Isolation and Characterization of Phenylpropanoid and Lignan Compounds from Peperomia pellucida [L.] Kunth with Estrogenic Activities

Abstract

Extracts of Peperomia pellucida [L.] Kunth have previously been demonstrated to have in vivo estrogenic-like effects, thereby functioning as an anti-osteoporotic agent. However, the compounds responsible for these effects have not yet been determined. Therefore, the aim of this study is to isolate and elucidate potential compounds with estrogenic activity. The structures of the isolated compounds were identified using 1D ¹H and ¹³C-NMR and confirmed by 2D FT-NMR. The estrogenic activity was evaluated using the E-SCREEN assay, and a molecular docking study was performed to predict the binding affinity of the isolated compounds to estrogen receptors. In this experiment, we successfully isolated three phenylpropanoids and two lignan derivatives, namely, 6-allyl-5-methoxy-1,3-benzodioxol-4-ol (1), pachypostaudin B (2), pellucidin A (3), dillapiole (4), and apiol (5). Among these compounds, the isolation of 1 and 2 from P. pellucida is reported for the first time in this study. Activity assays clearly showed that the ethyl acetate extract and its fractions, subfractions, and isolated compounds exerted estrogenic activity. Methanol fraction of the ethyl acetate extract produced the highest estrogenic activity, while 1 and 2 had partial agonist activity. Some compounds (derivates of dillapiole and pellucidin A) also had, in addition, anti-estrogenic activity. In the docking study, the estrogenic activities of 1-5 appeared to be mediated by a classical ligand-dependent mechanism as suggested by the binding interaction between the compounds and estrogen receptors; binding occurred on Arg 394 and His 524 of the alpha receptor and Arg 346 and His 475 of the beta receptor. In summary, we reveal that P. pellucida is a promising anti-osteoporotic agent due to its estrogenic activity, and the compounds responsible for this activity were found to be lignan and phenylpropanoid derivatives. The presence of other compounds in either the extract or fraction may contribute to a synergistic effect, as suggested by the higher estrogenic activity of the methanol fraction. Hence, we suggest further research on the osteoporotic activity and safety of the identified compounds, especially regarding their effects on estrogen-responsive organs.

Keywords: E-SCREEN; Peperomia pellucida; docking; phenylpropanoid and lignan; phytoestrogen.

Figure 1

Chemical structures of compounds 1–5 isolated from the aerial part of P. pellucida as described in materials and methods.

Figure 2

Key COSY, NOESY, and HMBC Correlations of Pachypostaudin B (2).

Figure 3

The estrogenic activity of the ethyl acetate extract and its fractions, subfractions, and isolated compounds from P. pellucida was measured using the E-SCREEN assay. Cells were treated with 1 µg/mL of the samples for 144 h, and the proliferative effects relative to cells in the presence of E₂ (10⁻⁹ M, 100%) were then investigated using sulforhodamine b (SRB) assays. The data are expressed as the mean ± SD of at least two separate experiments with duplicates or triplicates for each group. The symbol ** represents statistically significant differences compared with the methanol fraction, and ## represents statistically significant differences compared with subfraction 1, analyzed by the Kruskal–Wallis test using Bonferroni adjustment; ** p < 0.01, # p < 0.05, ## p < 0.01.

Figure 4

The estrogenic activity of five compounds isolated from the ethyl acetate extract of P. pellucida using the E-SCREEN assay. Cells were treated with the compounds in a dose range of 0.001–10 µg/mL for 144 h, and relative proliferative effects on the cells were then investigated using sulforhodamine b (SRB) assays, with 10⁻⁹ M E₂ as the standard control. The data are expressed as the mean ± SD of at least two separate experiments with duplicates or triplicates for each group. The symbols * and ** represent statistical differences compared with the vehicle group: * p < 0.05, ** p < 0.01, analyzed by the Kruskal–Wallis test using Bonferroni adjustment.

Figure 5

Estrogenic effects of the tested compounds on MCF-7/BUS cells in the presence of 10⁻⁹ M E₂. Cells were treated with estradiol alone or estradiol in combination with 10 µg/mL of the tested samples for 144 h, and relative proliferative effects on the cells were then investigated using SRB assays. The data are expressed as the mean ± SD of at least two separate experiments in duplicate or triplicate for each group. The symbols * and ** represent statistical differences compared with the estradiol group: * p < 0.05, ** p < 0.01, analyzed by the Kruskal–Wallis test using Bonferroni adjustment.

Figure 6

Estrogenic effects of estradiol and the tested compounds in the presence of 10⁻⁶ M tamoxifen on MCF-7/BUS cells. Cells were treated with estradiol and samples alone or in combination with tamoxifen for 144 h, and then relative proliferative effects on the cells were investigated using SRB assays. The data are expressed as mean ± SD of at least two separate experiments in duplicate or triplicate for each group. Black bars indicate a testing of a single compound in isolation while white bars indicate their combination with tamoxifen. The symbols * and ** represent statistical differences compared with the estrogenic effect of the samples alone: * p < 0.05, ** p < 0.01, analyzed by the independent-samples t-test or Mann–Whitney U test.