:917–20. officinal Chinese pharmacopoeia species of Glycyrrhiza based on

828 Cancer Prev Res; 11(12) December 2018 Cancer Prevention Research

Chemopreventive Activities of Licorice In Vivo

separation and quantification of triterpene saponins and chemo- 31. Prochaska HJ, Santamaria AB. Direct measurement of NAD(P)H: metrics analysis. Food Chem 2013;141:1681–9. quinone reductase from cells cultured in microtiter wells: a 16. Farag MA, Porzel A, Wessjohann LA. Comparative metabolite screening assay for anticarcinogenic enzyme inducers. Anal profiling and fingerprinting of medicinal licorice roots using a Biochem 1988;169:328–36. multiplex approach of GC-MS, LC-MS and 1D NMR techniques. 32. Ansell PJ, Espinosa-Nicholas C, Curran EM, Judy BM, Philips BJ, Phytochemistry 2012;76:60–72. Hannink M, et al. In vitro and in vivo regulation of antioxidant 17. Mersereau JE, Levy N, Staub RE, Baggett S, Zogovic T, Chow response element-dependent gene expression by estrogens. S, et al. Liquiritigenin is a plant-derived highly selective Endocrinology 2004;145:311–7. estrogen receptor beta agonist. Mol Cell Endocrinol 2008; 33. Wang R, Zhang CY, Bai LP, Pan HD, Shu LM, Kong A-NT, et al. 283:49–57. Flavonoids derived from liquorice suppress murine macrophage 18. Simmler C, Hajirahimkhan A, Lankin DC, Bolton JL, Jones T, activation by up-regulating heme oxygenase-1 independent of Soejarto DD, et al. Dynamic residual complexity of the isoliquir- Nrf2 activation. Int Immunopharmacol 2015;28:917–24. itigenin-liquiritigenin interconversion during bioassay. J Agric 34. Wang X, Zhang H, Chen L, Shan L, Fan G, Gao X. Liquorice, a Food Chem 2013;61:2146–57. unique "guide drug" of traditional Chinese medicine: a review of 19. Shull JD, Spady TJ, Snyder MC, Johansson SL, Pennington KL. its role in drug interactions. J Ethnopharmacol 2013;150:781–90. Ovary-intact, but not ovariectomized female ACI rats treated 35. Madak-Erdogan Z, Gong P, Zhao YC, Xu L, Wrobel KU, Hartman with 17b-estradiol rapidly develop mammary carcinoma. JA, et al. Dietary licorice root supplementation reduces diet- Carcinogenesis 1997;18:1595–601. induced weight gain, lipid deposition, and hepatic steatosis in 20. Nishida CR, Everett S, Ortiz de Montellano PR. Specificity deter- ovariectomized mice without stimulating reproductive tissues minants of CYP1B1 estradiol hydroxylation. Mol Pharmacol and mammary gland. Mol Nutr Food Res 2016;60:369–80. 2013;84:451–8. 36. Jiang Z-Q, Chen C, Yang B, Hebbar V, Kong A-NT. Differential 21. Hajirahimkhan A, Simmler C, Dong H, Lantvit DD, Li G, Chen responses from seven mammalian cell lines to the treatments of SN, et al. Induction of NAD(P)H:quinone oxidoreductase 1 detoxifying enzyme inducers. Life Sci 2003;72:2243–53. (NQO1) by Glycyrrhiza species used for women's health: differ- 37. Dietz BM, Hagos GK, Eskra JN, Wijewickrama GT, Anderson JR, ential effects of the Michael acceptors isoliquiritigenin and lico- Nikolic D, et al. Differential regulation of detoxification enzymes chalcone A. Chem Res Toxicol 2015;28:2130–41. in hepatic and mammary tissue by hops (Humulus lupulus) in vitro 22. Simmler C, Jones T, Anderson JR, Nikolic DC, van Breemen RB, and in vivo. Mol Nutr Food Res 2013;57:1055–66. Soejarto DD, et al. Species-specific standardisation of licorice by 38. Luo Y, Eggler AL, Liu D, Liu G, Mesecar AD, van Breemen RB. Sites metabolomic profiling of flavanones and chalcones. Phytochem of alkylation of human keap1 by natural chemoprevention Anal 2014;25:378–88. agents. J Am Soc Mass Spectrom 2007;18:2226–32. 