2,3-cis-2R,3R-(−)-epiafzelechin-3-O-p-coumarate, a novel flavan-3-ol isolated from Fallopia convolvulus seed, is an estrogen receptor agonist in human cell lines
© Brennan et al.; licensee BioMed Central Ltd. 2013
Received: 5 July 2012
Accepted: 22 May 2013
Published: 14 June 2013
The plant genus Fallopia is well-known in Chinese traditional medicine and includes many species that contain bioactive compounds, namely phytoestrogens. Consumption of phytoestrogens may be linked to decreased incidence of breast and prostate cancers therefore discovery of novel phytoestrogens and novel sources of phytoestrogens is of interest. Although phytoestrogen content has been analyzed in the rhizomes of various Fallopia sp., seeds of a Fallopia sp. have never been examined for phytoestrogen presence.
Analytical chemistry techniques were used with guidance from an in vitro estrogen receptor bioassay (a stably transfected human ovarian carcinoma cell line) to isolate and identify estrogenic components from seeds of Fallopia convolvulus. A transiently transfected human breast carcinoma cell line was used to characterize the biological activity of the isolated compounds on estrogen receptors (ER) α and β.
Two compounds, emodin and the novel flavan-3-ol, (−)-epiafzelechin-3-O-p-coumarate (rhodoeosein), were identified to be responsible for estrogenic activity of F. convolvulus seed extract. Absolute stereochemistry of rhodoeosein was determined by 1 and 2D NMR, optical rotation and circular dichroism. Emodin was identified by HPLC/DAD, LC/MS/MS, and FT/ICR-MS. When characterizing the ER specificity in biological activity of rhodoeosein and emodin, rhodoeosein was able to exhibit a four-fold greater relative estrogenic potency (REP) in breast cells transiently-transfected with ERβ as compared to those transfected with ERα, and emodin exhibited a six-fold greater REP in ERβ-transfected breast cells. Cell type-specific differences were observed with rhodoeosein but not emodin; rhodoeosein produced superinduction of reporter gene activity in the human ovarian cell line (> 400% of maximum estradiol [E2] induction) but not in the breast cell line.
This study is the first to characterize the novel flavan-3-ol compound, rhodoeosein, and its ability to induce estrogenic activity in human cell lines. Rhodoeosein and emodin may have potential therapeutic applications as natural products activating ERβ, and further characterization of rhodoeosein is necessary to evaluate its selectivity as a cell type-specific ER agonist.
KeywordsFallopia convolvulus Phytoestrogens Bioassay-directed fractionation HPLC/MS/NMR (−)-epiafzelechin-3-O-p-coumarate (rhodoeosein) Emodin Transfection Estrogen receptor Relative estrogenic potency
The plant genus Fallopia (Polygonacae) is well known in traditional medicines, and extracts have been used to treat hepatitis, liver damage, inflammation, and postmenopausal diseases [1–4]. Compounds isolated from rhizomes of Fallopia sp. have demonstrated vasorelaxant, anti-oxidant, anti-bacterial, anti-inflammatory, and anti-tumor properties [4–7], which have likely led to the Fallopia genus being widely used in traditional Chinese medicines. Several polyphenolic compounds exhibiting estrogenic activity (phytoestrogens) have also been isolated from the roots and rhizomes of numerous Fallopia (recently Polygonum) species such as F. multiflorum, F. cuspidatum, and F. japonica[8–12]. Many phytoestrogens exhibit preferential activation of estrogen receptor beta (ERβ) over estrogen receptor alpha (ERα) , and diets high in phytoestrogen content have been correlated with lower incidence of hormone-related cancers, namely breast and prostate . ERβ activation has an anti-proliferative effect in breast cells and is viewed as a protective balance against ERα activation (associated with proliferation) [15–17]. As such, there is interest in identifying plant sources rich in phytoestrogen content as well as discovering novel ERβ-selective phytoestrogens.
Recently, the rhizomes of Fallopia convolvulus (L.) Á.Löve (black bindweed, Polygonum convolvulus L. ) were examined for their inhibitory effects on nitric oxide production in lipopolysaccaharide-activated macrophages. Seventeen known and three novel phenolic compounds were identified in the active extract . However, extracts of F. convolvulus have not been examined for estrogenic activity nor has the polyphenolic content of its seeds been studied. Additionally, despite the wealth of information on the polyphenolic content and/or bioactive properties in the genus Fallopia, no attention has been paid to the content of the seeds. F. convolvulus is a widely distributed species, native throughout Asia, Europe, and northern Africa and invasive in the Americas and Australia . Study of ancient herb consumption in northern Europe indicates that the seeds of F. convolvulus were consumed by humans in early pre-Roman Iron Age and the Roman Iron Age (500 BC-400 AD) [21, 22].
Several major classes of phytoestrogens exist including isoflavones, lignans, stilbenes such as resveratrol, and anthraquinones such as emodin and emodin-glycoside. Flavanols, a class rich in biologically active compounds, may undergo metabolism into ligands with estrogenic activity . For identifying phytoestrogens in the Fallopia genus, mass spectrometry (MS), rather than diode array detection (DAD) or ultra-violet (UV) absorption, has become the method of choice due to its high specificity and ability to characterize unknowns through fragmentation, with electrospray ionization (ESI) being the predominant ionization source. The bulk of estrogenic compounds in Fallopia identified by MS are anthraquinones, stilbenes, and phenylpropanoids [6, 9, 10, 24]. Fourier-transform ion cyclotron resonance (FTICR) MS has been used to determine accurate mass (and elemental composition), of estrogenic compounds . 1H-NMR and 13C- NMR, have been used to elucidate the structure of many polyphenolic components from the genus Fallopia[19, 26, 27]. However, if the polyphenols contain chiral centers, NMR analysis will only yield the relative stereochemistry of the compound, and it is then necessary to use either X-ray crystallography or optical rotation combined with circular dichroism to determine the absolute stereochemistry . To identify compounds with certain biologic activity in a complex matrix, toxicant identification evaluation (TIE) combines chromatography separation and bioassay analysis to achieve rapid screening, isolation, and identification of compounds of interest. TIE studies have been applied successfully to the genus Fallopia to isolate and identify compounds with estrogenic, antibacterial, anti-HIV, or anti-inflammatory properties ([10, 29–31]). Use of the estrogen-sensitive carcinoma cell line MCF-7 guided separation of the phytoestrogens emodin and emodin 8-Ο-β-D-glucopyranoside from a methanolic root extract of F. cuspidatum. These two compounds in addition to citreorosein were isolated from F. cuspidatum using a recombinant yeast screening assay (YES) . Our objective was to determine whether the seeds of F. convolvulus contain compounds which display estrogenic activity (phytoestrogens), and, if so, the identity of the responsible compounds and whether they displayed ERβ-selectivity. In this study the estrogenic activity of F. convolvulus seed extract was evaluated using the stably transfected recombinant human ovarian carcinoma BG1Luc4E2 cell line which contains an estrogen-responsive reporter gene . Through TIE, active (estrogenic) components were isolated from F. convolvulus seed and identified by instrumental analyses, and the transiently-transfected human breast carcinoma SKBR3 cell line was used to assess ER subtype-selectivity of the isolated estrogenic components.
