Anti-Osteoporotic Potential of Water Extract of Anethum graveolens L. Seeds

Anethum graveolens L., known as European dill, is a versatile herb widely used in both traditional medicine and culinary practices. Despite its long-standing history, the potential impact of the water extract of A. graveolens seeds (WEAG) on bone health remains unexplored. In this study, we investigated the influence of WEAG on osteoclast differentiation and assessed its potential as an anti-osteoporotic agent. WEAG hindered osteoclast differentiation through the suppression of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoclast-supporting cells and by directly targeting osteoclast precursor cells. WEAG significantly reduced the expression of key osteoclastogenic transcription factors, namely c-Fos and NFATc1, typically induced by RANKL in osteoclast precursors. This reduction was attributed to the suppression of both MAPKs and NF-κB pathways in response to RANKL. In vivo experiments further revealed that WEAG administration effectively reduces trabecular bone loss and weight gain triggered by ovariectomy, mimicking postmenopausal osteoporosis. Furthermore, our comprehensive phytochemical analysis of WEAG identified a range of phytochemical constituents, associated with bone health and weight regulation. Notably, we discovered a specific compound, isorhamnetin-3-O-glucuronide, within WEAG that exhibits anti-osteoclastogenic potential. Overall, this research elucidated the beneficial effects and mechanistic basis of WEAG on osteoclast differentiation and bone loss, indicating its potential as a viable alternative to address bone loss in conditions like postmenopause.


Introduction
Osteoporosis, a metabolic disorder, is characterized by diminished bone mass and microarchitectural deterioration, leading to weakened bone strength and an elevated susceptibility to fractures [1]. The process of bone remodeling is crucial for replacing old and damaged bone with new bone through the cooperation of osteoclasts and osteoblasts. During normal bone remodeling, the creation of new bone is intricately linked with the removal of old bone to ensure the maintenance of bone mass and quality. However, in various pathological conditions, bone resorption outpaces bone formation, resulting in excessive skeletal deterioration. One of the most common bone disorders, postmenopausal osteoporosis, arises from an increase in the formation and activity of osteoclasts due to the decrease in estrogen levels [2,3]. Heightened osteoclast formation and function are also associated with bone erosion in conditions such as rheumatoid arthritis and osteolysis seen in the osteolytic complications of metastatic tumors [4,5]. Osteoclasts, multinucleated giant cells, are derived from precursor cells within the monocyte/macrophage lineage through a series of processes including commitment, differentiation, fusion, and activation. The receptor activator of nuclear factor-κB ligand (RANKL) is a crucial cytokine that activates all phases of osteoclast development. RANKL is primarily synthesized by mesenchymal

Cell Viability Assay
Cells were seeded into a 96-well plate at the density of 2 × 10 4 cells/well. The medium was removed after cells became adherent, and cells were treated with or without WEAG and cultured for 24 h. Cell viability assessments were carried out by incubating the cells with a Cell Counting Kit-8 reagent (CCK-8; Dojindo Molecular Technologies Inc., Rockville, MD, USA) for 2 h.

Osteoclast Differentiation
In order to induce osteoclast differentiation within an osteocyte-BMMs co-culture system, MLO-Y4 osteocyte cells (1 × 10 3 cells/well) were initially seeded into 96-well plates, followed by the addition of BMMs (4 × 10 4 cells/well) on the subsequent day. The co-cultures were treated with WEAG along with 1α,25-dihydroxyvitamin D3 (VitD3; 10 nM) or VitD3 and RANKL (50 ng/mL) for a duration of 5 days. To stimulate osteoclast differentiation in BMMs, BMMs (1 × 10 4 cells/well) were treated with WEAG or with phytochemicals identified in WEAG in the presence of RANKL (50 ng/mL) and M-CSF (60 ng/mL) in 96-well plates for 4 days. All cultures were substituted with fresh medium together with supplements on day 3.

