Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats

Author Manuscript Published OnlineFirst on October 4, 2018; DOI: 10.1158/1940-6207.CAPR-18-0178 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Title: Evidence for chemopreventive and resilience activity of licorice: Glycyrrhiza glabra and 2 G. inflata extracts modulate estrogen metabolism in ACI rats 3 4 Shuai Wang1, Tareisha L. Dunlap1, Lingyi Huang1, Yang Liu1,4, Charlotte Simmler1, 2, Daniel D. 5 Lantvit1, Jenna Crosby1, Caitlin E. Howell1, Huali Dong1, Shao-Nong Chen1, 2, Guido F. Pauli1, 2, 6 Richard B. van Breemen3, Birgit M. Dietz1, Judy L. Bolton1 7 8 Authors’ Affiliations: 1UIC/NIH Center for Botanical Dietary Supplements Research, 2Center for 9 Natural Product Technologies, Department of Medicinal Chemistry and Pharmacognosy, College of 10 Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA; 3Linus Pauling Institute, Oregon 11 State University, Corvallis, OR 97331, USA. 12 13 4Current Address: Research and Innovation, United States Pharmacopeia, Rockville, MD 20852, 14 USA. 15 16 S. Wang and T. Dunlap contributed equally to this article. 17 18 19 Corresponding Authors: 20 Judy L. Bolton, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, 21 University of Illinois at Chicago, Chicago, IL 60612, USA. Phone: (312) 996-5280, Email: 22 [email protected] 23 24 Birgit M. Dietz, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, 25 University of Illinois at Chicago, Chicago, IL 60612, USA. Phone (312) 996-2358, Email: 26 [email protected] 27 28 Running Title: Chemopreventive activities of licorice in vivo 29 30 Keywords: Chemoprevention, licorice, estrogen metabolism, Glycyrrhiza inflata, licochalcone A 31 32 Financial Support: P50 AT000155 by NCCIH and ODS to the UIC/NIH Center for Botanical Dietary 33 Supplements Research and U41 AT008706 by NCCIH/ODS to the Center for Natural Product 34 Technologies. 35 36 Conflict of Interest: The authors declare no potential conflicts of interest. 37 38 Word count of text: 4859; Number of figures: 6; Number of tables: 1; abstract word count: 248 39 40 41 Abbreviations: Arylhydrocarbon receptor (AhR), botanical dietary supplements (BDS), hormone 42 therapy (HT), catechol-O-methyltransferase (COMT), estrogen receptor (ER), estradiol benzoate 43 (EB), Glycyrrhiza glabra (GG), Glycyrrhiza inflata (GI), isoliquiritigenin (LigC), licochalcone A (LicA), 44 liquiritigenin (LigF), NAD(P)H quinone oxidoreductase 1 (NQO1), SULT sulfotransferases, traditional 45 Chinese Medicine (TCM), UDP-glucuronosyltransferase (UGT), xenobiotic response element (XRE).

Downloaded from cancerpreventionresearch.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 4, 2018; DOI: 10.1158/1940-6207.CAPR-18-0178 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

46 Abstract 47 Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot

48 flashes. While licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDSs, the exact

49 plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A),

50 Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1

51 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity,

52 respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker

53 compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1. The objective of this study was

54 to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether

55 GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In

56 addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention

57 (CYP1A1) as well as carcinogenesis (CYP1B1) were studied. LicA was extensively glucuronidated

58 and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after

59 oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In

60 mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased

61 CYP1A1. LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration.

62 In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1. These studies highlight

63 the polypharmacological nature of botanicals and the importance of standardization of licorice BDSs

64 to specific Glycyrrhiza species and to multiple constituents.

66 Introduction 67 In 2018, breast cancer will account for nearly one-third of new cancer cases in women (1). A

68 decline in HT (hormone therapy) usage was observed after the Women’s Health Initiative study in

69 2002 due to the increased breast cancer risk caused by the estrogen + progestin HT regimen (2).

70 Besides the well-known hormonal estrogen carcinogenesis pathway, breast cancer risk is also

71 influenced by changes in estrogen oxidative metabolism (3). In the breast, P450 1A1 and P450 1B1

72 catalyze the metabolism of estrogens to 2- and 4-hydroxylated catechols, 2-OHE1/E2 and 4-OHE1/E2,

73 respectively (Fig. 1) (4). The 2-hydroxylated metabolites are strongly associated with reduced breast

74 cancer risk (5) because they inhibit E2-induced proliferation (6) and are converted to non-toxic

75 quinones (Fig. 1). On the other hand, P450 1B1 is linked to carcinogenesis because it is

76 overexpressed in malignant tissues (7). P450 1B1 produces 4-OHE1/E2 which are oxidized by

77 peroxidases/P450s to genotoxic quinones (4-OHE1/E2-Q) that oxidize and alkylate DNA and generate

78 depurinating adducts (estrogen chemical carcinogenesis, Fig. 1) (4,8). Hence, upregulation of the 2-

79 hydroxylation (P450 1A1) and downregulation of the 4-hydroxylation pathway (P450 1B1) may

80 significantly inhibit estrogen chemical carcinogenesis in the breast. Additionally, NAD(P)H quinone

81 oxidoreductase 1 (NQO1) decreases depurinating estrogen-DNA adducts (4) due to reduction of the

82 reactive 4-OHE1/E2 -Q to its catechol. Catechol-O-methyltransferase (COMT) also prevents quinone

83 formation through methylation of estrogen catechols to produce stable metabolites, 2-MeOE1/E2 and

84 4-MeOE1/E2 (Fig. 1) (9).