Chemicals and standards
Restriction enzymes were purchased from New England Biolabs (Ipswich, MA), and antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Translation grade L-[35S]-methionine (>400 Ci/mmol) was purchased from MP Biomedical (Solon, OH). Standards 17-β-estradiol (E2), emodin, and genistein were obtained from Sigma Aldrich Chemical Company (St. Louis, MO). Molecular grade dimethyl sulfoxide (DMSO) was obtained from OmniPur. Molecular grade ethanol (EtOH) and HPLC-grade solvents ethyl acetate (EtOAc) and n-hexane (n-hex) were obtained from Sigma (St. Louis, MO); HPLC-grade solvents acetonitrile (ACN), water, glacial acetic acid (HOAc), and methanol (MeOH) were obtained from Fisher Scientific (Waltham, MA). Deuterated dimethyl sulfoxide (DMSO, 99.9%) was obtained from Cambridge Isotope Laboratories (Andover, MA), and silica gel (170–400 mesh) was obtained from Fisher Scientific. Premium and charcoal-stripped fetal bovine serum (FBS) were obtained from Atlanta Biologicals (Lawrenceville, GA), Alpha Minimal Essential Medium (α-MEM) was obtained Invitrogen (San Diego, CA), Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Sigma (St. Louis, MO), Lipofectamine 2000 transfection reagent was from Invitrogen (San Diego, CA), and Cell Culture Lysis Buffer and Passive Lysis Buffer were from Promega (Madison, WI). Protein Assay Dye Reagent Concentrate was from Bio-Rad (Hercules, CA). Seeds (catalogue numbers 11716 and 11717 for F. convolvulus and F. dumetorum, respectively) were purchased from Herbiseed Company (Twyford, United Kingdom), and species authenticity was verified by Dr. Martin Parham (Herbiseed). SKBR3 cells were purchased from ATCC (Manassas, VA). All standards were stored at 4°C +in borosilicate amber vials (Fisher Scientific) with PTFE-lined caps.
General experimental procedures
HPLC for DAD and purification was performed using an Agilent series 1100 HPLC instrument equipped with a quaternary pump, autosampler, degasser, and Agilent Chemstation software for LC 3D systems. A Phenomenex Luna C18 column (150 mm × 4.6 mm I.D., 5 μm) with an Alltech guard column (Econosphere C18, 5 μm) was used at 23°C. LC/MS/MS analysis was performed using a triple quadrupole mass spectrometer API 2000 (PE Sciex, Concord, Ontario, Canada) operated in negative mode electrospray ionization (ESI) and a Perkin-Elmer (PE) series 200 equipped with a series 200 pump (PE), series 200 autosampler (PE), a CT0-10A column oven (Shimadzu), and DGU-14A degasser (Shimadzu) with an injection volume of 10 μL in split-injection mode. Gradient elution with a constant flow-rate of 1 mL/min was carried out with the mobile phase program outlined in Additional file 1. Ion source settings and potentials are shown in Supplementary Information. The instrument was operated with Analyst Software (v 1.3.1). FT-ICR MS analysis was performed using direct infusion into an FT-ICR MS (ThermoFisher) equipped with nano-ESI ion source, and spectra were acquired and processed with MassWorks software. Elemental composition of peaks was processed with Molecular Weight Calculator for Windows 9x/NT/2000/ME/XP (©Matthew Monroe). Emodin standard (10 μg/mL) was used as an external standard for calibrating mass accuracy of peaks in samples. NMR spectra were recorded on Bruker 600 MHz and 800 MHz Avance III spectrometers equipped with 5mm CPTXI cryogenic probes operating at 297 K. Sample volume in deuterated DMSO was 550 μL. The chemical shift (d) values are given in ppm and coupling constants (J) in Hz. Further NMR parameters are in Additional file 1. ECD spectrum was collected in MeOH using an Olis DSM 20 CD spectrophotometer and optical rotations were obtained on a Rudolph AUTOPOL IV polarimeter at wavelengths of 365, 405, 436, 546, 589, and 633 nm using a 1.0 dm cell. Specific rotations are reported in degrees per decimeter at 23°C and the concentrations are given in grams per 100 mL of solvent. Solvent used for optical rotations was MeOH (100%).
Construction and validation of ER plasmids
The human ERβ expression plasmid (ERβ/pcDNA3) was constructed by PCR amplification of the human ERβ segment (530 aa, GenBank: AF051427.1) out of pCMV5 (kindly provided by Dr. John Katzenellenbogen, University of Illinois) and insertion of this fragment at BstE II and Afl II restriction sites into the modified pcDNA3 construct, which was previously described . This construct contains a short 5′-untranslated sequence from the β-globin promoter and a 3′-untranslated region (~1.2 kb) from the mouse AhR gene that in previous studies significantly increased the in vitro expression of several proteins ( and data not shown). The human ERα expression plasmid, pcDNA3.1 + ERα, was purchased from Missouri S&T cDNA Resouce Center and was confirmed by restriction digestion. The human ERα fragment (444 aa, GenBank: AAD52984.1) from this plasmid was PCR-amplified with primers containing BstE II and Afl II sites, and the plasmid ERα/pcDNA3 was constructed by inserting this BstE II/Afl II fragment into the modified pcDNA3 vector. The generated plasmids were verified by DNA sequencing. Plasmids ERβ/pcDNA3 and ERα/pcDNA3were used as templates for in vitro expression (Additional file 2); ERα and ERβ were synthesized in vitro using the TNT Quick coupled transcription/translation rabbit reticulocyte lysate system (Promega). Briefly, 35S-Radiolabeled hERα and hERβ were synthesized in separate reactions in vitro according to manufacturer’s protocol, denatured, and subjected to SDS-polyacrylamide gel electrophoresis, and the kDa of each protein was determined by autoradiography of the dried gel.