Tartrate-Resistant Acid Phosphatase (TRAP) Activity and Staining
Cells were fixed and subsequently permeabilized with 0.1% Triton X-100. Next, the cells were incubated with TRAP solution at 37 • C for 20 min. The resulting supernatant was combined with 0.1 N NaOH in a 1:1 ratio, and absorbance was measured at 405 nm. Following TRAP activity measurement, the cells were stained to a violet color using a solution comparing fast red violet LB salt and naphthol AS-MX phosphate.

Western Blot Analysis
Cultured cells were lysed in ice-cold protein lysis buffer containing protease and phosphatase inhibitors and then cleared via centrifugation. Equivalent amounts of extracted proteins were separated using 10% SDS-PAGE and then transferred to the PVDF membranes (Millipore, MA, USA). After being blocked at room temperature in 5% skim-milk for 1 h, the membranes were incubated overnight at 4 • C with the primary antibodies. After washing with TBS-T buffer, the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The target bands were detected using SuperSignal ® West Pico Chemiluminescent Substrate.

Quantitative Real-Time Polymerase Chain Reaction (PCR)
Total RNA was extracted on the indicated days using the RNA-spin TM Total RNA Extraction Kit (iNtRON Biotechnology, Sungnam, Republic of Korea), and purified total RNA was used for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The target mRNAs were quantified using the TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA, USA) along with TaqMan probes on an ABI 7500 Real-Time PCR system (Applied Biosystems). Data analysis was conducted following the comparative cycle threshold method [15], with normalization to 18S expression in each sample.

Animal Experiment
Six-week-old female C57BL/6J mice (Central Lab. Animal Inc., Seoul, Republic of Korea) were acclimatized under controlled laboratory conditions. After a one-week acclimatization period, bilateral OVX was performed to induce an osteoporosis model. The sham control group consisted of mice whose ovaries were not removed. Previous studies have shown that the oral administration of an aqueous extract of A. graveolens seeds resulted in a dose-dependent reduction in HCl-induced gastric lesions in mice, with effective doses ranging from 50 to 450 mg/kg [16]. Building upon these findings, we opted to investigate the impact of WEAG on bone loss in OVX mice using doses of 30 mg/kg/day (referred to as WEAG-L) and 100 mg/kg/day (referred to as WEAG-H). One week after surgery, we established the following four groups (n = 6 each): sham, OVX, OVX treated with WEAG at 30 mg/kg/day (WEAG-L), and OVX treated with WEAG at 100 mg/kg/day (WEAG-H). The mice were provided with a normal-fat diet (10 kcal%; Research Diet, New Brunswick, NJ, USA) and had access to water ad libitum, while also receiving daily oral gavage administration of WEAG for a duration of 5 weeks.

Micro-Computed Tomography (µ-CT) Analysis
For the analysis of trabecular bone microstructure, we employed the micro-CT SkyScan 1276 system (Bruker, Kontich, Belgium) to perform scans on the distal femoral bone. The reconstruction of the scanned images was carried out using the NRecon software (version 1.7.42, Bruker). Three-dimensional images of the distal femoral bone were generated using CTvol software (version 2.3.2.0, Bruker). The volume of interest for trabecular bone analysis in the distal femur spanned from 50 µm below the growth plate to the proximal direction, covering a total length of 1.0 mm. Bone mineral density (BMD) and bone morphometric parameters were analyzed using CTAn software (version 1.20.3.0, Bruker).

Statistical Analysis
In vivo data are presented as the mean ± standard error of the mean (SEM), while in vitro data are represented as mean ± standard deviation (SD). The values were assessed via one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test or two-way ANOVA followed by Sidak's post hoc test.

WEAG Inhibits Osteoclastogenesis In Vitro
Osteocytes are terminally differentiated cells of the osteoblastic lineage, distributed throughout the mineralized bone matrix [19]. They regulate the activities of osteoclasts and osteoblasts, making them pivotal in bone remodeling. Recent research has unveiled osteocytes as a primary source of RANKL [6,20]. The osteocyte-like cell line, MLO-Y4, which exhibits many characteristics of osteocytes [21], expresses high levels of RANKL and can support osteoclast formation when co-cultured with osteoclast precursor cells [22]. To assess the influence of WEAG on osteoclast formation in vitro, our initial investigation focused on its effect in a co-culture system comprising MLO-Y4 osteocyte-like cells and BMMs. Consistent with previous reports [23], VitD3 stimulated osteoclast formation in the co-culture, as evidenced by the presence of TRAP-positive multinucleated cells containing more than three nuclei. The inhibitory effect of WEAG on osteoclast formation was observed in a dose-dependent manner ( Figure 1A). Notably, even when exogenous RANKL was introduced under identical co-culture conditions, WEAG continued to exert its inhibitory effect, implying that WEAG inhibits osteoclast formation independently of RANKL production ( Figure 1A). Supporting these findings, we additionally validated that WEAG exhibited a dose-dependent reduction in RANKL-induced osteoclast differentiation in isolated BMMs ( Figure 1B). Importantly, WEAG treatment did not induce cytotoxic effects on BMMs, regardless of the concentration used ( Figure 1C). Next, we explored whether WEAG has any influence on the expression of RANKL and the decoy receptor osteoprotegerin (OPG). The treatment of MLO-Y4 cells with VitD3 significantly increased RANKL mRNA levels while decreasing OPG mRNA levels. In contrast, WEAG inhibited VitD3-stimulated RANKL mRNA expression but did not influence OPG mRNA expression ( Figure 1D). These results collectively suggest that WEAG is capable of inhibiting osteoclast formation by directly targeting osteoclast precursors and simultaneously suppressing RANKL expression in osteoclast-supporting MLO-Y4 cells.
and BMMs. Consistent with previous reports [23], VitD3 stimulated osteoclast formation in the co-culture, as evidenced by the presence of TRAP-positive multinucleated cells containing more than three nuclei. The inhibitory effect of WEAG on osteoclast formation was observed in a dose-dependent manner ( Figure 1A). Notably, even when exogenous RANKL was introduced under identical co-culture conditions, WEAG continued to exert its inhibitory effect, implying that WEAG inhibits osteoclast formation independently of RANKL production ( Figure 1A). Supporting these findings, we additionally validated that WEAG exhibited a dose-dependent reduction in RANKL-induced osteoclast differentiation in isolated BMMs ( Figure 1B). Importantly, WEAG treatment did not induce cytotoxic effects on BMMs, regardless of the concentration used ( Figure 1C). Next, we explored whether WEAG has any influence on the expression of RANKL and the decoy receptor osteoprotegerin (OPG). The treatment of MLO-Y4 cells with VitD3 significantly increased RANKL mRNA levels while decreasing OPG mRNA levels. In contrast, WEAG inhibited VitD3-stimulated RANKL mRNA expression but did not influence OPG mRNA expression ( Figure 1D). These results collectively suggest that WEAG is capable of inhibiting osteoclast formation by directly targeting osteoclast precursors and simultaneously suppressing RANKL expression in osteoclast-supporting MLO-Y4 cells.