85 Due to fear of increased breast cancer risk with HT, many women have turned to botanical

86 dietary supplements (BDS) as a complementary approach therapy for menopausal symptom relief.

87 Female consumers generally perceive BDSs as safer modalities because BDSs often contain

88 constituents that are reported to be anti-inflammatory, antioxidant, and induce detoxification enzymes

89 (10). However, the effects of most BDSs on estrogen oxidative metabolism is unknown. While licorice

90 belongs to one of the most popular botanicals contained in BDSs used for women’s health issues,

91 several licorice species (Glycyrrhiza sp., Fabaceae) are used to source these BDSs without

92 discrimination (11). Also, clinical evidence for efficacy is generally lacking (10).

93 Botanically, over 30 species of licorice exist (12,13). From a pharmacopoeial perspective,

94 three species are currently used in BDSs interchangeably: Glycyrrhiza glabra (GG), G. inflata (GI),

95 and G. uralensis. Notably, these three species have distinctive and very different chemical profiles, as

96 already demonstrated in previous studies (Table 1) (12,14). Cultivated in China, GI is naturally more

97 popular in Asia. The roots from this species contain an abundant species-specific chemopreventive

98 Michael acceptor, licochalcone A (LicA) (Fig. 2). GG is the most popular licorice species in the United

99 States and Europe and its species-specific compound is glabridin (Table 1 and Fig. 2) (15,16). Both

100 licorice species contain isoliquiritigenin (LigC, C for chalcone), also a chemopreventive Michael

101 acceptor, and liquiritigenin (LigF, F for flavanone), a phytoestrogen with estrogen receptor (ER) β

102 preferential properties (13,17) (Fig. 2). Both LigC and LigF are spontaneously interconvertible

103 isomers (Fig. 2) (18), found mainly as glycosides (e.g. liquiritin and isoliquiritin) in the roots (Table 1).

104 Due to the species-specific compound profile, each licorice species has a unique bioactivity that could

105 lead to differential clinical activity.

106 Previously, we showed that GG and LigC increased estrogen oxidative metabolism (2- and 4-

107 hydroxylation), whereas GI and LicA decreased metabolism in MCF-10A “normal” breast epithelial

108 cells (11). The purpose of the current study was to determine the chemopreventive effects of GI and

109 GG on estrogen oxidative metabolism in the ACI rat model, which is frequently used for in vivo

110 estrogen carcinogenesis studies (19). LicA was administered in parallel to determine its role in GI’s

111 bioactivity and its metabolic profile. LicA’s distribution in liver and mammary tissues was determined

112 and compared to LigC and LigF. Levels of 2-MeOE1 in serum were quantified by LC-MS/MS, as a

113 biomarker for overall estrogen oxidative metabolism, because rat P450 1B1 predominantly performs

114 estrogen 2-hydroxylation (20). CYP1A1 and CYP1B1 expression were determined in mammary

115 tissues and NQO1 activity was measured in liver and mammary tissues. Studies in MCF-10A cells

116 with GI revealed different results between in vitro and in vivo studies and identified potential bioactive 4

117 compounds. The current experiments carried out in ACI rats provide crucial information to continue

118 studying these licorice species and their effects on estrogen carcinogenesis in long-term studies with

119 ACI rats and eventually estrogen metabolism in women. The data herein explore additional

120 chemopreventive modes to help develop more beneficial and safer standardized licorice dietary

121 supplements for women’s health.

122

123 Materials and Methods 124

125 Materials Chemicals and reagents. All chemicals and reagents were purchased from Fisher Scientific

126 (Hanover Park, IL) or Sigma (St. Louis, MO) except for the following: 2-methoxyestrone (MeOE1)-

127 1,4,16,16-d4 was obtained from CDN isotope (Pointe-Claire, Quebec), E2 and 2-/4-MeOE1/E2

128 reference compounds were acquired from Steraloids Inc. (Newport, RI), LigF and LigC were obtained

129 from ChromaDex (Irvine, CA), and rat serum for initial standardization studies was purchased from

130 BioreclamationIVT (Chestertown, MD).

131

132 Plant material, extraction, and characterization. GI was provided as a gift to SNC by Dr. Liang

133 Zhao (Lanzhou Institute of Chemical Physics CAS) and GG was purchased from Mountain Rose

134 Herbs (OR, USA). Raw materials were identified by macroscopic/microscopic analyses and DNA

135 barcoding, as previously described (14). Ground roots of GG and GI were extracted by maceration

136 and percolation at room temperature with a solvent mixture [ethanol (200 USP proof), isopropanol,

137 and water (90:5:5, v/v)] at the ratio of botanical to solvent as 1:15 (g/mL). The extracts were

138 concentrated and freeze dried to yield 12% w/w of the initial ground roots. Extracts were analyzed by

139 UHPLC-UV to quantify major chalcone and flavanone constituents, as previously reported (21,22).

140 Briefly, a standard curve containing the following 11 reference standards was used for their

141 quantitation in both licorice extracts. The area under the curve (AUC) was taken at 360 nm for all

142 chalcones (isoliquiritin, isoliquiritin apioside, licuraside, LigC, LicA), and at 275 nm for all flavanones

143 (liquiritin, liquiritin apioside, liquiritigenin 7-O-apiosylglucoside, LigF) and for glabridin. Quantitative

144 results obtained for each LigF glycoside or LigC glycoside were converted by their molecular weight,

145 thereby leading to their concentration as LigF or LigC equivalents, respectively (Table 1).

146

147 Preparation and characterization of Licochalcone A. The crude LicA sample, enriched from GI

148 (eLicA, purity of LicA, ~ 50%) was a gift to SNC from Qinghai Lake Medicinal CO., Ltd. A loss-free 6

149 countercurrent separation was implemented for the purification of LicA from the eLicA, as follows:

150 TLC-based solvent system strategy (23) was performed for screening a proper solvent system.

151 Among screened solvent systems (Supplementary Table S1), LicA was eluted by the organic phase

152 of n-hexane-ethyl acetate-methanol-water (HEMWat, 4:6:5:5, v/v) to Rf = 0.43 on a pre-coating

153 normal-phase Si TLC plate (MACHEREY-NAGEL, Bethlehem, PA, USA). Considering the main

154 principles of the GUESS method (Generally Useful Estimate of Solvent Systems) (24), this data

155 suggested that the corresponding solvent system may elute the target analyte into a high resolution

156 separation range (the sweet spot, partition coefficient, K value from 0.25 to 4 of a countercurrent

157 separation method) (25). Regarding verification of the selected solvent system, an analytical scale of

158 high-speed countercurrent chromatography (HSCCC) (16 mL, Tauto Biotech, Shanghai, China) was

159 applied, which achieved a higher purity of LicA than that from the literature reported solvent system

160 on the same instrument (Supplementary Fig. S1) (26). The scaled-up separation is described in the

161 Supplementary information. The purity of LicA used in this animal study was determined as 95%

162 (w/w) by qHNMR,

163

164 Animal experiment. Female August-Copenhagen Irish (ACI) rats were purchased from Harlan

165 Laboratories (Indianapolis, IN) at 5 weeks of age, acclimated for one week, fed a phytoestrogen free

166 diet (AIN-76A), and randomly divided into five groups with 6 rats each: vehicle, estradiol benzoate

167 (EB, 1 mg/kg/day), LicA (80 mg/kg/day) + EB, GG (2 g/kg/day, gavage) + EB, and GI (2 g/kg/day,

168 containing 141 mg LicA) + EB. At 6 weeks of age, one vehicle was applied subcutaneously (sc,

169 sesame oil) and one by gavage (50% corn oil with 50% PEG/H2O), EB and LicA were given sc, and

170 the licorice extracts were administered by gavage for four days. The rats were sacrificed by CO2

171 asphyxiation on day 5. Blood was collected, and serum prepared immediately after collection;

172 mammary tissues, uterus, and liver were collected and snap frozen in liquid nitrogen and stored at -

173 80 °C until analysis. The animal protocol complied with the Guide for the Care and Use of Laboratory

174 Animals and all procedures were approved by UIC’s Institutional Animal Care and Use Committee

175 (Protocol No. 16-033).

176

177 Preparation of serum and tissue samples for LicA metabolism profile analysis and for LicA

178 quantitation in vivo. LicA was quantified in serum, liver, and mammary gland samples from the LicA

179 + EB and GI + EB treatment groups (Fig. 3C and Supplementary Table 2). After thawing at room

180 temperature, serum (50 μL) was transferred to a 1.5 mL Eppendorf tube and mixed with 10 μL of

181 ACN containing naringenin (500 nM) as the internal standard (IS). Liver and mammary tissues (500-

182 800 mg for liver and 50-100 mg for mammary gland) were weighed and homogenized in 70%

183 aqueous methanol (containing 0.1% formic acid) at 5 mL for liver and 1 mL for mammary tissues. The

184 homogenate (200 μL) was taken and spiked with 20 μL naringenin (500 nM). Ice-cold ACN (600 μL)

185 was added and the mixture was centrifuged for 15 min at 13000 x g at 4 °C for protein precipitation.

186 The supernatant (400 μL) was transferred to a new Eppendorf tube and evaporated to dryness under

187 a stream of nitrogen. The residue was reconstituted in 100 μL of 20% ACN, 5 µL was injected into

188 LC-MS/MS for analysis. The same rat samples were analyzed for LicA metabolites.

189

190 UHPLC-MS/MS analyses. UHPLC-MS/MS analysis was performed as described previously (27,28).

191 Briefly, a Shimadzu (Kyoto, Japan) LCMS-8060 triple quadrupole mass spectrometer equipped with a

192 Shimadzu Nexera UHPLC system was used for analysis. For quantitative analysis, analytes were

193 separated on a Waters (Milford, MA) Acquity UPLC BEH C18 2.1 × 50 mm column (1.7 μm particle

194 size). Mass-spectrometer parameters were as follows: nebulizing gas flow: 2.5 L/min; heating gas

195 flow: 10 L/min; interface temperature: 300 °C; DL temperature: 250 °C; heating block temperature:

196 400 °C; drying gas flow: 10 L/min. The data were acquired using selected reaction monitoring (SRM)

197 with positive ion electrospray as follows: naringenin ([M+H]+ m/z 273 to 153 and m/z 273 to 147,

198 internal standard); and LicA ([M+H]+ m/z 339 to 121 and m/z 339 to 297).

199 For determination of LicA metabolites, the positive ion electrospray SRM transitions for each

200 analyte were established as m/z 515 to 339 for LicA monoglucuronides, m/z 646 to 339 for the

201 glutathione conjugate of LicA, m/z 419 to 339 for LicA sulfate, m/z 531 to 339 for monooxygenated

202 LicA glucuronides, and m/z 545 to 369 for catechol-O-methylated LicA glucuronides.

203 For qualitative determination of LigC, LigF, LicA, and glabridin in the GI and GG extracts,

204 serum, and tissues, the licorice extracts (GG and GI) were dissolved in 50% aqueous methanol at 10

205 μg/mL. Rat serum and tissue samples were prepared as described above. Samples were analyzed

206 using UHPLC-MS/MS with negative ion electrospray and SRM (28) as follows: liquiritin and

207 isoliquiritin, m/z 417 to 255; liquiritin apioside, isoliquiritin apioside and licuraside, m/z 549 to 255;

208 LigF and LigC, m/z 255 to 119; glabridin, m/z 323 to 201; and glycyrrhetinic acid, m/z 469 to 425.

209 Analysis of estrogen oxidative metabolism (2-MeOE1) and E1/E2 in serum. LC-MS/MS analysis 210 was performed as previously described with minor modifications (29). Serum samples (150 µl) were 211 incubated at 37 °C for 4 h after adding glucuronidase & sulfatase hydrolysis buffer (300 µl) and the

212 internal standard, 2-methoxyestrone-d4. After the incubation, sample preparation and analysis were 213 conducted as previously described (29). The SRM transitions were as follows: m/z 534.3 to 171.2,

214 m/z 504.3 to 171.0, and m/z 506.3 to 171.0, for 2-MeOE1, E1, and E2, respectively. Results are 215 expressed as fold change from average amount of analytes of rats treated with EB alone.

216

217 Analysis of NQO1 activity in liver tissue and mammary gland. The NQO1 activity in frozen liver

218 and mammary tissue was determined as described previously (30). The NQO1 activity was

219 determined in a clear supernatant solution of the tissue homogenate (5 µg of liver protein and 30 µg

220 of mammary gland protein) as previously described (31). The absorbance was measured at 610 nm

221 and and the results were expressed as fold induction of NQO1 activity relative to the control group.

222

223 Quantification of P450 1A1/1B1 mRNA expression via RT-qPCR. Mammary tissues (100 mg)

224 were homogenized in TRIzol reagent. The total RNA was extracted using the RNeasy lipid tissue kit 9

225 (Qiagen, Valencia, CA) and RNA (5 μg) was reverse transcribed with Invitrogen’s SuperScript® III

226 First-Strand Synthesis System. RT-qPCR was performed using TaqMan rat CYP1A1, CYP1B1, and

227 ACTB primers with FAM/MGB probe (Applied Biosystems, Carlsbad, CA) as described previously

228 (29). MCF-10A cells were obtained from ATCC (Manassas, VA) and authenticated via STR profiling

229 (Promega). MCF-10A cells were cultured as described previously (29). For the in vitro

230 CYP1A1/CYP1B1 induction experiments, cells having approximately 15 through 20 passages were

231 plated in 96-well plates and treated with vehicle (DMSO), GG, GI, and licorice compounds for 24 h.

232 RT-qPCR was performed as previously described (29) using TaqMan® 1-Step RT-PCR Master Mix,

233 and CYP1A1 and CYP1B1 primer with FAM-MGB probe and GAPDH primer with VIC-MGB probe.

234 Data were analyzed with the comparative CT (ΔΔCT) method and expressed as fold induction relative

235 to the vehicle control group.

236

237 Statistical analysis. The data were expressed as mean ± SEM from 6 animals per group or ± SEM

238 for three independent experiments in MCF-10A cells. Significance was determined using student t-

239 test to compare two samples or one-way ANOVA with Dunnett’s post-test to compare multiple

240 samples with the control (*p < 0.05).

241

242 Results

243 Measurement of bioactive compounds in Glycyrrhiza species. Extracts were analyzed by

244 UHPLC-UV and all compounds were expressed as % w/w of each extract (Table 1). Glabridin (Fig. 2)

245 was present at 1.34% of total GG extract, while LicA represents one of the major compounds of GI

246 crude extract (7.07%). Glabridin and LicA exist as aglycones, but LigC and LigF occur primarily as

247 glycosylated compounds, which exceeded the corresponding aglycones by 15-fold. LigC glycosides

248 (isoliquiritin, isoliquiritin apioside, and licuraside) and LigF glycosides (liquiritin, liquiritin apioside, and

249 liquiritigenin-7-O-apiosylglucoside) (Fig. 2) together with the corresponding aglycones are

250 represented as total LigC and LigF equivalents, as the glycosides are deglycosylated in vivo (Fig. 3A

251 and B). GG extract contained 1.5-fold more LigC equivalents and over 2-fold more LigF equivalents

252 than GI. The most abundant LigF glycoside (>70%) in GG extract was liquiritin apioside (Table 1).

253

254 Distribution of LicA, LigF, LigC, and glabridin in serum, liver, and mammary tissue after GG

255 and GI administration. LigC/LigF, LigC/LigF glycosides, glabridin, and LicA were qualitatively

256 determined in crude extracts, serum, liver, and mammary tissues by UHPLC-MS/MS analysis. The

257 concentration of glabridin was low in the serum and it was not detected in liver and mammary tissues

258 (Fig. 3A). The three other licorice compounds, LicA, LigF, and LigC were all available in the liver and

259 mammary gland. The most striking observation was that most of the LigC/LigF glycosides were

260 hydrolyzed, which increased LigC/LigF aglycone concentrations in the serum and both tissue

261 samples. LigC and LigF concentrations were similar in mammary tissues, although LigC was notably

262 higher in serum but significantly lower than LigF in liver tissues (Fig. 3A and B). In the GI crude

263 extract, LicA levels were much higher than free LigC; however, because LigC equivalents were

264 hydrolyzed in vivo, the concentration of free LigC exceeded LicA concentrations in serum and

265 mammary tissues (Fig. 3B). After LicA sc and GI gavage administration for 4 days, LicA was also

266 quantified by UHPLC-MS/MS in serum, liver tissue, and mammary gland. Interestingly, after both

267 applications, free LicA was available in serum, liver, and mammary gland 24 h after the last dose (day 11

268 5) (Fig. 3C). Although LicA is highly glucuronidated in vivo (Fig. 1B, Supplementary Fig. 2),

269 considerable amounts of free LicA were observed in the target tissues after oral administration of the

270 extract (Supplementary Table S2, Figure 3C).

271

272 Metabolism of LicA in serum, liver tissue, and mammary gland. LicA metabolites were analyzed

273 in serum, liver, and mammary gland samples by UHPLC-MS/MS after sc administration of LicA. In

274 serum, liver, and mammary tissues, glucuronidation reactions dominated LicA metabolism and

275 resulted in two major (MG1 and MG2) and one minor (MG3) LicA glucuronide metabolites (Fig. 1B

276 and Supplementary Fig. S2A-D). LicA also formed a GSH conjugate, which was a major metabolite in

277 the liver (Supplementary Fig. S2, Fig. 1B), and a minor metabolite in the serum and mammary tissues

278 (Supplementary Fig. S2A and C). Sulfation was minor in both serum and liver (Supplementary Fig.

279 S2A and B, Fig. 1B) and not detectable in mammary tissue (Supplementary Fig. S2C). LicA also

280 formed phase I metabolites (M1, M2, and M3); however, these metabolites were only detectable in

281 vivo after their glucuronides were hydrolyzed with β-glucuronidase/sulfatase (Supplementary Fig.

282 S2D). These LicA phase I and II metabolites have been previously determined from incubations of

283 LicA in liver microsomes (27).

284

285 Licorice species and LicA increase NQO1 activity in the liver. NQO1 activity was measured in

286 liver and mammary tissues. EB treatment alone did not affect the NQO1 activity significantly in liver

287 tissue (Fig. 4). EB treatment slightly reduced NQO1 activity in the mammary gland which is consistent

288 with previous reports (Supplementary Fig. S3) (32).

289 Upon co-treatment of EB with botanicals, GG and GI significantly induced NQO1 activity to 2-fold of

290 the EB control groups in liver tissue. LicA significantly increased NQO1 activity to 1.5-fold (Fig. 4). No

291 induction in NQO1 activity by the licorice extracts or LicA were observed in mammary tissue

292 (Supplementary Fig. S3).