Cell culture and transient transfection assays
The recombinant BG1Luc4E2 cell-line containing a stably transfected estrogen-responsive luciferase reporter gene was grown and prepared for bioassay analysis as previously described . Briefly, cells in 10 cm plates of approximately 20% confluence were cultured in phenol-red free DMEM supplemented with 10% charcoal-stripped FBS, for six days with daily media replacement. Cells were then plated into white, clear-bottomed 96-well tissue culture plates at a density of 750,000 cells/mL and allowed to attach for 24 h. Cells were incubated with carrier solvent EtOH (1% final solvent concentration), the indicated concentration of E2, or respective chemical or fraction treatment for 24 h at 37°C. Method blank treatments were included if applicable. After incubation cells were washed twice with PBS, followed by addition of cell lysis buffer (Promega), the plates shaken for 20 min at room temperature to allow cell lysis, and luciferase activity in each well was measured using an Orion microplate luminometer as previously described . SKBR3 cells were grown and maintained in high-glucose DMEM and 10% premium FBS. SKBR3 cells were cultured in phenol red-free medium supplemented with 10% charcoal-stripped FBS for 48 h before transfection and seeded in 24-well plates at a density of 300,000 cells/mL. 24 h after plating, cells were transfected for 24 h using Lipofectamine reagent (2 μL/well) according to the manufacturer’s recommendations (0.2 μg of ER-responsive reporter plasmid pGudLuc7ere  and 0.05 μg receptor plasmids (ERα/pcDNA3 or ERβ/pcDNA3) or empty vector (pcDNA3.1+) per well, normalizing μg DNA/well to 0.8 μg/well with empty vector). 24 h post-transfection, SKBR3 cells were incubated for 24 h with chemical, the cells harvested, and both protein concentration and luciferase activity determined. Protein concentration was determined using the Bradford assay . Briefly, 5 μL/well SKBR3 lysate was incubated for 5 min at room temperature with 1X Bradford Reagent (200 μL). Protein concentration was calibrated against a standard curve of bovine serum albumin (0.05-0.5 mg/mL) on the same plate and was measured as mg protein/mL using a PhosphorImager (Molecular Dynamics).
Luciferase activity was expressed as relative light units (RLU) for assays using BG1Luc4E2 cells or as a ratio of luciferase RLU/mg protein for assays using SKBR3 cells. Protein amount (mg) was calculated from concentrations obtained in Bradford assay and the respective luciferase RLU was divided by mg protein to obtain luciferase RLU/mg protein. Average luciferase RLU (or luciferase RLU/mg protein) values were calculated from triplicate wells. Solvent luciferase RLU (or luciferase RLU/mg) was subtracted from luciferase RLU induced by chemical. In the case of extract and fractions, luciferase RLU induced by method blank was subtracted from luciferase RLU induced by extract or fraction. Negative values were reported as 0. Luciferase RLU and luciferase RLU/mg protein from rhodoeosein, emodin, and genistein, extracts, and fractions were calculated as a percent of maximum E2 activity (achieved with 1 nM E2 and 10 nM E2 in BG1Luc4E2 cells and SKBR3 cells, respectively, and set as 100%) as previously described . The Student’s t-test (2-tailed, paired) was used to establish significant difference of chemical/fraction/extract activity from that of solvent or method blank (p ≤ 0.05), and dose–response curves were fitted using a sigmoidal Hill’s 4-parameter algorithm (SigmaPlot v.9.0, Systat, San Jose, CA), as described previously [37, 38]. Half-maximal inductions of each treatment (EC50) were determined as described in .
Sample extraction and isolation of (−)-epiafzelechin-3-O-p-coumarate
Extraction and isolation to determine the estrogenic compounds in F. convolvulus is described in Additional file 1. Seed was extracted using the following optimized protocol. Triple aliquots of ground F. convolvulus seed (25 g) were prepared. A method blank was included. Samples were sonicated in EtOAc (25 mL) for 7 min and liquid portions were collected. This process was repeated three times with an additional final solvent rinse of the samples. Liquid portions for each sample were centrifuged and re-centrifuged (550 g force) for 10 min per centrifugation. Supernatants were collected and the solvent evaporated from each sample at room temperature using nitrogen evaporation. Residues were re-suspended in EtOH and vacuum filtered using 0.2 μm Millipore filters. Solvent was evaporated and residues were re-suspended in H2O/ACN (1:1) 0.1% HOAc for preparative HPLC (Program 2). An isocratic program was carried out with A, water with 0.1% HOAc, and B, acetonitrile with 0.1% HOAc. The elution program was as follows: 20-30% B in 2 min (1 mL/min), 30% B for 13 min (1 mL/min), 30-100% B in 5 min (1 mL/min), with a flush of 100% B for 2 min (2 mL/min), 100-20% B in 2 min (1 mL/min), and 20% B for 1 min (1 mL/min (run-time 23 min). Valve (direct to waste) timing was 1.8 min onwards. Fractions were collected in 0.25 min intervals and evaluated for activity in BG1Luc4E2 cells, and fractions 12–14 (containing compound 5) were combined and fractionated resulting in fractions 12–13.5 that were combined and re-fractionated to yield pure compound 5 (20.9 mg). Compound 5 ((−)-epiafzelechin-3-O-p-coumarate) was then evaluated for activity at different concentrations in BG1Luc4E2 cells and maximal ERα and ERβ activity in SKBR3 cells.
Estrogenic activity of seed extracts and crude fractions
Isolation and identification of estrogens from F. convolvulus seed
Chemical characterization of compound 5
1 H and 13 C NMR spectroscopic data of compound 5 (600 MHz, δ in ppm)
(dd, J = 17.6, 2.4 Hz)
(dd, J = 17.6, 4.9 Hz)
(d, J = 2.4 Hz)
(d, J = 2.4 Hz)
(d, J = 8.6 Hz)
(d, J = 8.6 Hz)
C = O
(d, J = 15.8 Hz)
(d, J = 15.8 Hz)
(d, J = 8.6 Hz)
(d, J = 8.6 Hz)
Translucent pink oily film from 70/30 H2O/ACN; pink in MeOH and EtOH; [α]23 D -151 (c 0.22, MeOH); For 1H and 13C NMR spectroscopic data, see Table 1; for CD data (MeOH), see Figure 4; ESIMS m/z: 840.5 [2M], 419.4 [M-1], 273, 254.9, 229.1, 164.9, 145: 419.11360 [M-H]- (C24H19O7, experimental 419.11322, calc 419.11307). For peak abundances, UV spectrum, HMBC, HSQC, NOESY, and COSY spectra, see Additional file 4: Table S1 and Additional file 7.
Biological characterization of rhodoeosein and emodin
Potency comparisons of phytoestrogens
1.8 × 10-11, 1
1.2 × 10-4, 3.5 × 10-7
1.3 × 10-6, 1.5 × 10-5
7.3 × 10-7, 2.5 × 10-5
2.0 × 10-11, 1
5.8 × 10-6, 4.0 × 10-6
1.3 × 10-6, 1.5 × 10-5
9.0 × 10-7, 2.2 × 10-5
2.7 × 10-10, 1
1.8 × 10-5, 1.8 × 10-5
3.0 × 10-6, 8.9 × 10-5
8.3 × 10-8, 3.3 × 10-3
Consumption of a diet rich in phytoestrogens has been strongly correlated with beneficial effects on human health [51, 52]. Flavan-3-ols (the subclass of compounds containing rhodoeosein) have been reported to have a wide array of positive health effects such as antioxidant, anti-viral, anticarcinogenic, antimicrobial, and cardiopreventation (reviewed in ). Aside from the current study, there is no report of phytoestrogenic activity associated with flavan-3-ols. However, flavonoids, the broad class of plant compounds under which flavan-3-ol is found, contains many phytoestrogens (genistein, biochanin A, daidzein). Thorough structure-function analyses of various flavonoids on estrogen-responsive cell lines have correlated hydroxyls at the 7 and 4′ positions in flavonoids to those at the 17 and 3 positions in E2 and concluded that the flavonoid hydroxyls are essential for estrogenic activity [54, 55]. The estrogenic flavonoids in these two studies contained between 2 to 4 hydroxyl substituents. Accordingly, it is not surprising that rhodoeosein is estrogenic since it contains hydroxyls at the 7 and 4′ positions (Figure 3) and displays the classic A-ring structure which is shared by all known estrogenic compounds . A further phenomenon that may allow for the estrogenic activity of rhodoeosein is increased acidity of a hydroxyl through in-plane hydrogen bonding as suggested for other compounds by Fang et al. . Rhodoeosein possesses a benzylic hydrogen, Hβ, that is in close proximity to an ester on C = Ο (in conjugation with a hydroxyl in the 4″ position). This ester may be able to form an intramolecular H-bond with the in-plane C4 Hβ (Figure 3); the increased electron-withdrawing capability of the C = Ο carbonyl from an H-bond interaction with the C4 Hβ would increase the acidity of the 4″ hydroxyl (favorable for ER activity). In Zand et al. , a double bond between carbons 2 and 3 was important for estrogenic activity of a flavonoid as the double bond at this position increased rigidity of the molecule and, therefore, ER-affinity ; interestingly, rhodoeosein lacks a double bond at this position in the molecule but contains, in addition to the A- and B-rings, a large conjugated system exhibiting rigidity (−O-p-coumarate) which may lend favorable ER-binding attributes to the molecule. It may be that the structurally-favorable aspects of rhodoeosein (hydroxyls at the 7 and 4′ positions, having 4 hydroxyl substituents, rigidity) outweigh the absence of a double bond between C2 and C3. Additionally, molecules exhibiting a certain measure of hydrophobicity (where the molecule is hydrophobic while containing a polar group on each end) bind to ER with greater affinity . Rhodoeosein contains not just one set of opposing polar groups but two (7-OH and 4′-OH, 7-OH and 4″-OH) which sandwich large hydrophobic regions (Figure 3).
Plants have a complex pathway for flavan-3-ol formation. Flavan-3-ols can be found in the seeds and fruits but not leaves of certain plants, and a pathway for flavonoid conversion in the plant from flavanone to flavan-3-ol has been proposed . The gene responsible for the formation of flavan-3-ol through this cascade is highly expressed in the seeds but not in the flowers or leaf tissues . Recently, F. convolvulus roots were examined for polyphenolic content . Except for emodin, none of the 20 polyphenolic compounds (3 new, 17 known) found in the roots seem to exist in the seeds of F. convolvulus (based on mass spectrometry supplementary data). This curious phenomenon of the polyphenolic content of the seeds being unrepresentative of the content in the roots (aside from emodin) may be explained through the multi-step flavonoid pathway illustrated in Xie et al. . It is thought that flavan-3-ols (known as tannins in plants) play a mainly defensive role in the plant against herbivorous animals, insects, fungi, and other harmful agents . However, as demonstrated here and in other studies, flavan-3-ols exhibit complex biological properties in human systems.
