Isolated Compounds from Turpinia formosana Nakai Induce Ossification

Abstract

Bone metabolism is a homeostatic process, imbalance in which leads to the onset of diseases such as osteoporosis and osteopenia. Although several drugs are currently available to treat such conditions, they are associated with severe side effects and do not enhance bone formation. Thus, identifying alternative treatment strategies that focus on enhancing bone formation is essential. Herein, we explored the osteogenic potential of Turpinia formosana Nakai using human osteoblast (HOb) cells. The plant extract was subjected to various chromatographic techniques to obtain six compounds, including one new compound: 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1). Compounds 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1), gentisic acid 5-O-β-d-(6'-O-galloyl) glucopyranoside (2), strictinin (3), and (-)-epicatechin-3-O-β-d-allopyranoside (6) displayed no significant cytotoxicity toward HOb cells, and thus their effects on various osteogenic markers were analyzed. Results showed that 1-3 and 6 significantly increased alkaline phosphatase (ALP) activity up to 120.0, 121.3, 116.4, and 125.1%, respectively. Furthermore, 1, 2, and 6 also markedly enhanced the mineralization process with respective values of up to 136.4, 118.9, and 134.6%. In addition, the new compound, 1, significantly increased expression levels of estrogen receptor-α (133.4%) and osteogenesis-related genes of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), bone morphogenetic protein (BMP)-2, bone sialoprotein (BSP), type I collagen (Col-1), and brain-derived neurotropic factor (BDNF) by at least 1.5-fold. Our results demonstrated that compounds isolated from T. formosana possess robust osteogenic potential, with the new compound, 1, also exhibiting the potential to enhance the bone formation process. We suggest that T. formosana and its isolated active compounds deserve further evaluation for development as anti-osteoporotic agents.

Keywords: 3,3′-di-O-methylellagic acid-4-O-α-l-arabinofuranoside; Turpinia formosana; alkaline phosphatase; bone formation; estrogen receptors; mineralization; osteoporosis.

Figure 1

Structure of compounds isolated from T. formosana.

Figure 2

Cytotoxicity and induction ALP activity by isolated compounds in HOb cells. Cells were seeded in a 96-well plate, and after 24 h, 100 µM samples were added. (A) After five days, an MTT assay was performed to analyze cell viability. (B) After three days, ALP activity was detected by performing an ALP assay using bicinchoninic acid (BCA) protein to normalize protein expression of cells. Data are expressed as the mean ± SD, * p ≤ 0.05, ** p ≤ 0.01 compared to the control, and all experiments were performed in triplicate.

Figure 3

Isolated compounds increase mineralization in HOb cells. Cells were seeded in a 48-well plate for three days, after which fresh osteoblast differentiation media (ODM) miner containing inducers and samples were added. Subsequently, every two days, fresh ODM miner, inducers, and samples were added until the 11th day. Mineralization was then detected by performing an alizarin red assay. (A) Pictures show mineral deposition after addition of the alizarin red dye, and (B) the graph shows quantitative data obtained after adding 10% cetylpyridinium chloride as a de-stain to dissolve the crystals. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate.

Figure 4

Effect of isolated compounds on ER expression in HOb cells. Cells were seeded in a 96-well plate and after 24 h, 100 µM of samples and 1 µM estradiol (positive control) were added. After five days, (A) ER-α/β expression levels were detected. (B) The dose-dependent effect of 1 on the expression of ER-α was analyzed. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate.

Figure 4

Effect of isolated compounds on ER expression in HOb cells. Cells were seeded in a 96-well plate and after 24 h, 100 µM of samples and 1 µM estradiol (positive control) were added. After five days, (A) ER-α/β expression levels were detected. (B) The dose-dependent effect of 1 on the expression of ER-α was analyzed. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate.

Figure 5

Effect of active compounds on bone formation-related genes in HOb cells. Cells were seeded in 6-cm dishes and after 24 h, fresh ODM containing (A) 100 µM of 1 and 1 µM puerarin (the positive control), or (B) 100 µM of 2, 3, and 6 was added to cells. This was followed by mRNA isolation and reverse transcription. Expression levels were detected by performing a real-time PCR. Data are expressed as the mean ± SD (* p ≤ 0.05, ** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate.