Estrogenic Effects of Extracts and Isolated Compounds from Belowground and Aerial Parts of Spartina anglica

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.


Introduction
Female hormones are produced by mature follicles in the ovary, promote the development of secondary sexual characteristics in women, and play a role in thickening the membrane for the implantation of the fertilized egg in the uterine wall [1,2]. Owing to the action of female hormones, the uterine wall collapses and thickens periodically, which is called menstruation [3]. The ovaries genetically regulate the fertility of women by the generation of female hormones; however, in menopause, this function is lost, which leads to various physiological changes [4,5]. Recently, the number of women affected by menopause (the period after menstruation ends) is increasing in line with an increase in lifespan. Reportedly, women now spend one-third of their lives in menopause [6]. Menopause presents with symptoms such as facial flushing, vaginal atrophy, vaginal dryness, insomnia, and osteoporosis owing to decreased estrogen production from the ovaries. It can lead to a lower quality of life and depression [7][8][9][10].

Estrogenic Effects of Different Extracts from Belowground and Aerial Parts of S. anglica on MCF-7 Cells
To evaluate the potential estrogenic activity of the extracts of S. anglica, Soto's Escreen assay using an ER-positive MCF-7 human breast cancer cell line was performed

Estrogenic Effects of Different Extracts from Belowground and Aerial Parts of S. anglica on MCF-7 Cells
To evaluate the potential estrogenic activity of the extracts of S. anglica, Soto's Escreen assay using an ER-positive MCF-7 human breast cancer cell line was performed [31]. Queens One tablet (tab.), which was used as a positive control, increased MCF-7 cell proliferation in a concentration-dependent manner (25 µg/mL: 115.2 ± 6.3%, 50 µg/mL: 129.0 ± 4.6%, 100 µg/mL: 132.5 ± 5.9%; Figure 2). Moreover, because proliferation was significantly suppressed in the presence of ICI 182,780, which is an ER antagonist, the proliferation caused by Queens One Table was regarded as estrogen-responsive proliferation.

Estrogenic Effects of Different Extracts from Belowground and Aerial Parts of S. anglica on MCF-7 Cells
To evaluate the potential estrogenic activity of the extracts of S. anglica, Soto's Escreen assay using an ER-positive MCF-7 human breast cancer cell line was performed [31]. Queens One tablet (tab.), which was used as a positive control, increased MCF-7 cell proliferation in a concentration-dependent manner (25 μg/mL: 115.2 ± 6.3%, 50 μg/mL: 129.0 ± 4.6%, 100 μg/mL: 132.5 ± 5.9%; Figure 2). Moreover, because proliferation was significantly suppressed in the presence of ICI 182,780, which is an ER antagonist, the proliferation caused by Queens One Table was regarded as estrogen-responsive proliferation.  25,50, and 100 μg/mL extracts in the presence or absence of 0.5 μM ICI 182,780 for 144 h in a charcoal-dextran stripped media condition. Proliferation was determined using an EZ-Cytox kit. Data are presented as mean ± standard error of mean (SEM) of at least three independent experiments. * p < 0.05 versus untreated cells. ** p < Figure 2. Estrogenic effects of the different extracts from belowground and aerial parts of S. anglica on MCF-7 cells. MCF-7 cells were challenged with 25, 50, and 100 µg/mL extracts in the presence or absence of 0.5 µM ICI 182,780 for 144 h in a charcoal-dextran stripped media condition. Proliferation was determined using an EZ-Cytox kit. Data are presented as mean ± standard error of mean (SEM) of at least three independent experiments. * p < 0.05 versus untreated cells. ** p < 0.01 versus untreated cells. # p < 0.05 versus each group without ICI 182,780. (SA-bg-W: water extract from belowground part of S. anglica, SA-bg-E30: 30% ethanol extract from belowground part, SA-bg-E50: 50% ethanol extract from belowground part, SA-bg-E80: 80% ethanol extract from belowground part, SA-a-W: water extract from aerial part, SA-a-E30: 30% ethanol extract from aerial part, SA-a-E50: 50% ethanol extract from aerial part, SA-a-E80: 80% ethanol extract from aerial part).