Although emodin’s behavior in ERα-transfected SKBR3 cells and BG1Luc4E2 cells was consistent (Table 2), rhodoeosein showed marked cell-specific differences in the potency and magnitude of luciferase induction between BG1Luc4E2 cells and ERα-transfected SKBR3 cells (Table 2). Luciferase activity was induced by rhodoeosein in BG1Luc4E2 cells to a level greater than that maximally induced by E2 and this superinduction response suggests that rhodoeosein is also affecting other signaling pathways that impact on ER-dependent activation of luciferase expression from the reporter plasmid. Numerous studies have not only demonstrated that the functional activities of ER and other steroid hormone receptors can be altered by posttranslational modifications (i.e. phosphorylation, acetylation and others), but phosphorylation can also activate the transcriptional activity of these receptors in the absence of ligand binding [57–60]. Binding of ligand to a cytosol-membrane ERs or to GPR30 (a G-coupled protein receptor) can activate protein-kinase cascades which phosphorylate and activate nuclear ERs as well as other signaling pathways and transcription factors . For example, tectoridin, a flavonoid phytoestrogen, was found to exert its estrogenic effects not through the ER but through an extracellular signal-regulated kinase pathway . Additionally, direct or indirect stimulation of growth factor receptors can lead to activation of protein-kinase cascades and hormone receptor-dependent and independent gene expression responses. The enhancement of reporter gene expression observed with rhodoeosein treatment in BG1Luc4E2 cells but not SKBR3 cells could result from a rhodoeosein-dependent stimulation of an additional signaling pathway(s) that enhances the transcription of the luciferase reporter gene promoter. This pathway(s) may not present or affected in the SKBR3 cells. Although superinduction has been previously observed in the BG1Luc4E2 cell line , the molecular mechanism responsible for superinduction of ER-dependent gene expression in BG1Luc4E2 cells remains to be elucidated.
Regarding half-maximal concentration differences between the two ER subtypes, we did not find that rhodoeosein or emodin had lower EC50 values in ERβ-transfected SKBR3 cells than in ERα-transfected SKBR3 cells which does not agree with previous literature on phytoestrogens [13, 62]. However, the ERβ-transfected SKBR3 cells appear to be an order of magnitude less responsive to ER-ligands than the ERα-transfected SKBR3 cells as shown by the EC50 values of E2, rhodoeosein, and emodin in both transfections (Table 2). Therefore, we choose to assess potency by comparing the EC50 values of emodin and rhodoeosein directly to those of the E2 standards in the ERα-transfected cells and the ERβ-transfected cells (Table 2). This method of using relative estrogenic potency (REP) has been previously established in  where REP was defined as the ratio between E2 EC50 and EC50 of the chemical. We have also defined REP = EC50 of E2/EC50 of phytoestrogen, and when compared to the half-maximal concentration of the E2 standard, both emodin and rhodoeosein were more potent in ERβ-transfected SKBR3 cells than in ERα-transfected SKBR3 cells (Table 2). REP of rhodoeosein in ERα-transfected SKBR3 cells was 3.4 × 10-6 but in ERβ-transfected SKBR3 cells was 1.5 × 10-5, a 4-fold increase in REP. The difference between emodin’s REP values in ERα-transfected SKBR3 cells and ERβ-transfected SKBR3 cells was similar to that of rhodoeosein; REP of emodin in ERβ-transfected SKBR3 cells was 8.9 × 10-5 whereas in ERα-transfected SKBR3 cells, the REP was 1.5 × 10-5, a 6-fold decrease. This is in agreement with  where A YES assay transfected separately with ERα or ERβ was used to examine emodin’s estrogenic activity; emodin had greater REP in ERβ-transfected yeast cells than in ERα-transfected yeast cells by approximately one order of magnitude. Our findings are also in agreement with a study examining binding affinity of several phytoestrogens to either ERα or ERβ; compared to the E2 standard (set as 100%), relative binding affinity of genistein, biochanin A, coumestrol, and diadzein were greater to ERβ than to ERα . In our assay, the control phytoestrogen genistein showed a 146-fold increase in REP for ERβ compared to ERα (REP values 3.3 × 10-2 and 2.2 × 10-5, respectively), in agreement with . ERα and ERβ have unique and overlapping tissue distribution in the human body, and the roles of the ER subtypes in the human body are now becoming more clear (reviewed in  and ). Although ERβ activation may be associated with certain deleterious effects (i.e. potential involvement in metabolic disorders leading to diabetes ), beneficial roles of ERβ in the human body include development and maintenance of the brain, ovulation, prostate health, and anti-proliferative roles in certain breast cancers. The effectiveness of phytoestrogens, which exist in nature not as single compounds but as complex mixtures in food matrices, as therapeutic agents for existing diseases is still unclear, but epidemiological studies indicate diets containing a significant amount of phytoestrogens seem to correlate with ERβ-mediated benefits (such as decreased incidence of breast and prostate cancers). The higher REP values displayed by rhodoeosein and emodin in ERβ-transfected cells may indicate use of foods containing these compounds as part of a comprehensive plan for maintaining tissue health through ERβ-mediated activity.
Seeds of F. convolvulus were identified as a novel source of phytoestrogens from which we have isolated and chemically characterized a novel phytoestrogen rhodoeosein. Estrogenic activity of rhodoeosein was evaluated in two human cell-lines in which we were able to demonstrate cell-type specific effects of rhodoeosein. To our knowledge, rhodoeosein is the first published flavan-3-ol to demonstrate estrogenic properties in vitro. In addition to rhodoeosein, we also found that F. convolvulus seed contains emodin (a known and potent estrogen), and these compounds were also identified in the closely-related species F. dumetorum. By comparing relative estrogenic potencies (REP) of emodin and rhodoeosein in SKBR3 cells transfected with either ERα or ERβ we found that both phytoestrogens are more potent in ERβ-transfected cells than in ERα-transfected cells and that emodin is more potent than rhodoeosein on both ER subtypes. Similar differences in potency were observed in the BG1Luc4E2 cell line. The estrogenic compounds in this study may regulate reproduction to some degree in wildlife consuming these seeds . Effects in vivo have not yet been assessed, but the complex polyphenolic content of F. convolvulus seed may indicate therapeutic potential.
Diode array detection
Fourier transform ion cyclotron resonance
Nuclear magnetic resonance
Electronic circular dichroism
Toxicant identification evaluation
Effective concentration 50
Multiple reaction monitoring
Heteronuclear Multiple Bond Correlation
Nuclear Overhauser effect spectroscopy
Heteronuclear Single Quantum Coherence
Relative estrogenic potency.
