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

Abstract

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.

Keywords: 2-phenylacetamide; ERα; ERβ; GPR30; Lepidium apetalum Willd; estrogen-like effects.

Figure 1

Effect of Lepidium apetalum Willd (LA) on MCF-7 cell proliferation (x ± SD, n = 3). * p < 0.05; ** p < 0.01 compared to controls.

Figure 2

Western blot analysis of the expression of ERα, ERβ and GPR30 in the uterus of immature female Swiss mice and MCF-7 cells (n = 3). Figure (A), (B), (C) and (D) show the expression of ERα, ERβ and GPR30 in the uterus. Figure (E), (F), (G) and (H) show the expression of ERα, ERβ and GPR30 in MCF-7 cells. * p < 0.05; ** p < 0.01 compared to controls.

Figure 3

Effect of estrogen receptor antagonists on LA aroused MCF-7 cell proliferation (n = 3). (A) Effects of unspecific ER antagonist (ICI182780) on LA stimulated MCF-7 cell proliferation. (B) Effects of methylpiperidino-pyrazole (MPP) on LA stimulated MCF-7 cell proliferation. (C) Effects of Delta (9)-tetrahydrocannabinol (THC) on LA stimulated MCF-7 cell proliferation. (D) Effects of specific GPR30 antagonist G-15 (G-15) on LA stimulated MCF-7 cell proliferation. * p < 0.05; ** p < 0.01 compared to controls.

Figure 4

Effect of 2-phenylacetamide (PA) on MCF-7 cell proliferation (x ± SD, n = 3). ** p < 0.01 compared to control using a Student’s t-test.

Figure 5

Western blot analysis of the expression of estrogen receptors in the uterus of immature female Swiss mice and MCF-7 cells (n = 3). Figure (A), (B), (C) and (D) show the expression of ERα, ERβ and GPR30 in the uterus. Figure (E), (F), (G) and (H) show the expression of ERα, ERβ and GPR30 in MCF-7 cells. * p < 0.05; ** p < 0.01 compared to controls.

Figure 6

Effect of estrogen receptor antagonists on PA aroused MCF-7 cell proliferation (n = 3). (A) Effects of unspecific ER antagonist (ICI182780) on PA stimulated MCF-7 cell proliferation. (B) Effects of methylpiperidino-pyrazole (MPP) on PA stimulated MCF-7 cell proliferation. (C) Effects of Delta (9)-tetrahydrocannabinol (THC) on PA stimulated MCF-7 cell proliferation. (D) Effects of specific GPR30 antagonist G-15 (G-15) on PA stimulated MCF-7 cell proliferation. * p < 0.05; ** p < 0.01 compared to controls.

Figure 7

The ¹H-NMR (A) and ¹³C-NMR (B) spectrums of PA (in CD₃OD). The data of NMR are as follows: ¹H-NMR (CD₃OD, 500 MHz) δ:7.29 (5H, m, H-2,3,4,5,6), 3.50 (2H, s, H-7); ¹³C-NMR (CD₃OD, 125 MHz) δ:136.9 (C-1), 130.1 (C-2,6), 129.6 (C-3,5), 127.9 (C-4), 43.4 (C-7), 177.0 (C-8).

Figure 8

Representative HPLC chromatogram of PA (A) and crude water extract of LA (B). Waters Alliance 2695 separations module equipped with Empower software hyphened with quaternary pumps, an automatic injector, a Waters 2998 photodiode array (PDA) detector at 190–800 nm, and a 250 mm × 4.6 mm × 5 μm Platisil ODS C18 column was used for the separation. HPLC analysis conditions: Acetic acid water ((A), 0.1%)-acetonitrile (B), 0~60 min (5~25% (A)); flow velocity: 1 mL/min, 5 μL injection; wavelength of detection: 254 nm; column temperature: 30 °C.