Evaluation of Estrogenic Activity of Extract from the Herbal Mixture Cynanchum wilfordii Hemsley , Phlomis umbrosa Turczaninow, and Angelica gigas Nakai

Abstract

Hormone replacement therapy (HRT) consists of highly effective prescription medications for treating menopausal symptoms; however, these agents have exhibited side effects including the risk of estrogen-induced carcinogenesis. Therefore, interest in phytotherapy-based materials as a natural source of alternatives to estrogen therapy has increased. However, some of these herbal medicines have been reported to increase the risk of estrogen-induced cancer. Herbal formulations composed of a combination of Cynanchum wilfordii Hemsley (CW), Phlomis umbrosa Turczaninow (PU), and Angelica gigas Nakai (AG) extracts (CPAE) have been used for treating menopausal symptoms. Therefore, in this study, we aimed to examine the safety of CPAE by determining its potential adverse estrogenic activity using the Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455) in a stably transfected transcriptionally activated human estrogen receptor α (hERα)-HeLa9903 cell model. We found that CPAE did not how any estrogenic activity or stimulate promoters containing estrogen response elements in MCF-7 cells. In addition, CPAE showed no significant selective activity against hERα and hERβ, non-selective activity against the ER, or effects on ER target gene expression. Furthermore, CPAE did not significantly induce MCF-7 cell proliferation and uterine weight increase in ovariectomized rats. These results demonstrate that CPAE can be used as beneficial herbal drug for prevention and therapeutic intervention of estrogen carcinogenesis in menopausal women.

Keywords: Angelica gigas Nakai; Cynanchum wilfordii Hemsley; Estrogen receptor; Estrogenic activity; Phlomis umbrosa Turczaninow.

Fig. 1

Concentration-response curve of transcriptional activation by Cynanchum wilfordii Hemsley, Phlomis umbrosa Turczaninow, and Angelica gigas Nakai extracts (CPAE) using HeLa9903 cells. Estradiol (E2, 10^−13.6–10^−7.6 g/L, 10⁻⁴–10² pM), genistein (10^−8.6–10^−2.6 g/L, 10⁻⁶–10 μM), and CPAE (10⁻⁷–10⁻¹ g/L). Cells were harvested and assayed for luciferase activity. All experiments were performed in triplicate. Bars represent mean ± SD.

Fig. 2

Effect of estrogen response element (ERE) luciferase promoter activity induced by Cynanchum wilfordii Hemsley, Phlomis umbrosa Turczaninow, and Angelica gigas Nakai extract (CPAE) in MCF-7 cells. MCF-7 cells were transfected with pERE-Luc, treated with CPAE (100 μg/mL), estradiol (E2; 10 nM), ICI 182,780 (ICI, 10 μM) or a combination of these compounds for 24 hr. Cells were harvested and assayed for luciferase activity, which was normalized to β-galactosidase activity. All experiments were performed in triplicate. Bars represent mean ± SD. ^#P < 0.05 vs. control and *P < 0.05 vs. E2.

Fig. 3

Gene expression of progesterone receptor (PGR) and trefoil factor 1 (TFF1) in MCF-7 cells. Effect of CPAE (100 μg/mL) on PGR and TFF1 mRNA levels determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). All experiments were performed in triplicate. Bars represent mean ± SD. ^#P < 0.05 vs. control and *P< 0.05 vs. E2.

Fig. 4

Effects of Cynanchum wilfordii Hemsley, Phlomis umbrosa Turczaninow, and Angelica gigas Nakai extract (CPAE) on MCF-7 cell growth. MCF-7 cells were exposed to CPAE (100 μg/mL), estradiol (E2; 10 nM), ICI 182,780 (ICI, 10 μM) or a combination of these compounds for 24 hr. Cell proliferation was measured using water-soluble tetrazolium (WST)-1 assay. Bars represent mean ± SD. ^#P < 0.05 vs. control and *P < 0.05 vs. E2.

Fig. 5

Effects of Cynanchum wilfordii Hemsley, Phlomis umbrosa Turczaninow, and Angelica gigas Nakai extract (CPAE) in ovariectomized (OVX) rat uterotrophic assay. Each compound was administered subcutaneously at indicated concentration (125 and 250mg/kg per day) for 12 weeks. ^#P < 0.05 vs. sham group and *P < 0.05 vs. OVX group.