*3.8. Osteocalcin Content*

We investigated the effects of *G. littoralis* extracts on the degree of osteocalcin production during the late stage of osteoblast differentiation. As Figure 9 shows, *G. littoralis* extract significantly increased osteocalcin production in MC3T3-E1 cells. This is the first report describing the inhibitory osteoporotic properties of *G. littoralis* using the MC3T3- E1 in vitro system. Osteocalcin content levels varied with different concentrations of the tested plant parts. Except in the case of GLAE, an increase in the sample concentration

resulted in reduced osteocalcin content. Comparatively, 0.5 μg/mL GLSE resulted in a higher osteocalcin content, indicating that the phytochemical responsible for producing the protein in osteoblast cells is more present in this extract. However, increasing the GLFE concentration resulted in a decrease in osteocalcin content in a concentration-dependent manner. Comparatively, 20 μg/mL GLFE displayed lower osteocalcin production than the negative control, indicating that a higher GLFE concentration is cytotoxic and inhibits osteoblast formation.

**Figure 8.** Mineralization contents of extracts from each part of *G. littoralis* in osteoblastic MC3T3-E1 cell line. Each value is the mean ± standard deviation of nine replicate tests. Mean values followed by the same letter are not significantly different based on the DMRT (*p* < 0.05). P.C.: Positive control (ascorbic acid (50 μg/mL), β-glycerophosphate (100 mM). GLSE: *G. littoralis* leaf, stem extracts, GLFE: *G. littoralis* fruit extracts, GLAE: *G. littoralis* all extracts, GLRE: *G. littoralis* root extracts.

**Figure 9.** Osteocalcin contents of extracts from each part of *G. littoralis* in osteoblastic MC3T3-E1 cell line. Each value is mean ± standard deviation of nine replicate tests. The mean values followed by the same letter are not significantly different based on the DMRT (*p* < 0.05). P.C.: Positive control (ascorbic acid (50 μg/mL), β-glycerophosphate (100 mM). GLSE: *G. littoralis* leaf, stem extracts, GLFE: *G. littoralis* fruit extracts, GLAE: *G. littoralis* all extracts, GLRE: *G. littoralis* root extracts.

#### *3.9. mRNA Expression Rate*

We confirmed how *G. littoralis* extracts affect osteoblast differentiation by analyzing the expression patterns of prominent osteoblast marker genes Table 3. After seven days of treatment with different *G. littoralis* extracts, a real-time polymerase chain reaction (PCR) was performed to investigate the effect on osterix and RUNX2 mRNA expression. As the results show, the expression patterns of both osteoblastic genes changed when treated with the *G. littoralis* extracts. The findings showed that, except in the case of GLFE extracts, *G. littoralis* extract treatment significantly elevated osterix gene expression, and RUNX2 gene expression was increased by all *G. littoralis* extracts. Comparatively, RUNX2 expression significantly increased after treatment with lower *G. littoralis* extract concentrations (ranging from 0.5 to 2 μg/mL).


**Table 3.** Effect of *G. littoralis* extracts on Osterix and RUNX2 mRNA expression in osteoblastic MC3T3-E1 cells.

<sup>1</sup> Each value is mean ± standard deviation of nine replicate tests. Mean values within a column followed by the same letter are not significantly different based on the DMRT (*p* < 0.05). P.C.: Positive control (ascorbic acid (50 μg/mL), β-glycerophosphate (100 mM), AA: Ascorbic acid, BGP: β-glycerophosphate. GLSE: *G. littoralis* leaf, stem extracts, GLFE: *G. littoralis* fruit extracts, GLAE: *G. littoralis* all extracts, GLRE: *G. littoralis* root extracts.

