2-Phenylacetamide Isolated from the Seeds of Lepidium apetalum and Its Estrogen-Like Effects In Vitro and In Vivo

The aim of this study was to investigate the estrogen-like effects of 2-phenylacetamide (PA), which is the main compound isolated from the seeds of Lepidium apetalum Willd (LA). Results showed that LA and PA could promote the proliferation of MCF-7 cells. The mouse uterine weight test showed that, LA and PA could increase the uterus index of immature female mice, and the levels of luteinizing hormone (LH) and estrogen (E2). LA could increase the expression of ERα and ERβ, while PA could increase the expression of ERα, ERβ and GPR30 in the uterus and MCF-7 cells. In addition, co-incubation of the estrogen receptor blocker with LA or PA abolished the inductive effect of the proliferation. PA has estrogenic activities and was the material basis of LA that played the estrogenic effect. LA and PA might be used for the treatment of perimenopause syndrome in a novel application.


Introduction
Estrogen is an important regulatory hormone in women's bodies. It is a class of steroids, mainly produced by the ovary and placenta. After women enter menopause, estrogen levels rapidly decline, leading to postmenopausal osteoporosis and perimenopausal syndrome. Estrogen replacement therapy (ERT) is used for treatment in clinical studies [1]. However, the long-term use of this therapy has obvious side effects [2]. Therefore, the safer and more effective phytoestrogen (PE) has received increasing attention from researchers. In addition, estrogen receptors also mediate the effects of PE which include the genomic nuclear estrogen receptors (ERα and ERβ) and the nongenomic estrogen receptor (GPR30) [3].
The dry mature seeds of Lepidium apetalum Willd (LA) which belong to the Brassicaceae family have been used to relieve coughs, prevent asthma, reduce edema and promote urination in traditional Chinese medicine (TCM). Oils, flavonoids, sterols and cardiac glycosides [4][5][6] are contained in the seeds of L. apetalum Willd. In addition, it shows anti-oxidant, antibacterial and cardiac activities [7]. Clinical research suggests that it can be used for heart failure [8,9], pigmentation [10] and oral cancer [11]. In our preliminary study, we found that several Chinese herbal medicines showed estrogen-like effects, including LA. Then, we isolated the main compounds from LA and obtained

Effects of LA on Immature Female Swiss Mice
As shown in Table 1, EV (estradiol valerate, the positive drug of the animal experiment) significantly increased the uterus coefficient in mice, as well as the levels of LH, follicle stimulating hormone (FSH) and E2. Compared with the control group, LA-L (446 mg/kg) and LA-H (892 mg/kg) significantly increased the uterus coefficient. LA-L and LA-H significantly increased the levels of E2 and LH.

Effects of LA on the Expression of Estrogen Receptors
LA-L (446 mg/kg) and LA-H (892 mg/kg) enhanced the expression of estrogen nuclear receptors (ERα and ERβ) in the uterus (Figure 2A-D). LA (10 −2 mg/mL) enhanced the expression of estrogen nuclear receptors in MCF-7 cells ( Figure 2E-H). EV (0.33 mg/kg) and 17β-E2 (10 −6 mol/L) enhanced the expression of estrogen nuclear receptors and GPR30 in uterus and MCF-7 cells separately.

Effects of LA on Immature Female Swiss Mice
As shown in Table 1, EV (estradiol valerate, the positive drug of the animal experiment) significantly increased the uterus coefficient in mice, as well as the levels of LH, follicle stimulating hormone (FSH) and E2. Compared with the control group, LA-L (446 mg/kg) and LA-H (892 mg/kg) significantly increased the uterus coefficient. LA-L and LA-H significantly increased the levels of E2 and LH.

Effects of LA on the Expression of Estrogen Receptors
LA-L (446 mg/kg) and LA-H (892 mg/kg) enhanced the expression of estrogen nuclear receptors (ERα and ERβ) in the uterus (Figure 2A

Effect of Estrogen Receptor Antagonists on LA Aroused MCF-7 Cell Proliferation
As shown in Figure 3, ICI 182780 (unspecific ER antagonist, 1 μM), methylpiperidino-pyrazole (MPP; specific ERα antagonist; 1 μM), Delta (9)-tetrahydrocannabinol (THC; specific ERβ antagonist; 1 μM) could block the effect of LA (10 −2 mg/mL) on MCF-7 cell proliferation. ICI 182780 (1 μM), MPP (1 μM), THC (1 μM) and G-15 (specific GPR30 antagonist G-15, 1 μM) could block the effect of 17β-E2 on MCF-7 cells.      Table 2 shows that EV and LA significantly promoted the uterus coefficient, LH and E2. Compared with the control group, PA-L and PA-H significantly promoted the uterus coefficient, LH and E2.       Table 2 shows that EV and LA significantly promoted the uterus coefficient, LH and E2. Compared with the control group, PA-L and PA-H significantly promoted the uterus coefficient, LH and E2.  Table 2 shows that EV and LA significantly promoted the uterus coefficient, LH and E2. Compared with the control group, PA-L and PA-H significantly promoted the uterus coefficient, LH and E2.

