Complex Extract of Polygonatum sibiricum and Nelumbinis semen Improves Menopause Symptoms via Regulation of Estrogen Receptor Beta in an Ovariectomized Rat Model

Abstract

Menopause is a hormone-deficiency state that causes facial flushing, vaginal dryness, depression, anxiety, insomnia, obesity, osteoporosis, and cardiovascular disease as ovarian function decreases. Hormone-replacement therapy is mainly used to treat menopause; however, its long-term use is accompanied by side effects such as breast cancer and endometriosis. To identify the effect of a complex extract of Polygonatum sibiricum (PS) and Nelumbinis semen (NS) on improving menopause without side effects, an ovariectomized rat model was established to analyze several menopause symptoms. Compared to single extracts, the complex extract restored vaginal epithelial cell thickness and decreased serotonin concentration by increasing the estrogen receptors ERα (ESR1) and ERβ (ESR2), depending on the ratio. Although the complex extract exerted a lower weight-loss effect than the single extracts, improved blood-lipid metabolism was observed after increasing high-density lipoprotein cholesterol levels and decreasing low-density lipoprotein cholesterol and triglyceride levels, and ovariectomy-induced osteoporosis was alleviated by suppressing osteoclast production. Thus, by increasing only ERβ expression without regulating ERα expression in the uterus, the complex extract of PS and NS may be a natural treatment for improving menopause symptoms without side effects, such as endometriosis.

Keywords: Nelumbinis semen; Polygonatum sibiricum; estrogen receptor; herbal medicine; menopause; menopause symptoms.

Figure 1

Changes in the tail skin temperature of OVX rats. Data are expressed as the mean ± SD (n = 10). Two-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Asterisks (*) indicate statistical significance (* p < 0.05, ** p < 0.005, and *** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; LJ, leonurus japonicus; SD, standard deviation; ANOVA, analysis of variance.

Figure 2

Effects of complex extract of PS and NS on vaginal epithelial tissue. (A) Vaginal epithelium cell smears of OVX rats treated with PS, NS, or the mixture for 6 weeks. Cornified cells, nucleated epithelial cells, and leukocytes were observed via methylene blue staining (n = 6). Scale bar = 200 μm. (B) Quantitative analyses of the percentage of cornified cells. (C) Vaginal tissue slides with H&E staining. (D) Quantitative analyses of vaginal epithelial layer thickness. Values are expressed as the mean ± SD. One-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Pound signs (#) indicate statistical significance (### p < 0.0005) relative to the sham-operated group. Asterisks (*) indicate statistical significance (** p < 0.005, and *** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; H&E, hematoxylin and eosin; SD, standard deviation; ANOVA, analysis of variance.

Figure 3

Confirmation of ERα and ERβ expression in vaginal tissues of rats treated with complex extract of PS and NS. (A,C) Immunostaining of ERα (A) and ERβ (C) protein expression levels in the vagina of extract-treated OVX and sham-operated rats (n  =  6). Scale bar = 100 μm. (B,D) Quantitative analyses of protein expression levels of ERα and ERβ, respectively. Values are expressed as the mean ± SD. One-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Pound signs (#) indicate statistical significance (### p < 0.0005) relative to the sham-operated group. Asterisks (*) indicate statistical significance (** p < 0.005, and *** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; SD, standard deviation; ANOVA, analysis of variance.

Figure 4

Effects of PS, NS, and mixtures on menopause-related obesity regarding body weight and lipid profile. (A) Body weight and (B) HDL-C, (C) LDL-C, and (D) triglyceride levels (n = 6). Values are expressed as the mean ± SD. One-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Pound signs (#) indicate statistical significance (## p < 0.005 and ### p < 0.0005) relative to the sham-operated group. Asterisks (*) indicate statistical significance (* p < 0.05, ** p < 0.005, and *** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; HDL-C, high-density lipoprotein; LDL-C, low-density lipoprotein; SD, standard deviation; ANOVA, analysis of variance.

Figure 5

Effect of PS, NS, and mixtures on bone loss in OVX rats. (A) Representative H&E staining images of tibial serial sections (n = 6). Scale bar = 500 μm. (B) Serum ALP level. (C) Representative TRAP staining images of tibial serial sections (n = 6). Scale bar = 50 μm. (D) Osteoclast numbers per bone surface (OC.N/mm) are shown. Values are expressed as the mean ± SD. One-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Pound signs (#) indicate statistical significance (### p < 0.0005) relative to the sham-operated group. Asterisks (*) indicate statistical significance (* p < 0.05, ** p < 0.005, and *** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase; OC, osteoclast; SD, standard deviation; ANOVA, analysis of variance.

Figure 6

Inhibitory effect of PS, NS, and mixtures on endometrial hyperplasia side effects caused by estradiol. (A–D) Protein expression confirmation using immunohistochemical analyses of ERα (A,B) and ERβ (C,D) in the endometria (n = 6). Scale bar = 50 μm. Values are expressed as the mean ± SD. One-way ANOVA was performed, followed by Tukey’s multiple comparisons test. Pound signs (#) indicate statistical significance (## p < 0.005 and ### p < 0.0005) relative to the sham-operated group. Asterisks (*) indicate statistical significance (*** p < 0.0005) relative to the OVX group. OVX, ovariectomized; E2, β-Estradiol; PS, polygonatum sibiricum; NS, nelumbinis semen; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; SD, standard deviation; ANOVA, analysis of variance.