#### **4. Discussion**

Plants contain various antioxidants; these antioxidants play an important role in protecting plants from oxidative stress and signals, when ingested, and they can act as natural antioxidants to help prevent disease [45]. Among these compounds, the phenolic compounds present in the plants are mostly responsible for the antioxidant properties [46]. In the present study, the DPPH and ABTS radical scavenging assays presented wide variations in antioxidant activity values for different plant parts. We studied the relationship between the different parameters by obtaining Pearson's correlation coefficients. The antioxidant potentials estimated by both assays were somewhat different and showed a high correlation between them (*p* < 0.05, r = 0.894). The variation in antioxidant potential could be due to the various antioxidant compound responses to the different radicals present in each assay. Excessive reactive oxygen species (ROS) production can cause lipid and protein oxidation, damage DNA integrity, and simultaneously cause tissue damage [47]. Several previous studies have shown the involvement of ROS in bone remodeling by enhancing osteoclastic bone resorption and decreasing osteoblast cell formation [48,49]. In the present study, all *G. littoralis* extracts effectively scavenged the DPPH and ABTS radicals, indicating that the *G. littoralis* extracts had potential antioxidant activity and protected the MC3T3-E1 cells from degeneration and death. Moreover, Pearson's correlation analysis revealed a significant correlation between DPPH and TPC and TFC in *G. littoralis* extracts. A simi-

lar correlation was also observed between ABTS and TFC. Phenolic compounds, such as coumarins and their derivatives, have been reported as dominant *G. littoralis* phytochemical components [48,49]. In *G. littoralis* extracts, phenolic compounds, including caffeic acid, vanillic acid, ferulic acid, chlorogenic acid, rutin, quercetin, kaempferol, and coumarins and their derivatives, have been identified [50–52]. In the present study, coumarin-based flavonoids such as scopoletin, umbelliferone, imperatorin, phellopterin have been detected in *G. littoralis* extracts. These compounds are mostly responsible for the antioxidant properties. For instance, scopoletin has been involved in considerable antioxidant activities by scavenging ROS, especially hydrogen peroxide (H2O2) scavenging activity, ferrous ion (Fe2+) chelating activity, and activity against superoxide anion radicals (O2 •−), and OHradicals [53]. Moreover, Um et al. [53] isolated scopoletin and umbelliferone from *Glehnia littoralis* and demonstrated a reactive oxygen species (ROS) scavenging ability of about 60% or more compared to a control. Imperatorin (IMP) has been reported in several plants with antioxidant properties [54–57]. Methanolic extracts containing umbelliferone have been shown to exhibit an efficient pro-oxidant activity [58] and inhibit lipid peroxidation [59]. Others observed that the treatment of umbelliferone has been shown to inhibit the intracellular ROS production in irradiated lymphocytes and effectively restore the mitochondrial membrane and inhibited gamma radiation-induced DNA damage [60].

Numerous studies observed that oxidative stress enhances the differentiation and function of osteoclasts [61]. ROS-induced oxidative stress has been shown to involve the suppression of bone formation and the stimulation of osteoclast resorption [62]. Present results indicate that phenolic compounds such as scopoletin, umbelliferone, imperatorin, and phellopterin present in the *G. littoralis* extracts inhibited ROS formation to suppress the excessive bone breakdown by osteoclasts. Moreover, several studies have reported the antiosteoporotic properties of coumarins by suppressing the interaction of advanced glycation end-products (AGE) and their receptors [63]. Treatment using scopoletin prevented bone loss in diabetic mice by increasing bone turnover of bone-degrading osteoclasts and boneforming osteoblasts. It has been shown that treatment with imperatorin in rats promoted osteogenesis and suppressed the osteoclast differentiation [64]. The authors found that the imperatorin activates AKT that leads to the inactivation of GSK3β that causes the activation of β-catenin and accumulation of β-catenin in the nucleus [65–68]. It was believed that the activation of β-catenin plays an important role in the suppression of osteoblast differentiation [69]. Thus, it can be inferred that imperatorin could induce osteogenesis via the AKT/GSK3β/β-catenin pathway [64], indicating that imperatorin present in the GL extracts could be responsible for bone growth and inhibition of resorption. Furthermore, umbelliferone prevented trabecular bone matrix degradation and osteoclast formation in bone tissue [70]. The authors reported that umbelliferone is closely associated with the dysfunction of osteoclasts attributed to defects in osteoclast survival and differentiation [65]. In addition, Li et al. [71] reported that phellopterin inhibits Ca2+ influx induced by the stimulation of voltage-gated and receptor-dependent calcium channels [72,73]. Therefore, in the present study, these compounds, together with the other polyphenols present in the *G. littoralis* extracts, strongly favored MC3T3-E1 cell differentiation. Furthermore, it can be suggested that *G. littoralis* effectively contributes to the prevention of oxidative damage to bone tissues via antioxidant action and its phytochemicals.

ALP, a typical protein product, is associated with osteoblast growth and differentiation and is expressed and increased during the active matrix maturation phase immediately after the cell proliferation period [18,20,74]. Although the exact ALP mechanism of action is poorly understood, it is believed that these enzymes are responsible for bone mineralization [75]. Therefore, it is important to examine the effect of *G. littoralis* extracts on MS3T3-E1 cell ALP activity. In the present study, all *G. littoralis* extracts effectively accelerated ALP activity in a dose-dependent manner. Moreover, some *G. littoralis* extracts showed higher ALP activity than the positive control, indicating that the different phytochemicals present in the extracts may be necessary for osteoblast differentiation. We hypothesize that

*G. littoralis* extracts are associated with osteoblast proliferation and differentiation from a newly synthesized protein component.

In this study, maximum ALP activity was observed at the lowest *G. littoralis* extract concentration (0.5 μg/mL), which was confirmed in GLSE and GLAE extracts. Similar to our findings, the aqueous extracts of rooibos promoted ALP activity and mineralization [76]. Moreover, there is abundant evidence that dietary phytochemicals have osteoprotective effects. Caffeic acid regulates bone remodeling by inhibiting osteoclastogenesis, bone resorption, and osteoblast apoptosis [77]. Chlorogenic acid extracted from *Cortex Eucommiae* inhibited a decrease in bone mineral density [78]. Quercetin inhibits osteoblast apoptosis, osteoclastogenesis, and oxidative stress [79]. Jang et al. [80] reported similar results in *A. rugosa*, stating that some of the phenolic compounds present in *A. rugosa* effectively suppressed osteoclasts [81,82]. Flavonoids, such as orientin, quercetin, and luteolin, have shown blastogenic effects by increasing ALP activity and mineralization in rooibos [83,84]. Treating osteoblast cells with various phenolic compounds increases ALP synthesis and decreases the expression of antigens involved in osteoblast immune functions, which may improve bone mineral density [85]. Flavonoids, such as icariin and naringin, were found to regenerate bone tissues by increasing ALP activity and osteopontin content [86–89]. Another study observed increases in osteoblast proliferation, and several other reports have provided convincing data about phytochemicals and their association with osteoclast formation in vitro [90]. Although this study did not determine the exact composition of phenolic compounds in *G. littoralis*, the phenolic compounds from *G. littoralis* could be crucial in modulating the bone formation process through the osteoblast formation process and ALP production.

It has been reported that osteoblasts produce biochemical markers, such as type I collagen, ALP, and osteopontin, which are important components for matrix maturation and mineralization [74]. In the present study, phenotypic markers, such as collagen and osteocalcin, mainly associated with the later stage of osteoblast differentiation and were elevated in the MS3T3-E1 cells treated with *G. littoralis* extract. This indicated that *G. littoralis* extracts were vital in osteoblast differentiation. GLSE showed greater collagen synthesis and upregulated osteoblastic MC3T3-E1 cell proliferation and differentiation by enhancing ALP activity and mineralization compared to the other extract types. There is increasing interest in both in vitro and in vivo research that phenolic compounds may favorably improve osteoporosis. Sparse experimental data show that phenolic acids may have in vitro estrogenic activity. Bioactive compounds, such as β-estradiol, reportedly significantly increase osteoblastic cell proliferation, DNA and protein content, and ALP activity [91]. Phenolic acids may act on osteoblasts by binding to their estrogen receptors, found in osteoblastic cells [92].

