**1. Introduction**

Osteoporosis is an osteometabolic disorder caused by an imbalance in bone formation by osteoblasts and bone resorption by osteoclasts that make up bones [1,2], which leads to fragility, increased risk of fractures, and threatens mobility in the elderly [3]. According to the International Osteoporosis Foundation (IOF), approximately 200 million people worldwide suffer from osteoporosis [4,5]. Recent studies suggest that increases in inflammation-related cytokine secretion, the number of macrophages, and leukotriene B4, an inflammation-inducing factor [6–9], cause this disease. In women, osteoporosis is associated with a sharp decline in estrogen secretion in the postmenopausal stage, known as postmenopausal osteoporosis [10]. Synthetic therapeutic agents, such as parathyroid hormones, bisphosphonates, selective estrogen receptor modulators (SERMs), and hormone replacement therapy (HRT), are used to treat osteoporosis [11]. However, these drugs can have side effects, including hypercalcemia and osteosarcoma in postmenopausal

**Citation:** Kim, C.J.; Ghimire, B.K.; Choi, S.K.; Yu, C.Y.; Lee, J.G. Sustainable Bioactive Composite of *Glehnia littoralis* Extracts for Osteoblast Differentiation and Bone Formation. *Processes* **2023**, *11*, 1491. https://doi.org/10.3390/pr11051491

Academic Editors: Iliyan Ivanov and Stanimir Manolov

Received: 20 February 2023 Revised: 31 March 2023 Accepted: 10 May 2023 Published: 15 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

women [12], esophageal gastric irritation, and cancer [13], and they can increase the chance of strokes, breast cancer, and coronary heart diseases [14]. Thus, it would be beneficial to discover natural anti-osteoporotic agents that minimize bone loss in postmenopausal women.

Osteoblast cells are critical in bone metabolism and are responsible for bone matrix synthesis and mineralization [15]. Cell culture is widely used to assess the activity of a substance in vitro, such as its osteoinductive potential. In particular, MC3T3 pre-osteoblasts have methodological advantages and facilities [16,17], meaning the differentiation of these cells into mature osteoblasts can be easily recognized by markers of osteoblastic metabolism, such as alkaline phosphatase (ALP), and by the degree of extracellular matrix (ECM) mineralization [18]. ALP is the most widely used biochemical marker for estimating osteoblastic activity [19]. This enzyme is associated with the ECM mineralization process and osteoblastic differentiation, as it is responsible for the maturation of the matrix, which will later be mineralized [18,20].

Current research focuses on natural materials and phytoestrogens for bone formation and resorption pathways related to bone metabolism. There is a constant search for alternatives that can help bone healing, in cases of injury, or favor rehabilitating the quality of the bone tissue. Several recent reports have indicated that phytoestrogen compounds in food and plants can effectively suppress the secretion of inflammation-inducing factors and inflammation-related cytokines associated with osteoporosis [21,22], and enhance or stimulate osteoblast activity [23]. Phytoestrogens act like estrogen and exist in the form of flavonoids, lignins, isoflavones, and coumestans, which share structural and functional similarities with synthetic estrogens [24,25]. The present study aimed to research therapeutic alternatives to treat osteoporosis based on drugs obtained from natural sources, mainly by observing their lower cost and incidence of adverse effects when compared to synthetic drugs.

*Glehnia littoralis* Fr. Schmidt et Miquel, a perennial marine herbaceous plant belonging to the Apiaceae family, is native to the sandy coastal area of eastern Asia, mainly Japan, Korea, China, Manchuria, Sakhalin, Okhotsk, the Kuril Islands, and North America from California to Alaska [26,27]. The leaves and flower buds are edible, their rhizomes and roots are used traditionally to treat lung diseases, tuberculosis, coughs, hemoptysis, and dyspnea [28,29], and they have diaphoretic, antipyretic, and analgesic effects [30,31]. Previous studies have reported the presence of phytochemicals such as phenolic acids, flavonoids, pyranocoumarins, and polysaccharides in *G. littoralis* extracts, which have various biological activities, including antitumor, antimicrobial, antioxidant, blood circulation-promoting and immunomodulatory properties [29]. Moreover, several previous studies have shown that these phytochemicals are effective in bone formation [32]. To our knowledge, no studies have reported the use of *G. littoralis* extracts as potential natural therapeutic agents to prevent osteoporosis.

Therefore, the main objectives of the present study were to identify and select the toxicity of different *G. littoralis* plant parts for use in the differentiation analysis of an MC3T3-E1 cell culture through a cell viability assay. In addition, this study investigated the proliferative potential of MC3T3-E1 cells treated with *G. littoralis* using a 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This study also compared the degree of osteoblastic differentiation between control cells and those treated with different *G. littoralis* concentrations by quantifying ALP. Finally, we evaluated the degree of matrix mineralization formed by MC3T3-E1 cells treated with different *G. littoralis* plant parts and concentrations to confirm its applicability as a natural material for osteoblast differentiation and bone formation.