Uterotrophic Activity of 50% Ethanol Extract from Belowground Part of S. anglica in the Immature Rat
To determine the potential of SA-bg-E50 to have estrogenic effects, we evaluated the estrogenic effect of SA-bg-E50 in an immature female rat model. 17β-Estradiol (10 µg/kg) was subcutaneously injected for 3 days in the control group, and SA-bg-E50 (200 mg/kg) was orally administered for 3 days in the SA-bg-E50 group.

Uterotrophic Activity of 50% Ethanol Extract from Belowground Part of S. anglica in the Immature Rat
To determine the potential of SA-bg-E50 to have estrogenic effects, we evaluated the estrogenic effect of SA-bg-E50 in an immature female rat model. 17β-Estradiol (10 μg/kg) was subcutaneously injected for 3 days in the control group, and SA-bg-E50 (200 mg/kg) was orally administered for 3 days in the SA-bg-E50 group.
The uterine weight of the positive control group was significantly increased, to 50.3 ± 3.1 mg, compared with that of the normal group (41.5 ± 1.6 mg). After the administration of SA-bg-E50, the uterine weight was 49.0 ± 2.5 mg. The results indicate that SA-bg-E50 hypertrophies the uterus owing to its estrogenic activity ( Figure 3).  . Uterotrophic activity of 50% ethanol extract from belowground part of S. anglica (SAbg-E50) in the immature rat. 17β-Estradiol (10 µg/kg) was subcutaneously injected for 3 days. SA-bg-E50 (200 mg/kg) was orally administered for 3 days. Rats were euthanized 24 h after the final administration and the uterus was removed and weighed. Data are presented as mean ± SEM of at least three independent experiments. * p < 0.05 versus normal group. (Normal: corn oil and water treatment group, Positive control: 10 µg/kg 17β-estradiol injected group, SA-bg-E50: 50% ethanol extract from belowground part of S. anglica administered group).  ppm. One feruloyl residue was connected to the C-1 of quinic acid, as shown by the downfield shift in 13 C NMR at δ C 81.12 (C-1) ppm. The position of the other ferulate unit was determined by HMBC from H-3 to C-9" (Figure 4). The numbering system of acyl-quinic acid was critical because of the confusing nomenclature caused by mixing IUPAC with non-IUPAC numbering [32]. A number of reported publications related to acyl-quinic acid were identified with inconsistencies in numbering and/or structure with IUPAC numbering [33,34]. The planar structure of 1 was determined to be 1,3-di-O-transferuloyl quinic acid by the IUPAC nomenclature of the quinic acid moiety. The two of vicinal coupling constants of H-5 were observed to be large ( 3 J H-4,H-5 = 9.6 Hz, 3 J H-5,H-6ax = 11.3 Hz) which supported the three protons of H-4, H-5 and H-6 ax as axial position ( Figure 4). In addition, other coupling constants, including long-range coupling through a 1,3-diequatorial interaction (W-coupling) between H-2 eq and H-6 eq , were well-matched with those of quinic acid, rather than with those of epi-quinic acid ( Figure 4) [35]. The comparison of the specific rotation of 1 with that of similar chemical compounds (cynarine; 1,3-dicaffeoylquinic acid) supported the absolute configuration of quinic acid in 1 as (−)-
The results indicate that compound 1 induces phosphorylation of serine residues of ERα; thus, compound 1 may be considered an ERα ligand. Compound 1, which contains phenolic hydroxyl groups similar to 17β-estradiol, seems to result in the activation of ERα via interactions with the hydroxyl groups and ligand-binding domains of ERα. The results indicate that compound 1 induces phosphorylation of serine residues of ERα; thus, compound 1 may be considered an ERα ligand. Compound 1, which contains phenolic hydroxyl groups similar to 17β-estradiol, seems to result in the activation of ERα via interactions with the hydroxyl groups and ligand-binding domains of ERα.

Anti-proliferative Effect of Extracts and Compounds from S. anglica on MCF-7 Cells
Breast cancer is divided into ER-positive and ER-negative, depending on the presence or absence of ER expression. More than 70% of breast cancers are ER-positive, and high estrogen levels have a close impact. High levels of estrogen in the blood increase the risk of breast cancer development. Moreover, HRT, which involves the administration of female hormones to compensate for the lack of estrogen in menopausal women, increases the risk of breast cancer development [43,44]. HRT acts as a ligand in ER-positive breast cancer cells and increases cell proliferation.
Several previous studies on the safety of estrogen-like active compounds regarding ER-positive MCF-7 breast cancer cell proliferation under normal conditions have been reported [45][46][47]. Therefore, to evaluate the safety regarding the adverse effects associated with HRT treatment, the cytotoxic effects of extracts and compounds from S. anglica were measured in MCF-7 breast cancer cells using a cell viability assay. control. Subsequently, the protein levels were determined by western blotting. Data are presented as mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus untreated cells.

Anti-Proliferative Effect of Extracts and Compounds from S. anglica on MCF-7 Cells
Breast cancer is divided into ER-positive and ER-negative, depending on the presence or absence of ER expression. More than 70% of breast cancers are ER-positive, and high estrogen levels have a close impact. High levels of estrogen in the blood increase the risk of breast cancer development. Moreover, HRT, which involves the administration of female hormones to compensate for the lack of estrogen in menopausal women, increases the risk of breast cancer development [43,44]. HRT acts as a ligand in ER-positive breast cancer cells and increases cell proliferation.
Several previous studies on the safety of estrogen-like active compounds regarding ER-positive MCF-7 breast cancer cell proliferation under normal conditions have been reported [45][46][47]. Therefore, to evaluate the safety regarding the adverse effects associated with HRT treatment, the cytotoxic effects of extracts and compounds from S. anglica were measured in MCF-7 breast cancer cells using a cell viability assay.
Therefore, we investigated the proliferation of ER-positive MCF-7 human breast cancer cells under normal conditions. None of the extracts and compounds from the belowground and aerial parts of S. anglica resulted in an increase in cell proliferation. Furthermore, p-hydroxybenzaldehyde (3) and N-trans-feruloyltyramine (4) reduced MCF-7 cell proliferation (Figure 7). The results indicate that extracts of S. anglica and its compounds have estrogenic effects, without increasing the risk of breast cancer development. Therefore, we investigated the proliferation of ER-positive MCF-7 human breast cancer cells under normal conditions. None of the extracts and compounds from the belowground and aerial parts of S. anglica resulted in an increase in cell proliferation. Furthermore, p-hydroxybenzaldehyde (3) and N-trans-feruloyltyramine (4) reduced MCF-7 cell proliferation (Figure 7). The results indicate that extracts of S. anglica and its compounds have estrogenic effects, without increasing the risk of breast cancer development.  Proliferation was determined using the EZ-Cytox kit. Data are presented as mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus untreated cells. (SA-bg-W: water extract from belowground parts of S. anglica, SA-bg-E30: 30% ethanol extract from belowground parts of S. anglica, SA-bg-E50: 50% ethanol extract from belowground parts of S. anglica, SA-bg-E80: 80% ethanol extract from belowground parts of S. anglica, SA-a-W: water extract from aerial parts of S. anglica, SA-a-E30: 30% ethanol extract from aerial parts of S. anglica, SA-a-E50: 50% ethanol extract from aerial parts of S. anglica, SA-a-E80: 80% ethanol extract from aerial parts of S. anglica).