Bone mineralization refers to the deposition of calcium and minerals in cells. It acts as a reservoir for calcium and phosphorus in the bone, maintains bone elasticity and flexibility, and provides compactness and mechanical resistance to the bone [93]. During the postmenopausal period, estrogen deficiency causes a decrease in the absorption of micronutrients in the body [94]. In the present study, different GL extracts showed various degrees of elevation in calcium levels, possibly due to affecting calcium absorption and contributing to matrix deposition during osteogenesis [95]. In this test, the maximum mineralization content was observed at the lowest concentration (0.5 μg/mL) of GLSE, GLFE, and GLRE extracts. Our results are consistent with those reported by Yun et al. [96], who observed an increase in calcium deposition in osteoblast MC3T3-E1 cells treated with lower concentrations of chrysanthemum extract. Prak et al. [97] reported similar results in 10 μg/mL of seaweed extracts. Osteocalcin is a non-collagenous protein in the bone secreted into osteoblasts and used as a biochemical marker for bone formation [98]. Osteocalcin is associated with changes in bone turnover rate in bone metabolism and is reflected in the rate of bone formation. The osteocalcin carboxyl group is removed and released into the circulation due to pH acidification of the bone when osteoclasts resorbed it [99].

Several transcription factors are involved in osteoblast differentiation. Most importantly, RUNX2 and osterix are genes that differentiate mesenchymal stem cells into immature osteoblasts and are osteoblast-specific transcription factors required for osteoblast differentiation and bone formation [100]. RUNX2, the earliest identifiable marker, is known as "a master gene" for osteoblast differentiation and is associated with ALP and osterix upregulation [101,102]. It has been argued that RUNX2 triggers osteocalcin expression by binding to the cis-acting elements of the osteocalcin promoter region of osteogenic genes to initiate the expression of ALP, osteopontin, bone sialoprotein (BSP), and osteocalcin [103,104]. Osterix is an osteoblast-specific transcription factor containing a zinc finger. It maintains strong expression in mesenchymal cells and the periosteum and is expressed in cells, such as chondrocytes and the bone matrix [105].

In the present study, we determined the gene expression patterns of osteoblast differentiation markers to understand how *G. littoralis* extracts induce mineralization. The results showed that *G. littoralis* extract treatments significantly elevated osterix and RUNX2 gene expression and enhanced the production of proteins involved in osteoblast production, such as type 1 collagen and osteocalcin. RUNX2 gene upregulation by the cells and their ALP activity and mineralization have also been reported in *Davallia formosana* extracts [106]. Previous studies have shown that phenolic compounds of different plant species extracts can induce the proliferative capacity and maturation of osteoblastic cells by improving ALP activity and increasing calcium ion deposition in the ECM [107,108]. It has been reported that changes in osteoblastic cell activity by phenolic compounds occur through the modulation of different transcription factors, such as osterix, osteocalcin, and bone morphogenic proteins (BMPs), by activating the genes involved [108]. The phenolic compounds of various plant species induce osteoblast cell differentiation through the expression of osterix and RUNX2 markers, which are associated with bone maturation [109–113]. In another study, daidzein, present in soy, acted as a phytoestrogen via osteoblast proliferation and differentiation by activating the BMP/Smad signaling pathway [114]. In the present study, all the GL extracts showed higher expression levels of mRNA expression rate of RUNX2 than control. Furthermore, it has been shown that imperatorin promotes the maturation and differentiation of osteoblast by increasing the expression of RUNX2; thus, it is closely associated with early stage osteogenic differentiation [115–117]. In the present study, all *G. littoralis* extracts increased RUNX2 gene expression. The results described here indicate that *G. littoralis* ethanolic extracts can effectively prevent osteoporosis. These results indicate that the phenolic compounds in GL extracts may synergistically induce osteoblastic cell proliferation to a greater extent than a single compound. Moreover, the results further suggest that phytochemicals other than phenolic compounds may be present in the *G. littoralis* extracts, causing the osteoblastic proliferation of MC3T3-E1 cells. Because the extracts of GLAE with stronger antioxidant activity show stronger anticancer activities, it is implied that the contents of flavonoids in GL are responsible not only for its antioxidant activities but also effectively prevent osteoporosis.