We thank Dr. John Katzenellenbogen for the pCMV5-ERβ construct. We also thank Charles Grove for optical rotation data collection, Dr. Annaliese Franz for advice and expertise on circular dichroism, and Dr. Dean Tantillo for expertise and advice on intramolecular forces. We thank the National Science Foundation (grant #0314510 to JM.), the National Institute of Environmental Health Sciences (grant ESO04699 to MD and the T32 training grant T32 ES007058-33 to JB), and the University of California, Davis (Jastro-Shields to JB) for funding. We wish to acknowledge equipment grants NIH RR11973 for the 600 MHz NMR and NSF DBIO 727538 for the 800 MHz NMR.
- Chen HS, Liu Y, Lin LQ, Zhao JL, Zhang CP, Jin JC, Wang L, Bai MH, Wang YC, Liu M: Anti-proliferative effect of an extract of the root of Polygonum multiflorum Thunb. on MCF-7 human breast cancer cells and the possible mechanisms. Molecular Medicine Reports. 2011, 4 (6): 1313-1319.PubMed
- Zhang QH, Yang JY, Wang DF, Wu CF: Genotoxic studies on Panax Ginseng, Polygonum multiflorum and their compatibility in mouse peripheral lymphocyte cells. Acta Pharmacol Sin. 2006, 27: 288-
- Zhang YZ, Shen JF, Xu JY, Xiao JH, Wang JL: Inhibitory effects of 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D-glucosid on experimental inflammation and cyclooxygenase 2 activity. J Asian Nat Prod Res. 2007, 9 (4): 355-363. 10.1080/10286020600727772.View ArticlePubMed
- Liao SG, Zhang LJ, Sun F, Zhang JJ, Chen AY, Lan YY, Li YJ, Wang AM, He X, Xiong Y: Antibacterial and anti-inflammatory effects of extracts and fractions from Polygonum capitatum. J Ethnopharmacol. 2011, 134 (3): 1006-1009. 10.1016/j.jep.2011.01.050.View ArticlePubMed
- Fremont L: Minireview - Biological effects of resveratrol. Life Sci. 2000, 66 (8): 663-673. 10.1016/S0024-3205(99)00410-5.View ArticlePubMed
- Li-Shuang LV, Gu XH, Ho CT, Tang J: Stilbene glycosides from the roots of Polygonum multiflorum Thunb and their in vitro antioxidant activities. Journal of Food Lipids. 2006, 13 (2): 131-144. 10.1111/j.1745-4522.2006.00039.x.View Article
- Noda T, Yamada T, Ohkubo T, Omura T, Ono T, Adachi T, Awaya T, Tasaki Y, Shimizu K, Matsubara K: Hot-water-extracts of Polygonum Multiflorum Do Not Induce Any Toxicity but Elicit Limited Beneficial Effects on the Liver in Mice. Journal of Health Science. 2009, 55 (5): 720-725. 10.1248/jhs.55.720.View Article
- Matsuda H, Shimoda H, Morikawa T, Yoshikawa M: Phytoestrogens from the roots of Polygonum cuspidatum (Polygonaceae): structure-requirement of hydroxyanthraquinones for estrogenic activity. Bioorg Med Chem Lett. 2001, 11 (14): 1839-1842. 10.1016/S0960-894X(01)00318-3.View ArticlePubMed
- Avula B, Josfh VC, Wang YH, Khan KA: Simultaneous identification and quantification of anthraquinones, polydatin, and resveratrol in Polygonum mutiflorum, various Polygonum species, and dietary supplements by liquid chromatography and microscopic study of Polygonum species. J AOAC Int. 2007, 90 (6): 1532-1538.PubMed
- Shen MY, Liu YJ, Don MJ, Liu HY, Chen ZW, Mettling C, Corbeau P, Chiang CK, Jang YS, Li TH: Combined Phytochemistry and Chemotaxis Assays for Identification and Mechanistic Analysis of Anti-Inflammatory Phytochemicals in Fallopia japonica. PLoS One. 2011, 6: 11-
- Zhang CZ, Wang SX, Zhang Y, Chen JP, Liang XM: In vitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms. J Ethnopharmacol. 2005, 98 (3): 295-300. 10.1016/j.jep.2005.01.033.View ArticlePubMed
- Zhang CN, Zhang XZ, Zhang Y, Xu Q, Xiao HB, Liang XM: Analysis of estrogenic compounds in Polygonum cuspidatum by bioassay and high performance liquid chromatography. J Ethnopharmacol. 2006, 105 (1–2): 223-228.View ArticlePubMed
- Harris DM, Besselink E, Henning SM, Go VLW, Heber D: Phytoestrogens induce differential estrogen receptor alpha- or beta-mediated responses in transfected breast cancer cells. Exp Biol Med. 2005, 230 (8): 558-568.
- Adlercreutz H: Phyto-oestrogens and cancer. Lancet Oncol. 2002, 3 (6): 364-373. 10.1016/S1470-2045(02)00777-5.View ArticlePubMed
- Covaleda AMS, Van den Berg H, Vervoort J, Van der Saag P, Strom A, Gustafsson JA, Rietjens I, Murk AJ: Influence of cellular ER alpha/ER beta ratio on the ER alpha-agonist induced proliferation of human T47D breast cancer cells. Toxicol Sci. 2008, 105 (2): 303-311. 10.1093/toxsci/kfn141.PubMed CentralView Article
- Sotoca AM, Ratman D, van der Saag P, Strom A, Gustafsson JA, Vervoort J, Rietjens I, Murk AJ: Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ER alpha/ER beta ratio. J Steroid Biochem Mol Biol. 2008, 112 (4–5): 171-178.View ArticlePubMed
- Williams C, Edvardsson K, Lewandowski SA, Strom A, Gustafsson JA: A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells. Oncogene. 2008, 27 (7): 1019-1032. 10.1038/sj.onc.1210712.View ArticlePubMed
- Klein H: black bindweed Fallopia convolvulus (Linnaeus) A. Love or Polygonum convolvulus. 2011, Alaska, USA: University of Alaska, Anchorage, 1-3.