293 12

294 GG, GI, and LicA significantly down-regulate overall estrogen oxidative metabolism in serum.

295 As rat P450 1B1 preferentially catalyze estrogen 2-hydroxylation (20), the levels of 2-MeOE1 were

296 measured in serum samples as a biomarker for overall estrogen oxidative metabolism. The results

297 showed that all three groups, GG, GI, and LicA significantly reduced 2-MeOE1 by almost 60%, 70%,

298 and 70% (Fig. 5A, Supplementary Fig. S4) of the EB treatment group, respectively. To analyze the

299 influence of the botanicals on the overall amount of E2/E1 levels, the E2/E1 concentrations were also

300 determined in serum. GG and GI slightly reduced E2/E1 levels; however, the difference did not reach

301 significance (Supplementary Fig. S5).

302

303 CYP1A1 and CYP1B1 expression in mammary tissues. To assess the effect on P450 1A1 and

304 1B1 expression in the mammary gland, CYP1A1 and CYP1B1 mRNA expression levels were

305 measured. Treatment with EB significantly suppressed the expression of both, CYP1A1 and CYP1B1,

306 to 3- and 5-fold lower than the vehicle control group, respectively (Fig. 5B). GG, GI, and LicA were

307 administered with EB, therefore their effects were compared to the EB treatment group to determine

308 statistical significance. Interestingly, both GG, but also GI upregulated CYP1A1 to 7- and 2-fold,

309 respectively. Only GI caused significant down-regulation of CYP1B1 expression, reducing it 3-fold

310 (Fig. 5B); however, LicA dosed rats showed no statistical difference from the EB dosed rats.

311

312 CYP1A1 and CYP1B1 expression after treatment with GG, GI, and licorice compounds in MCF-

313 10A cells. To identify potential compounds that might be responsible for the observed upregulation of

314 CYP1A1 in mammary tissue by GG and especially GI, CYP1A1 and CYP1B1 expression were

315 determined in MCF-10A cells after treatment with GI, GG, LicA, LigC, and LigF. As expected from

316 previous results (11), GG caused an increase in both genes, CYP1A1 (3-fold) and CYP1B1 (1.5-fold);

317 however, after GI treatment, CYP1A1 and CYP1B1 expression were reduced to nearly 13-fold and

318 6.5-fold less than basal levels, respectively (Fig. 6A). LicA (20 µM) was responsible for decreases in

319 both CYP1A1 and CYP1B1 expression seen by GI (Fig. 6B), which were 34-fold and 10-fold lower 13

320 than basal levels, respectively. In contrast, LigC (20 µM) caused an increase in CYP1A1 and

321 CYP1B1 expression. Interestingly, LigC preferentially increased CYP1A1 to 4-fold compared to a 2.5-

322 fold induction of CYP1B1, and LigF significantly increased CYP1B1 to 2-fold of control (Fig. 6B).

323

324 Discussion

325 Licorice is contained in very popular BDSs utilized for women’s health and regarded as a

326 potential chemopreventive agent (10,33). It is also one of the prevalent plants in Traditional Chinese

327 Medicine (TCM) as about 1/3 of TCM formulae contain licorice (Gan Cao) (34). In the USA , GG is the

328 most frequently utilized species. Besides GG, GI and also G. uralensis can be found in European

329 dietary supplements. In China, all three species are cultivated, and utilized without discrimination as

330 Gan Cao. GG, GI, and their compounds demonstrated differential effects on estrogen oxidative

331 metabolism in MCF-10A cells due to their distinct chemical profiles (11). In our previous studies, LigF

332 had no effect and GG and LigC treatments led to an increase in estrogen oxidative metabolism, while

333 GI and LicA inhibited estrogen metabolism (11). In the ACI rat model, we explored GI’s effect on

334 detoxification (CYP1A1) and genotoxic (CYP1B1) pathways involved in estrogen chemical

335 carcinogenesis and compared it to GG’s effect. In addition, the contribution of LicA to GI’s bioactivity

336 and LicA’s metabolism and distribution were analyzed. Although LicA was significantly conjugated

337 with glucuronic acid (Fig. 1B and Supplementary Fig. S2A-D), free LicA was still detected in serum,

338 liver, and mammary tissues 24 h after the last LicA injection and oral administration of the GI extract

339 (Supplementary Table S2, Fig. S2, Fig. 3). This demonstrated that LicA was bioavailable in its free,

340 bioactive form in target tissues. The present study corroborated previous reports (30,35) that LigC

341 and LigF were bioavailable (Fig. 3A and B).

