Anti-Menopausal Effects of Cornus officinalis and Ribes fasciculatum Extract In Vitro and In Vivo

Abstract

: Natural herbal medicines have been developed for the treatment and prevention of women's menopausal symptoms. In this study, we investigated the anti-menopausal effects of Cornus officinalis (CO) and Ribes fasciculatum (RF) extracts in 3T3-L1 preadipocytes, MC3T3-E1 preosteoblasts, and COV434 granulosa cells in vitro and ovariectomized (OVX) ddY mice in vivo. Combination treatment of CO and RF extract at 7:3 ratio inhibited lipid accumulation via Plin1 and Adipoq downregulation in a cocktail of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin (DMI)-induced differentiated 3T3-L1 cells. In addition, CO + RF treatment significantly enhanced osteoblastic differentiation, with mineralized nodule formation occurring through the upregulation of osteoblast-inducing markers in osteoblastic MC3T3-E1 cells. Increased production of estradiol and mRNA expression of ERα (ESR1) were observed in androstenedione-induced COV434 granulosa cells treated with the CO + RF extract. In CO + RF-treated mice, fatty hepatocyte deposition and abdominal visceral fat tissues reduced with OVX-induced uterine atrophy. Furthermore, bone mineral density and bone mineral content were significantly enhanced by CO + RF in mouse models of ovariectomy-induced femoral bone loss. Taken together, our findings suggested that CO + RF promoted estrogenic activity and had anti-obesity and anti-osteoporotic effects in vitro and in vivo. Thus, a combination of CO and RF extracts may be a good therapeutic strategy for managing women's menopausal syndromes.

Keywords: Cornus officinalis; Ribes fasciculatum; obesity; osteoporosis; women’s menopause.

Figure 1

Effects of Cornus officinalis (CO) and Ribes fasciculatum (RF) extract on mRNA expression of adipogenesis-related genes and the lipid accumulation in 3T3-L1 cells. The cocktail of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin (DMI)-induced 3T3-L1 cells were treated with combined CO and RF of different ratios (6:4, 7:3, or 8:2) and single extracts of CO or RF, and the mRNA expression level of adipogenesis-inducing genes, including Plin1 (A) and Adipoq (B), was calculated quantitatively by qRT-PCR. (C) Lipid accumulation was assessed by oil red O staining and visualized under a microscope. *: p < 0.05 vs. control, #: p < 0.05 vs. 7:3 (50).

Figure 2

Effects of the CO + RF extract on osteoblast differentiation and mRNA expression of bone remodeling markers in preosteoblastic MC3T3-E1 cells. (A and B) Cells were treated with CO or RF extract or their combination (6:4, 7:3, and 8:2 ratios). After induction of osteoblast differentiation, the alkaline phosphatase (ALP) activity was assessed. (C) Osteoblast differentiation and mineralized nodule formation were assessed using the ALP assay and by Alizarin Red S staining after treatment with the CO and RF extracts in combination at a 7:3 ratio (50 μg/mL). (D) The relative mRNA levels of Alpl, Runx2, and Bglap in the combined CO and RF extract-treated cells were analyzed by RT-PCR. *: p < 0.05 vs. control, # p < 0.05 vs. 7:3 (50).

Figure 3

Anti-obesity effects of CO + RF in ovariectomized (OVX) mice. The OVX mice were administered with combined CO + RF extract (75 (L), 150 (M) or 300 (H) mg/kg/day) for 12 weeks. Sham: Sham operated, OVX: Non-CO + RF-administered mice. At the end of animal experiments, total % fat (A) and total body weight were measured (B) and adipose cell diameters (C) were measured using the CaseViewer program (3DHISTECH Ltd.). Histological images of liver and abdominal fat tissues were assessed using hematoxylin and eosin (H&E) staining (D). The serum levels of leptin and insulin (E) were measured using enzyme-linked immunosorbent assay (ELISA). *: p < 0.05 vs. OVX.

Figure 4

The effects of CO and RF on uterine weight and morphology and estrogen receptor ESR1 expression in OVX mice. After CO and RF mixture treatment (75 (L), 150 (M) or 300 (H) mg/kg/day) for 12 weeks, morphological uterine atrophy was assessed by taking uterine photos and histological images of H&E staining (A) and measuring uterine weight (B). (C) mRNA expression of ESR1 was measured by qRT-PCR. *: p < 0.05 vs. OVX, #: p < 0.05 vs. CO + RF (L).

Figure 5

The effects of CO and RF on estradiol secretion and mRNA expression of estrogen receptor ESR1 gene in androstenedione (ADD)-induced COV434 granulosa cells. Cells were co-treated with CO or RF extract or their combination at a 7:3 ratio (50 μg/mL) and 10 µM ADD for 48 h, and the estradiol level in the supernatant of the cell culture medium was measured by ELISA kit (A), and the mRNA expression level of ESR1 gene (B) was calculated quantitatively by qRT-PCR. *: p < 0.05 vs. control, #: p < 0.05 vs. CO.

Figure 6

Anti-osteoporotic effects of CO + RF in OVX mice. After 12 weeks treatment, OVX-induced bone loss was evaluated by bone mineral density (BMD) (A) and bone mineral content (BMC) (B) with micro-CT image of right femur (C). *: p < 0.05 vs. OVX.