- Zhang CF, Chen J, Zhao LQ, Zhang D, Zhang MA, Wang ZT: Three new flavonoids from the active extract of Fallopia convolvulus. J Asian Nat Prod Res. 2011, 13 (2): 136-142. 10.1080/10286020.2010.546357.View ArticlePubMed
- USDA, ARS, National Genetic Resources Program: Fallopia convolvulus (L.). Germplasm Resources Information Network (GRIN). 2011, Beltsville, Maryland: National Germplasm Resources Laboratory, http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?40459. Web. 6 2 2013
- Helbaek H: Prehistoric food plants and weeds in Denmark. A survey of archaeobotanical research 1923–1954. Danmarks Geol Undersogelse 2 Raekke. 1954, 80: 250-261.
- Behre KE: Collected seeds and fruits from herbs as prehistoric food. Vegetation History and Archaeobotany. 2008, 17 (1): 65-73.View Article
- Arroo RRJ, Androutsopoulos V, Beresford K, Ruparelia K, Surichan S, Wilsher N, Potter GA: Phytoestrogens as natural prodrugs in cancer prevention: dietary flavonoids. Phytochemistry Reviews. 2009, 8 (2): 375-386. 10.1007/s11101-009-9128-6.View Article
- Fan PH, Marston A, Hay AE, Hostettmann K: Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high-speed countercurrent chromatography. J Sep Sci. 2009, 32 (17): 2979-2984. 10.1002/jssc.200900057.View ArticlePubMed
- Pickford R, Miguens-Rodriguez M, Afzaal S, Speir P, Pergantis SA, Thomas-Oates JE: Application of the high mass accuracy capabilities of FT-ICR-MS and Q-ToF-MS to the characterisation of arsenic compounds in complex biological matrices. Journal of Analytical Atomic Spectrometry. 2002, 17 (3): 173-176. 10.1039/b109842j.View Article
- Yao S, Li Y, Kong LY: Preparative isolation and purification of chemical constituents from the root of Polygonum multiflorum by high-speed counter-current chromatography. J Chromatogr A. 2006, 1115 (1–2): 64-71.View ArticlePubMed
- Xu ML, Zheng MS, Lee YK, Moon DC, Lee CS, Woo MH, Jeong BS, Lee ES, Jahng Y, Chang HW: A new stilbene glucoside from the roots of Polygonum multiflorum Thunb. Arch Pharm Res. 2006, 29 (11): 946-951. 10.1007/BF02969276.View ArticlePubMed
- Kwit M, Rozwadowska MD, Gawronski J, Grajewska A: Density Functional Theory Calculations of the Optical Rotation and Electronic Circular Dichroism: The Absolute Configuration of the Highly Flexible trans-Isocytoxazone Revised. J Org Chem. 2009, 74 (21): 8051-8063. 10.1021/jo901175s.View ArticlePubMed
- Zhang CN, Wang XM, Zhang XZ, Zhang Y, Xiao HB, Liang XM: Bioassay-guided Separation of Citreorosein and Other Oestrogenic Compounds From Polygonum cuspidatum. Phytother Res. 2009, 23 (5): 740-741. 10.1002/ptr.2619.View ArticlePubMed
- Shan B, Cai YZ, Brooks JD, Corke H: Antibacterial properties of Polygonum cuspidatum roots and their major bioactive constituents. Food Chem. 2008, 109 (3): 530-537. 10.1016/j.foodchem.2007.12.064.View Article
- Lin HW, Sun MX, Wang YH, Yang LM, Yang YR, Huang N, Xuan LJ, Xu YM, Bai DL, Zheng YT: Anti-HIV Activities of the Compounds Isolated from Polygonum cuspidatum and Polygonum multiflorum. Planta Med. 2010, 76 (9): 889-892. 10.1055/s-0029-1240796.View ArticlePubMed
- Rogers JM, Denison MS: Recombinant cell bioassays for endocrine disruptors: Development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. In Vitro & Molecular Toxicology-a Journal of Basic and Applied Research. 2000, 13 (1): 67-82.
- Soshilov A, Denison MS: Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor. J Biol Chem. 2008, 283 (47): 32995-33005. 10.1074/jbc.M802414200.PubMed CentralView ArticlePubMed
- Baston DS, Denison MS: Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: Normalization of superinduction results for improved sample potency estimation. Talanta. 2011, 83 (5): 1415-1421. 10.1016/j.talanta.2010.11.035.PubMed CentralView ArticlePubMed
- Bradford MM: Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem. 1976, 72 (1–2): 248-254.View ArticlePubMed
- Seidel SD, Li V, Winter GM, Rogers WJ, Martinez EI, Denison MS: Ah receptor-based chemical screening bioassays: Application and limitations for the detection of Ah receptor agonists. Toxicol Sci. 2000, 55 (1): 107-115. 10.1093/toxsci/55.1.107.View ArticlePubMed
- Natarajan K, Overstreet JW, Rogers JM, Denison MJ, Chen JG, Lohstroh PN, McConnell DS, Lasley BL: Detection of xenoestrogens in serum after immunoprecipitation of endogenous steroidal estrogens. Environ Health Perspect. 2002, 110 (8): 791-795. 10.1289/ehp.02110791.PubMed CentralView ArticlePubMed
- Rochester JR, Klasing KC, Stevenson L, Denison MS, Berry W, Millam JR: Dietary red clover (Trifolium pratense) induces oviduct growth and decreases ovary and testes growth in Japanese quail chicks. Reprod Toxicol. 2009, 27 (1): 63-71. 10.1016/j.reprotox.2008.11.056.PubMed CentralView ArticlePubMed
- Kang SC, Lee CM, Choi H, Lee JH, Oh JS, Kwak JH, Zee OP: Evaluation of oriental medicinal herbs for estrogenic and antiproliferative activities. Phytother Res. 2006, 20 (11): 1017-1019. 10.1002/ptr.1987.View ArticlePubMed
- Klein KO, Janfaza M, Wong JA, Chang RJ: Estrogen bioactivity in fo-ti and other herbs used for their estrogen-like effects as determined by a recombinant cell bioassay. J Clin Endocrinol Metab. 2003, 88 (9): 4077-4079. 10.1210/jc.2003-030349.View Article
