*Article* **Sustainable Bioactive Composite of** *Glehnia littoralis* **Extracts for Osteoblast Differentiation and Bone Formation**

**Chul Joong Kim 1,† , Bimal Kumar Ghimire 2,† , Seon Kang Choi 3, Chang Yeon Yu <sup>4</sup> and Jae Geun Lee 1,\***


**\*** Correspondence: leejeakun@hanmail.net; Tel.: +82-01091584996

† These authors contributed equally to this work.

**Abstract:** Different bone-related diseases are mostly caused by the disruption of bone formation and bone resorption, including osteoporosis. Traditional medicinal literature has reported the possible anti-osteoporotic properties of *Glehnia littoralis*. However, the chemical compounds in extracts that are responsible for bone metabolism are poorly understood. The present study aimed to explore and compare the coumarin-based compounds present in *G. littoralis* extracts, the antioxidant activities, and the anti-osteoporotic properties of different extracts of *G. littoralis* (leaf and stem, fruit, whole plant, and root extracts) on bone metabolism. This study analyzed *G. littoralis* extract effects on the proliferation and osteoblastic differentiation of MC3T3-E1 osteoblasts. Among the different tested samples, stem extracts had the highest scopoletin (53.0 mg/g), and umbelliferone (1.60 mg/g). The significantly (*p* < 0.05) highest amounts of imperatorin (31.9 mg/g) and phellopterin (2.3 mg/g), were observed in fruit and whole plant extracts, respectively. Furthermore, the results confirmed alkaline phosphatase activity, collagen synthesis, mineralization, osteocalcin content, and osterix and RUNX2 expression. *G. littoralis* extracts at concentrations greater than 20 μg/mL had particularly adverse effects on MC3T3-E1 cell viability and proliferation. Notably, cell proliferation was significantly elevated at lower *G. littoralis* concentrations. Comparatively, 0.5 μg/mL stem had a higher osteocalcin content. Of the four extract types, stem showed a higher collagen synthesis effect at concentrations of 0.5–5 μg/mL. Except for fruit extracts, *G. littoralis* extract treatment significantly elevated osterix gene expression. All *G. littoralis* extracts increased RUNX2 gene expression. The results described here indicate that *G. littoralis* ethanolic extracts can effectively prevent osteoporosis.

**Keywords:** *Glehnia littoralis*; osteoblast differentiation; collagen synthesis; alkaline phosphatase activity; osteoblast; osterix expression; RUNX2 expression