342 GG, GI, and LicA induced NQO1 activity in the liver, but not in mammary tissue (Fig. 4 and

343 Supplementary Fig. S3). The lack of NQO1 activity in the mammary gland is consistent with lower

344 LicA levels in mammary tissue compared to the liver (Supplementary Table S2 and Fig. 3C). In

345 addition, the inducibility of NQO1 in mammary epithelial cells has been demonstrated to be much

346 lower than in liver cells (36). In support of this, other known NQO1 inducers, 4’bromoflavone, hops,

347 and xanthohumol, caused only low (4’bromoflavone) or no NQO1 induction in the mammary gland

348 compared to significant NQO1 induction in liver tissues of Sprague-Dawley rats (37). In comparison to

349 vehicle, EB moderately reduced NQO1 activity in the mammary gland in this study (Supplementary 15

350 Fig. S3). Estradiol has been shown before to reduce NQO1 in ER positive cells and estrogen

351 sensitive tissue (32). However, EB did not change NQO1 activity in the liver in the present and

352 previous investigations (Fig. 4) (32).

353 The increase in NQO1 activity observed in liver tissues from GG, GI, and LicA groups

354 confirmed the observed in vitro NQO1 inducing properties in liver cells (21). LicA (Fig. 2) contains a

355 Michael acceptor group that reacts with sulfhydryl groups, such as in Keap1. LicA GSH conjugates

356 were detected in liver tissues which shows reaction with sulfur nucleophiles (Fig. 1B and

357 Supplementary Fig. S2B and D). Activation of the Keap1/Nrf2 and ARE pathway by LicA and also

358 LigC has been demonstrated previously in in vitro studies (21,38) leading to reduction of oxidative

359 stress (39,40). However, although LigC formed GSH conjugates in vivo (Sprague-Dawley rats) similar

360 to LicA, LigC didn’t induce NQO1 activity in in vivo models (21,30). In previous in vitro studies, LigF

361 had no effect on NQO1 activity (21). In the case of GG, these data suggest that other

362 phytoconstituents other than LigC or LigF must contribute to the observed NQO1 inducing activity

363 (Fig. 4).

364 GG, GI, and LicA caused a significant decrease in 2-MeOE1 levels (Fig. 5A) in serum. P450

365 1B1 in rats primarily catalyzes estrogen 2-hydroxylation, therefore 2-MeOE1 was used as a general

366 biomarker for estrogen oxidative metabolism (20). Estrogen hydroxylation in the liver is higher than in

367 any other tissue and is predominantly performed by P450 3A4 and P450 1A2 (41). P450 1A1/2 and

368 P450 1B1 are regulated by the aryl hydrocarbon receptor (AhR) and the xenobiotic response element

369 (XRE) (Fig. 1). LicA is an AhR antagonist (Fig. 1) (11) and since P450 1A2 is regulated by AhR (42) it

370 can be downregulated by LicA similarly to P450 1A1 (Fig. 6B) leading to reduced formation of 2-

371 MeOE1 levels in serum. It is interesting that GG, which led to an induction of 2-MeOE1 in MCF-10A

372 cells (Fig.6), downregulated 2-MeOE1 levels in serum in this study (Fig. 5A). Literature data

373 demonstrates that GG as well as GI, and licorice compounds inhibit P450 3A4 and P450 1A2 activity

374 leading to reduction of estrogen oxidative metabolism as demonstrated by reduced 2-MeOE1 levels

375 (Fig 5A) (43,44). Specifically, P450 1A2 and P450 3A4 activity was moderately inhibited by GI and

376 LicA and only weakly by GG, glabridin, and LigC (43,44). LicA inhibited P450 3A4 irreversibly as a

377 mechanism-based inhibitor (44) and decreased P450 3A4 gene expression in HepG2 cells (45).

378 These results suggest that GG, GI, and LicA might interfere with general P450 metabolism (44);

379 however, it is unclear at this point if these two licorice species lead to clinically relevant drug-botanical

380 interactions. Clinical studies to analyze the influence of GG and GI on P450 enzymes and its drug

381 interaction potential are warranted. In contrast to the 2-MeOE1 levels, E1/E2 levels were only slightly

382 influenced by GG, GI and LicA, suggesting that the decrease in estrogen oxidative metabolism did

383 not lead to an increase in E1/E2 levels (Supplementary Fig. S5).

384 To gauge the effects of GG, GI, and LicA on estrogen metabolism in mammary tissues,

385 CYP1A1 and CYP1B1 expression, were analyzed. These data closely correlated with 2-MeOE1 and

386 4-MeOE1 levels in our previous estrogen metabolism studies in MCF-10A and MCF-7 cells

387 (11,29,46). A significant reduction in CYP1A1 and CYP1B1 by EB was observed in mammary tissues

388 (Fig. 5B). This estrogen effect on CYP1A1 has previously been described in MCF-7 cells (46). As GG

389 and GI caused upregulation of CYP1A1 and GI additionally caused CYP1B1 downregulation, GG and

390 GI promote estrogen detoxification and GI also reduces the estrogen genotoxic pathway in mammary

391 tissue (Fig. 5B). Our current study and literature reports indicated that LigC preferentially increased

392 biomarkers of the 2-hydroxylation pathway; 2-MeOE1 and CYP1A1, in MCF-10A cells (Fig. 6B) (11)

393 and increased CYP1A1 levels (6.84-fold) in mammary tissues from female Sprague-Dawley rats (30).