- Slade D, Ferreira D, Marais JPJ: Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry. 2005, 66 (18): 2177-2215. 10.1016/j.phytochem.2005.02.002.View ArticlePubMed
- Kofink M, Papagiannopoulos M, Galensa R: Enantioseparation of catechin and epicatechin in plant food by chiral capillary electrophoresis. European Food Research and Technology. 2007, 225 (3–4): 569-577.View Article
- Kpegba K, Agbonon A, Petrovic AG, Amouzou E, Gbeassor M, Proni G, Nesnas N: Epiafzelechin from the Root Bark of Cassia sieberiana: Detection by DART Mass Spectrometry, Spectroscopic Characterization, and Antioxidant Properties. J Nat Prod. 2011, 74 (3): 455-459. 10.1021/np100090e.View ArticlePubMed
- Korver O, Wilkins CK: Circular dichroism spectra of flavanols. Tetrahedron. 1971, 27 (22): 5459-10.1016/S0040-4020(01)91712-0.View Article
- Xie DY, Sharma SB, Paiva NL, Ferreira D, Dixon RA: Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science. 2003, 299 (5605): 396-399. 10.1126/science.1078540.View ArticlePubMed
- van Rensburg H, Steynberg PJ, Burger JFW, van Heerden PS, Ferreira D: Circular dichroic properties of flavan-3-ols. Journal of Chemical Research-S. 1999, 7: 450-451.View Article
- Couse JF, Korach KS: Estrogen receptor null mice: What have we learned and where will they lead us?. Endocr Rev. 1999, 20 (3): 358-417. 10.1210/er.20.3.358.View ArticlePubMed
- Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA: Estrogen receptor beta inhibits 17 beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci U S A. 2004, 101 (6): 1566-1571. 10.1073/pnas.0308319100.PubMed CentralView ArticlePubMed
- Helguero LA, Faulds MH, Gustafsson JA, Haldosen LA: Estrogen receptors alfa (ER alpha) and beta (ER beta) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11. Oncogene. 2005, 24 (44): 6605-6616. 10.1038/sj.onc.1208807.View ArticlePubMed
- Harris HA: Estrogen receptor-beta: Recent lessons from in vivo studies. Mol Endocrinol. 2007, 21 (1): 1-13.View ArticlePubMed
- Adlercreutz H: Lignans and human health. Crit Rev Clin Lab Sci. 2007, 44 (5–6): 483-525.View ArticlePubMed
- Hartman J, Strom A, Gustafsson JA: Estrogen receptor beta in breast cancer-Diagnostic and therapeutic implications. Steroids. 2009, 74 (8): 635-641. 10.1016/j.steroids.2009.02.005.View ArticlePubMed
- Aron PM, Kennedy JA: Flavan-3-ols: Nature, occurrence and biological activity. Mol Nutr Food Res. 2008, 52 (1): 79-104. 10.1002/mnfr.200700137.View ArticlePubMed
- Le Bail JC, Varnat F, Nicolas JC, Habrioux G: Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids. Cancer Lett. 1998, 130 (1–2): 209-216.View ArticlePubMed
- Zand RSR, Jenkins DJA, Diamandis EP: Steroid hormone activity of flavonoids and related compounds. Breast Cancer Res Treat. 2000, 62 (1): 35-49. 10.1023/A:1006422302173.View ArticlePubMed
- Fang H, Tong WD, Shi LM, Blair R, Perkins R, Branham W, Hass BS, Xie Q, Dial SL, Moland CL: Structure-activity relationships for a large diverse set of natural, synthetic, and environmental estrogens. Chem Res Toxicol. 2001, 14 (3): 280-294. 10.1021/tx000208y.View ArticlePubMed
- Gambino YP, Maymo JL, Perez AP, Calvo JC, Sanchez-Margalet V, Varone CL: Elsevier Trophoblast Research Award Lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells - Focus on leptin expression. Placenta. 2012, 33: S63-S70.View ArticlePubMed
- Bjornstrom L, Sjoberg M: Mechanisms of estrogen receptor signaling: Convergence of genomic and nongenomic actions on target genes. Mol Endocrinol. 2005, 19 (4): 833-842. 10.1210/me.2004-0486.View ArticlePubMed
- Ward RD, Weigel NL: Steroid receptor phosphorylation: Assigning function to site-specific phosphorylation. Biofactors. 2009, 35 (6): 528-536. 10.1002/biof.66.PubMed CentralView ArticlePubMed
- Weigel NL, Moore NL: Cyclins, cyclin dependent kinases, and regulation of steroid receptor action. Mol Cell Endocrinol. 2007, 265: 157-161.View ArticlePubMed
- Kang K, Lee S, Jung S, Cha K, Park W, Sohn Y, Nho C: Tectoridin, a poor ligand of estrogen receptor alpha, exerts its estrogenic effects via an ERK-dependent pathway. Mol Cells. 2009, 27 (3): 351-357. 10.1007/s10059-009-0045-8.View ArticlePubMed
- Chu WL, Shiizaki K, Kawanishi M, Kondo M, Yagi T: Validation of a New Yeast-Based Reporter Assay Consisting of Human Estrogen Receptors alpha/beta and Coactivator SRC-1: Application for Detection of Estrogenic Activity in Environmental Samples. Environ Toxicol. 2009, 24 (5): 513-521. 10.1002/tox.20473.View ArticlePubMed
- Bovee TFH, Helsdingen RJR, Rietjens I, Keijer J, Hoogenboom R: Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds with both receptor types. J Steroid Biochem Mol Biol. 2004, 91 (3): 99-109. 10.1016/j.jsbmb.2004.03.118.View ArticlePubMed
- Escande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P, Nicolas JC, Cavailles V, Balaguer P: Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem Pharmacol. 2006, 71 (10): 1459-1469. 10.1016/j.bcp.2006.02.002.View ArticlePubMed
- Nilsson S, Koehler KF, Gustafsson J-A: Development of subtype-selective oestrogen receptor-based therapeutics. Nat Rev Drug Discov. 2011, 10 (10): 778-792. 10.1038/nrd3551.View ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/133/prepub
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