Discussion
Irregular menstruation and amenorrhea may occur when estrogen levels have decreased. After entering menopause [13], there may be a series of diseases and symptoms, such as menstrual disorders, mood swings, hot flushes and cardiovascular disease. Estrogen replacement therapy is used for treatment in clinical studies [14]. However, the long-term use of this therapy has obvious side effects [15]. Therefore, safer and more effective phytoestrogen has received increasing attention from researchers. Phytoestrogens play a two-way role in changing and promoting estrogen activity. When the levels of estrogen are low in vivo, phytoestrogens have an estrogen-like effect; when the levels are high, they exert antiestrogenic activity by competitively binding to estrogen receptors [16,17]. Therefore, phytoestrogens are known as natural, selective estrogen receptor modulators. Nourishing plants and seed medicines are normally used as the phytoestrogens in traditional Chinese medicine. In a previous study from our laboratory, it was found that the water extract of LA, a kind of seed medicine, had an estrogen-like effect. In the following experiment, we investigated the estrogen-like effects of compounds isolated from LA, and found that PA, which is one of the main compounds of LA, was the material basis of LA that played the estrogenic effect. In addition, PA is the key pharmaceutical intermediate of atenolol [18] and penicillin [19].
Uterine coefficient of immature rabbits was used to detect estrogenic activity of drugs in some of the earliest studies [20,21]. This was until Astwood and others [22,23] determined that the uterine coefficient of immature mice is more sensitive than that of immature rabbits. Uterine coefficient (the

Discussion
Irregular menstruation and amenorrhea may occur when estrogen levels have decreased. After entering menopause [13], there may be a series of diseases and symptoms, such as menstrual disorders, mood swings, hot flushes and cardiovascular disease. Estrogen replacement therapy is used for treatment in clinical studies [14]. However, the long-term use of this therapy has obvious side effects [15]. Therefore, safer and more effective phytoestrogen has received increasing attention from researchers. Phytoestrogens play a two-way role in changing and promoting estrogen activity. When the levels of estrogen are low in vivo, phytoestrogens have an estrogen-like effect; when the levels are high, they exert antiestrogenic activity by competitively binding to estrogen receptors [16,17]. Therefore, phytoestrogens are known as natural, selective estrogen receptor modulators. Nourishing plants and seed medicines are normally used as the phytoestrogens in traditional Chinese medicine. In a previous study from our laboratory, it was found that the water extract of LA, a kind of seed medicine, had an estrogen-like effect. In the following experiment, we investigated the estrogen-like effects of compounds isolated from LA, and found that PA, which is one of the main compounds of LA, was the material basis of LA that played the estrogenic effect. In addition, PA is the key pharmaceutical intermediate of atenolol [18] and penicillin [19].
Uterine coefficient of immature rabbits was used to detect estrogenic activity of drugs in some of the earliest studies [20,21]. This was until Astwood and others [22,23] determined that the uterine coefficient of immature mice is more sensitive than that of immature rabbits. Uterine coefficient (the ratio of weight of the uterus to body weight) in immature mice was used to evaluate estrogen-like activity in our study. LA and PA could significantly increase the uterus coefficient, LH and E2, indicating that the LA and PA had an estrogen-like effect. MCF-7 cells are the most commonly used cell lines for detecting estrogen-like activity. Estrogen-like substances can promote the proliferation of MCF-7 cells which are estrogen receptor-positive cells [24]. In our study, LA and PA promoted the proliferation of MCF-7 cells and exhibited dose-dependent behavior. In another experiment, estrogen receptor antagonists (ICI182, 780, MPP, THC and G-15) [25][26][27][28] were used to investigate the mechanisms of LA and PA. Estrogen nuclear receptor antagonists could block the effect of LA on MCF-7 cells, demonstrating that the estrogen-like activity of LA was mediated by estrogen nuclear receptors (ERα and ERβ). Estrogen nuclear receptor antagonists and G-15 could block the effect of PA on MCF-7 cells indicating the estrogen-like activity of PA was mediated by estrogen nuclear receptors and GPR30.
Furthermore, Western blot showed that PA (5 µM) promoted the expression of ERα, ERβ and GPR30 in MCF-7 cells, and that PA (25, 50 mg/kg) promoted the expression of ERα, ERβ and GPR30 in uterine. LA (10 −2 mg/mL) promoted the expression of ERα and ERβ in MCF-7 cells, LA (446, 892 mg/kg) promoted the expression of ERα and ERβ in uterus. This indicates that PA might exert an estrogen-like effect through estrogen nuclear receptors and GPR30, while LA might exert an estrogen-like effect through estrogen nuclear receptors, EV (0.33 mg/kg) and 17β-E2 (1 µM) just as the positive control.
In addition, uterine edema and hyperplasia was found in the EV group which may be because estrogen can promote the expression of vascular endothelial growth factor (VEGF) in the uterus, enhance blood vessel permeability, induce overgrowth of endothelial cells and inhibit endothelial cells apoptosis [29]. In our study, LA and PA did not have the adverse reactions of EV.
In conclusion, our studies in vitro and in vivo all proved that LA has an estrogen-like effect, and PA, which is one of the main compounds of LA, was the material basis of LA that played the estrogenic effect. ICI182780, MPP, THC and G-15 estrogen receptor antagonist experiments, together with Western blot, revealed that the estrogen-like effects of LA are mediated by estrogen nuclear receptors, and the estrogen-like effects of PA are mediated by estrogen nuclear receptors and GPR30. In addition, LA and PA had no artificial side effects compared to synthetic estrogen. LA and PA may be used for the treatment of perimenopause syndrome in a novel application, and is easy to obtain and synthesize, making it suitable for industrial production.  1-F3.2). F3.2 (10.3 g) was dissolved in MeOH and after standing for 24 h, colorless crystals (4.3 g) were obtained. The purity of the colorless crystals was determined to be above 98% by HPLC. The structure was determined on the basis of NMR spectra and characterized as PA (Figure 7). The purity was estimated directly from the NMR spectrum and HPLC analysis to be greater than 99%. PA was verified as a genuine substance since it was detectable by assays of HPLC in the LA water extract prior to the separation procedure ( Figure 8).

Plant Material
Molecules 2018, 23, x 8 of 11 substance since it was detectable by assays of HPLC in the LA water extract prior to the separation procedure ( Figure 8).

Animals
The study was conducted in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China. Beijing Vital River Laboratory Animal Technology Co., Ltd. (Ethical approval reference number: SCXK2016-0011) provided us with immature female Swiss mice (4 weeks). All mice were housed in cages on a 12:12 h light-dark schedule at a controlled temperature (22 • C) with free access to food and water at the Laboratory Animal Research Center of Henan University of Chinese medicine. All the procedures for the care of the mice were in accordance with the institutional guidelines for animal use in research. A total of 40 mice (9-11 g) were divided into 4 groups: Control group, Estradiol Valerate group (EV; Bayer medical, Shanghai, China; 0.33 mg/kg), low dose of LA group (LA-L, 446 mg/kg), and high dose of LA group (LA-H, 892 mg/kg), continuous gavage for 7 d. Blood was collected by heart punctures. The uterus was peeled off quickly and weighed on a one millionth balance. The mouse uterine weight gain test of PA was the same as LA.

Enzyme-Linked Immuno Sorbent Assay
The levels of E2, FSH and LH in serum were detected by ELISA (R&D, Minneapolis, MN, USA) according to manufacturer's instructions.

Statistical Analysis
The results were expressed as the mean ± SD. The statistical differences between the control and the test fractions were assessed by analysis of variance (ANOVA) followed by a Student's t-test for multiple comparisons. The obtained data were compared with the negative control group and values of p < 0.05 were considered significant.

Conclusions
LA had an estrogen-like effect, and PA, which is one of the main compounds of LA, was the material basis of LA that played the estrogenic effect. Estrogen-like effects of LA are mainly mediated by estrogen nuclear receptors, and the estrogen-like effects of PA are mainly mediated by estrogen nuclear receptors and GPR30. Further studies on model rats (animals with induced menopause/perimenopause syndrome/osteoporosis), including clinical trials, will have to show whether LA or its constituent, PA, have a potential for clinical treatment of postmenopause syndrome.