23. Liu Y, Friesen JB, Grzelak EM, Fan Q, Tang T, Duric K, et al. Sweet 39. Dinkova-Kostova AT, Kostov RV, Canning P. Keap1, the cysteine- spot matching: a thin-layer chromatography-based countercur- based mammalian intracellular sensor for electrophiles and rent solvent system selection strategy. J Chromatogr A 2017; oxidants. Arch Biochem Biophys 2017;617:84–93. 1504:46–54. 40. Kuhnl€ J, Roggenkamp D, Gehrke SA, St€ab F, Wenck H, Kolbe L, 24. Liu Y, Friesen JB, Klein LL, McAlpine JB, Lankin DC, Pauli GF, et al. et al. Licochalcone A activates Nrf2 in vitro and contributes to The generally useful estimate of solvent systems (GUESS) method licorice extract-induced lowered cutaneous oxidative stress in vivo. enables the rapid purification of methylpyridoxine regioisomers Exp Dermatol 2015;24:42–7. by countercurrent chromatography. J Chromatogr A 2015;1426: 41. Tsuchiya Y, Nakajima M, Yokoi T. Cytochrome P450-mediated 248–51. metabolism of estrogens and its regulation in human. Cancer Lett 25. Liu Y, Friesen JB, McAlpine JB, Pauli GF. Solvent system selection 2005;227:115–24. strategies in countercurrent separation. Planta Med 2015;81: 42. Hankinson O. The role of AHR-inducible cytochrome P450s in 1582–91. metabolism of polyunsaturated fatty acids. Drug Metab Rev 26. Wang QE, Lee FSC, Wang X. Isolation and purification of infla- 2016;48:342–50. coumarin A and licochalcone A from licorice by high-speed 43. He W, Wu J-J, Ning J, Hou J, Xin H, He Y-Q, et al. Inhibition of counter-current chromatography. J Chromatogr A 2004;1048: human cytochrome P450 enzymes by licochalcone A, a naturally 51–7. occurring constituent of licorice. Toxicol Vitr 2015;29:1569–76. 27. Huang L, Nikolic D, van Breemen RB. Hepatic metabolism of 44. Li G, Simmler C, Chen L, Nikolic D, Chen SN, Pauli GF, et al. licochalcone A, a potential chemopreventive chalcone from lic- Cytochrome P450 inhibition by three licorice species and four- orice (Glycyrrhiza inflata), determined using liquid chromatogra- teen licorice constituents. Eur J Pharm Sci 2017;109:182–90. phy-tandem mass spectrometry. Anal Bioanal Chem 2017;409: 45. Chen H, Zhang X, Feng Y, Rui W, Shi Z, Wu L. Bioactive 6937–48. components of Glycyrrhiza uralensis mediate drug functions and 28. Li G, Nikolic D, van Breemen RB. Identification and chemical properties through regulation of CYP450 enzymes. Mol Med Rep standardization of licorice raw materials and dietary supplements 2014;10:1355–62. using UHPLC-MS/MS. J Agric Food Chem 2016;64:8062–70. 46. Dunlap TL, Howell CE, Mukand N, Chen S-N, Pauli GF, Dietz BM, 29. Wang S, Dunlap TL, Howell CE, Mbachu OC, Rue EA, Phansalkar et al. Red clover aryl hydrocarbon receptor (AhR) and estrogen R, et al. Hop (Humulus lupulus L.) extract and 6-prenylnaringenin receptor (ER) agonists enhance genotoxic estrogen metabolism. induce P450 1A1 catalyzed estrogen 2-hydroxylation. Chem Res Chem Res Toxicol 2017;30:2084–92. Toxicol 2016;29:1142–50. 47. Hajirahimkhan A, Mbachu O, Simmler C, Ellis S, Dong H, Nikolic 30. Cuendet M, Guo J, Luo Y, Chen S, Oteham CP, Moon RC, et al. D, et al. Estrogen receptor (ER) subtype selectivity identifies 8- Cancer chemopreventive activity and metabolism of isoliquir- prenylapigenin as an ERb agonist from Glycyrrhiza inflata and itigenin, a compound found in licorice. Cancer Prev Res 2010; highlights the importance of chemical and biological authenti- 3:221–32. cation. J Nat Prod 2018;81:966–75.

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Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats

Shuai Wang, Tareisha L. Dunlap, Lingyi Huang, et al.

Cancer Prev Res 2018;11:819-830. Published OnlineFirst October 4, 2018.

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