394 Thus, LigC is suggested to significantly contribute to the reversal of E2-mediated CYP1A1

395 downregulation as seen after GG and GI administration (Fig. 5B). Conversely, LicA, an AhR

396 antagonist that inhibited CYP1A1/CYP1B1 expression in MCF-10A cells (Fig. 6B) and XRE-luciferase

397 reporter activity in HepG2 cells (11), primarily downregulated CYP1B1 expression in vivo after GI

398 administration (Fig. 5B). Surprisingly, GI did not decrease CYP1A1 expression in ACI rats (Fig.5B),

399 although GI and LicA treatments caused a significant reduction in CYP1A1 well below basal levels in

400 MCF-10A cells (Fig. 6A). The GI crude extract used in MCF-10A cells and ACI rats (Table 1 and Fig. 17

401 3B) contained LigC glycosides that were hydrolyzed extensively in the gut/intestine, increasing the

402 concentration of LigC aglycone and consequently the LigC:LicA ratio and CYP1A1 induction in

403 mammary tissues (Fig. 3B, Fig. 5B). LigF did not affect CYP1A1 expression in MCF-10A cells in

404 these and previous studies (Fig. 6B) (11). Considering that GG exhibits a very complex

405 phytochemical profile other constituents of GG may add to the observed CYP1A1 induction (16).

406 In summary, this study investigated the effects of two popular licorice species on biomarkers of

407 detoxification and genotoxic estrogen metabolism pathways in ACI rats and breast epithelial cells. GG

408 and GI increased NQO1 activity in the liver (Fig. 4) and the detoxification estrogen 2-hydroxyation

409 pathway in the mammary tissues of ACI rats (Fig. 5B). In addition, GI also decreased CYP1B1

410 expression (genotoxic pathway), because it contains the chemopreventive AhR antagonist, LicA (Fig.

411 5B). Other studies suggest that GI has superior chemopreventive properties to GG (11)(47). GI had

412 the greatest anti-inflammatory activity among medicinal licorice species in macrophage cells because

413 it contains two major anti-inflammatory compounds, LicA and LigC (11). Also, GI contains the ERβ

414 preferential agonist 8-prenylapigenin (47), which may have a better safety profile. Long-term studies

415 are planned to compare the effect of GG and GI on E2-induced mammary carcinogenesis in ACI rats

416 and ultimately estrogen metabolism in women. This study highlights the fact that the chemopreventive

417 bioactivity of licorice species cannot be reduced to the activity of single bioactive compounds, but is

418 rather a result of multiple constituents leading to polypharmacological actions. Furthermore, knowing

419 that licorice species have very different chemical profiles (14) that profoundly impact their bioactivity

420 in vitro as well as in vivo should help design safer and more efficacious botanicals with the greatest

421 chemopreventive potential in women. These data suggest that GI containing the chemopreventive

422 compounds, LicA and LigC, might be the optimal licorice species used for women’s health.

423

424 Acknowledgement

425 This work was supported by NIH grant P50 AT000155 from the NIH Office of Dietary Supplements

426 (ODS) and the National Center for Complementary and Integrative Health (NCCIH) to the UIC/NIH

427 Center for Botanical Dietary Supplements Research and by NIH grant U41 AT008706 from

428 NCCIH/ODS to the Center for Natural Product Technologies. The construction of the UIC CSB NMR

429 facility and instrumentation was funded by NIGMS grant P41 GM068944 to Dr. Peter Gettins. We

430 thank Shimadzu for providing the LCMS-8060 mass spectrometer used in this investigation. We also

431 thank Dr. Liang Zhao at Lanzhou Institute of Chemical Physics, CAS, and Qinghai Lake Medicinal

432 CO., Ltd. for their generous gifts.

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Table 1. Concentration of bioactive compounds in licorice extracts determined by UHPLC-UV.

Species Compounds (% w/w crude extract) Glabridin LicA LigCa LigC LigF LigF equivalentsb 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

a b LigC was below the limit of detection in GG. The 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.

Figure Legends

Fig. 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) in the 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).

Fig. 2. Key Compounds in GG and GI and LigC and LigF equivalents (11).

Fig. 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 four days. C) Free LicA was quantified by UHPLC-MS/MS in rat serum, liver, and mammary gland after GI and LicA administration.

Fig. 4. NAD(P)H-quinone oxidoreductase (NQO1) induction by GG, GI, and LicA in the liver.

NQO1 activity was measured in the liver after administration of vehicle, EB (1 mg/kg/day), 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.

Fig. 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 four days with vehicle, EB (1 mg/kg/day), and EB (1 mg/kg/day) co-administered 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 to 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 to vehicle control and by comparing the treatment groups (EB plus GG, or GI, or LicA) to EB treatment.

Fig. 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 µg/mL), or B) licorice compounds, LicA, LigC, and LigF (20 µM), for 24 h before analysis of CYP1A1/CYP1B1 expression by qPCR.

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 Published OnlineFirst October 4, 2018.

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