Suppressive Effects of Geoje Raspberry (Rubus tozawae Nakai ex J.Y. Yang) on Post-Menopausal Osteoporosis via Its Osteogenic Activity on Osteoblast Differentiation

Abstract

Background: Osteoporosis is a metabolic bone disease with a high mortality rate due to non-traumatic fractures. The risk of osteoporosis is increasing globally due to an increasing aging population. Current therapies are limited to delaying disease progression. Recently, the need to discover foods with osteogenic activity for the prevention and treatment of osteoporosis has been emphasized. We focused on bone formation via osteoblast differentiation, considering bone formation and resorption during bone homeostasis. Rubus tozawae Nakai ex J. Y. Yang (RL, Geoje raspberry) is a deciduous subshrub that has been traditionally eaten for its fruit.

Methods and results: We identified the third subfraction of n-hexane fraction (RL-Hex-NF3) of RL, an endemic Korean plant with osteogenic activity, which increased bone density in ovariectomized mice, a representative animal model of osteoporosis, via the depletion of female hormones, which resulted from the increase in the osteoblast population. RL-Hex-NF3 induced osteoblast differentiation and the expression of osteogenic markers in MC3T3-E1 pre-osteoblasts. Seven compounds were identified from RL-Hex-NF3 using NMR spectroscopy. Of these, three compounds, namely, 3β-hydroxy-18α,19α-urs-20-en-28-oic acid, betulinic acid, and (1S,6R,7S)-muurola-4,10(14)-diene-15-ol, showed strong osteogenic activity.

Conclusions: RL-Hex-NF3 and its compounds suppress bone loss via their osteogenic properties, suggesting that they could be a potent candidate to treat osteoporosis.

Keywords: Rubus tozawae Nakai ex J.Y. Yang; osteoblast differentiation; osteocalcin; osteogenic activity; osteoporosis; ovariectomized mice.

Figure 1

Basic data of osteoporosis mouse experiment. (A) Design of the animal experiment. (B) Body weight and (C) uterus weight of OVX mice. (D) Serum osteocalcin levels measured using ELISA. Different letters represent significant differences (p < 0.05).

Figure 2

Effects of RL-Hex-NF3 on the bone status in OVX mice. (A) Micro-CT images and (B) bone microarchitecture indicators of the distal femoral region of mice from each group. Bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/bone volume (BS/BV), bone surface area/total volume (BS/TV), trabecular thickness (Tb.Th), and trabecular plate number (Tb.N) were determined using computed tomography. Different letters represent significant differences (p < 0.05).

Figure 3

Histological observation of the distal femoral region of bones. (A) H&E staining, (B,D) type 1 collagen staining, and (C,E) TRAP staining of the distal femoral region of mice from each group. Different letters represent significant differences (p < 0.05); n.s. means not significant.

Figure 4

Effects of RL-Hex-NF3 on the osteoblast population of bone marrow. (A) ALP activity, (B) protein expression, and (C) mRNA expression levels of osteoblastic markers in primary bone marrow cells. Different letters represent significant differences (p < 0.05).

Figure 5

Effects of the RL extract, fractions, and new subfractions (NF) on osteoblastic differentiation in vitro. (A) ALP activity of RL-Hex-NF3. (B) Protein and (C) mRNA expression levels of osteoblastic markers as determined using Western blotting and qRT-PCR. UNDIFF indicates MC3T3E1 cells in cultured media; DIFF indicates MC3T3E1 cells in differentiation media (DM); 10 and 20 indicate MC3T3E1 cells in DM with 10 μg/mL and 20 μg/mL of RL-Hex-NF3, respectively. Different letters represent significant differences (p < 0.05).

Figure 6

(A) ALP activity of compounds from RL-Hex-NF3. Different letters represent significant differences (p < 0.05). (B) Chemical structures of the compounds from RL-Hex-NF3: (1) 3β-hydroxy-18α,19α-urs-20-en-28-oic acid, (2) betulinic acid, and (3) (1S,6R,7S)-muurola-4,10(14)-diene-15-ol.