General Experimental Procedures
Optical rotation was measured using a Jasco DIP-1000 polarimeter (JASCO Inc., Tokyo, Japan) coupled with a sodium lamp (589 nm). Vacuum liquid chromatography (VLC) Mar. Drugs 2021, 19, 210 9 of 13 was performed using Silica Gel 60 (230-400 mesh, Merck KGaA, Darmstadt, Germany). LR-ESI-MS was performed on an Agilent 1260 series HPLC system coupled with a 6120 series single quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). HPLC was carried out on a Gilson HPLC system (321 pump, and UV/Vis-155, Gilson Inc., Middleton, WI, USA) and a Waters HPLC system (PDA 996, and 600 controllers, Waters Corporation, Milford, MA, USA). The 1D and 2D NMR spectra were obtained using a Bruker Avance DPX250 spectrometer (Bruker, Billerica, USA) and 600 MHz Fourier transform nuclear magnetic resonance spectrometer (VNS600, Agilent) at the Core Research Support Center for Natural Products and Medical Materials.

Plant Material
S. anglica was collected from a mudflat on Ganghwa Island in South Korea in July 2016. The plant was identified by Dr. Hyukjae Choi, College of Pharmacy, Yeungnam University. The collected S. anglica was washed with tap water and dried at 24 • C in the shade. The dried plant specimen was deposited at the College of Pharmacy in Yeungnam University in South Korea.
The belowground (dry wt. 10 kg) parts of S. anglica was extracted with 50% ethanol in deionized water to obtain crude extracts of belowground parts (590 g) for compound isolation.
A portion (150 g) of the 50% ethanolic extracts of the belowground parts was suspended in 1.0 L of deionized water and partitioned with hexanes, methylene chloride (MC), and ethyl acetate (EA) three times. Each layer was dried under reduced pressure to yield Hex-bg (472.5 mg), MC-bg (461.3 mg), and EA-bg (315.0 mg) fractions and a residual aqueous fraction (H 2 O-bg, 148 g).

MCF-7 Culture
The MCF-7 human breast cancer cell line was obtained from the American Type Culture Collection (Bethesda, MD, USA). Cells were maintained at 37 • C in a humidified, 5% CO 2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Corning, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Atlas, Fort Collins, CO, USA) and penicillin/streptomycin (Gibco, Grand Island, NY, USA).

Determination of Estrogenic Effect in MCF-7 Cells
Experiments were performed using an E-screen assay as previously described [31]. Briefly, MCF-7 cells were plated at a density of 1 × 10 4 cells/well in 48-well plates, allowed to adhere overnight, and challenged with the indicated concentrations of samples in phenol red-free RPMI (Gibco, Carlsbad, CA, USA) supplemented with 10% charcoal-dextran stripped serum (Innovative Research, Peary CourtNovi, MI, USA) for 144 h. Queens One Tablet was used as positive control in this study, it is constituted 200 mg of Red clover extract in tablet (360 mg), and its active compounds are isoflavones. It is commonly used for improvement of the symptoms about menopause such as hot flush, night sweats, emotional lability, agitation and insomnia. To confirm the estrogenic effect, the cells were maintained in the presence or absence of the estrogen receptor antagonist ICI 182,780 (Tocris Bioscience, Bristol, UK). Thereafter, Ez-Cytox (Daeil Lab Service Co., Seoul, Korea) was added into each well and the cells were incubated for 1 h. Subsequently, the absorbance was measured using a microplate reader (SPARK 10M; Tecan, Männedorf, Switzerland) at a wavelength of 450 nm. Data are presented as the mean ± SEM of three independent experiments performed in triplicate. Cell viability is expressed as a percentage of that in the untreated group (100%).

Determination of Anti-Proliferative Effect in MCF-7 Cells
Experiments were conducted as previously described [48]. Briefly, MCF-7 cells were plated at a density of 1 × 10 4 cells/well in 96-well plates, allowed to adhere overnight, and challenged with the indicated concentrations of samples for 24 h. Thereafter, Ez-Cytox was added into each well and the cells were incubated for 1 h. Subsequently, the absorbance was measured using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA) at a wavelength of 450 nm. Data are presented as the mean ± SEM of three independent experiments performed in triplicate. Cell viability is expressed as a percentage of that in the untreated group (100%).

Detection of Protein Expression in MCF-7 Cells
Experiments were conducted as previously described [48]. Briefly, MCFs were plated at a density of 2 × 10 5 cells/well in 6-well plates, allowed to adhere overnight, and challenged with the indicated concentrations of samples for 24 h. The levels of phospho-ERα (p-ERα), ERα, phosphor-ERβ (p-ERβ), ERβ and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the cells were determined by western blotting. Cell lysates were extracted using radioimmunoprecipitation assay buffer (Tech & Innovation, Gangwon, Korea). Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein separation was conducted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thereafter, proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany) and blocked with 5% skim milk. Afterward, the membrane was incubated with diluted primary antibodies against p-ERα, ERα, p-ERβ, ERβ, and GAPDH (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 • C. After washing the membrane, it was incubated at room temperature with a secondary rabbit IgG antibody for 1 h. The protein signal was measured using SuperSignal ® West Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce) and the Fusion Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Protein expression was normalized to that of GAPDH (reference protein). The analysis was performed using a Fusion Solo Chemiluminescence System. Relative protein expression was calculated and compared with that in an untreated group using ImageJ software (Version 1.51J; National Institutes of Health, Bethesda, MD, USA).

Animals
Crj:CD (SD) rats (female, 14-day-old pups) were obtained from DBL (Chungbuk, Korea). After adaptation for 1 week, the 21-day-old immature rats were individually housed in cages. The immature rats were weighed and randomly assigned to each group. Body weight was recorded daily throughout the study. The rats were provided free access to water and a commercial diet. The room was maintained at a temperature of 22 ± 2 • C and relative humidity of 50 ± 10% and artificially illuminated with a fluorescent lamp in a 12-h light/dark cycle. All animals were managed based on the animal experimental guidelines suggested by the Institutional Animal Care and Use Committee (GIACUC-R2019035, approval on 15 November 2019).

Uterotrophic Assay
The rat uterotrophic assay proposed by the OECD is a screening method for the determination of the estrogenic properties of endocrine-disrupting chemicals [49]. In the present study, to test the estrogenic properties of the sample, we performed an uterotrophic assay with immature rats. The rats were divided into three groups of three rats. Corn oil and water were administered to the normal group and estradiol in corn oil (0.01 mg estradiol/kg body weight in 4 mL corn oil/kg body weight) was injected subcutaneously in the back of the rats daily in the control group. In the SA-bg-E50 group, corn oil (4 mL/kg body weight) was injected subcutaneously and SA-bg-E50 was orally administered at a dosage of 200 mg/kg body weight per day for 3 days. The normal and control groups were orally administered water for 3 days. Rats were euthanized approximately 24 h after the final administration. During necropsy, the uterus was carefully removed and weighed without any attached fat or mesentery.

Statistical Analyses
All data are expressed as the mean ± standard error of mean (SEM). Statistical analyses were conducted using one-way analysis of variance (ANOVA) and Tukey's post-test to evaluate differences among the various experimental groups (GraphPad Prism 7.0, Graph-Pad Software Inc., San Diego, CA, USA). p < 0.05, p < 0.01, and p < 0.001 were considered significant.

Conclusions
Menopause presents with symptoms including facial flushing, vaginal atrophy, and osteoporosis, which are caused by decreased estrogen production. HRT, which is the administration of female hormones, is utilized to treat estrogen reduction, but it has been reported to increase the risk of breast cancer development. Therefore, we aimed to evaluate the estrogenic activities of S. anglica and its constituents and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic effects of extracts of S. anglica and its compounds in MCF-7 breast cancer cells. SA-bg-E50 and 1,3-di-O-trans-feruloyl-(−)-quinic acid (1) had significant estrogenic activity. Furthermore, the administration of SA-bg-E50 increased the uterine weight compared with that in the normal group in an immature female rat model. Compound 1 was regarded as an ERα ligand that induced the phosphorylation of serine residues of ERα. Additionally, compound 1 did not affect MCF-7 cell proliferation. Consequently, compound 1, as an ERα ligand, could be an active compound of S. anglica and have estrogenic effect without side effects, such as the developing risk of breast cancer.