WEAG Inhibits RANKL-Mediated Signaling Pathways
To elucidate the molecular mechanisms underpinning WEAG's inhibitory effect on osteoclast precursor differentiation, we conducted a thorough examination of WEAG's impact on the expression and activation of RANKL-induced signaling pathways central to osteoclastogenesis. Upon RANKL binding to RANK, it triggers the activation of NF-κB and MAPKs via TRAF6, culminating in the upregulation of c-Fos and NFATc1. During the osteoclastogenesis process, the heightened activity of NFATc1 serves to activate and induce the expression of osteoclast-specific genes encoding vital proteins necessary for osteoclast differentiation, fusion, and function. Atp6v0d2 and DC-STAMP play crucial roles in mediating osteoclast cell-cell fusion, while CtsK is intimately involved in bone resorption [24]. Concurrently, negative regulators that impede the transcriptional activity of NFATc1, such as interferon regulatory factor-8 (IRF-8) and v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (MafB), are downregulated by the transcriptional repressor B-lymphocyte-induced maturation protein 1 (Blimp1) during the course of osteoclast differentiation [25][26][27]. The treatment of BMMs with WEAG produced significant inhibition in RANKL-induced mRNA and the protein expression of c-Fos and NFATc1 (Figure 2A,B). Subsequently, WEAG also effectively dampened the mRNA expression of osteoclast-specific genes, including Atp6v0d2, DC-STAMP, and CtsK ( Figure 2B). Furthermore, WEAG hindered the reduction in the expression of IRF-8 and MafB, while simultaneously inhibiting the increase in Blimp1 expression observed during RANKLinduced osteoclastogenesis. These results collectively suggest that WEAG impedes osteoclast differentiation by concurrently inhibiting the upregulation of positive regulators and the downregulation of negative regulators crucial for osteoclastogenesis. Prior research has emphasized the critical role of p38 in osteoclastogenesis, as it is associated with RANKL-induced c-Fos and NFATc1 expression, and it facilitates CtsK expression through its interaction with NFATc1 [28,29]. Additionally, JNK activation and its downstream target c-Jun have been implicated in RANKL-induced c-Fos and NFATc1 expression in osteoclast precursors [30,31]. Conversely, the role of ERK in regulating osteoclast differentiation appears to be context-and stage-dependent [32]. In addition to the MAPK pathways, the NF-κB signaling pathway, a critical participant in inflammatory and immune responses, plays a central role downstream of RANKL in osteoclastogenesis [33,34]. In the inactive state, NF-κB forms a complex with the NF-κB inhibitor IκBα, which impedes the translocation of NF-κB, consisting of the p65 and p50 subunits, into the nucleus. Upon stimulation by RANKL, IKK triggers the phosphorylation and subsequent degradation of IκBα, releasing NF-κB for nuclear translocation [33]. To delve deeper into WEAG's inhibitory effect on NFATc1 expression and osteoclastogenesis, we investigated whether WEAG influences the activation of RANKL-induced MAPKs and NF-κB. Significantly, WEAG treatment exhibited the inhibition of the RANKL-induced phosphorylation of JNK and p38, with no discernible impact on ERK phosphorylation. Furthermore, it suppressed RANKL-induced NF-κB activation, as evidenced by the degradation of IκBα ( Figure 2C). Our findings collectively suggest that WEAG's anti-osteoclastogenic effect can be attributed to its ability to negatively regulate the induction of NFATc1 and the repressors IRF-8 and MafB by inhibiting the RANKL-induced activation of the JNK, p38, and NF-κB signaling pathways.

WEAG Inhibits Bone Loss
Given the observed inhibitory effect of WEAG on osteoclast differentiation, we examined the potential pharmacological impact of WEAG using an OVX mouse model. This model induces estrogen deficiency-induced osteoporosis, leading to an increase in RANKL expression that elevates bone resorption and bone turnover rate. Ultimately, it leads to a reduction in BMD and significant disruption of the trabecular microstructure [35]. µ-CT technology offers the capacity to provide both quantitative and visually informative data concerning bone microarchitecture. Three-dimensional µ-CT images provide a clear visualization of the trabecular bone and enable the acquisition of quantitative parameters, encompassing BMD, bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).
After 6 weeks of ovariectomy, µ-CT analysis of the femoral bone showed a notable reduction in trabecular bone in the OVX group when compared to the sham group. However, both WEAG-L (30 mg/kg/day) and WEAG-H (100 mg/kg/day) treatments effectively mitigated this bone loss. WEAG administration at both doses exhibited increased BMD, BV/TV, Tb.N, and Tb.Th, but decreased Tb.Sp compared to the OVX group ( Figure 3A,B). During the progression of osteoporosis induced by OVX surgery, uterine weight notably decreases while body weight increases [36]. In line with this, our study identified a substantial increase in body weight in the OVX group compared to the sham group. Remarkably, the elevated body weights observed in the OVX group were effectively restored to normal by administering two different doses of WEAG. However, in contrast to body weight changes, uterine atrophy induced by ovariectomy was not reversed in the WEAGtreated groups ( Figure 3C).
Our animal study collectively demonstrates that WEAG effectively mitigates bone loss and suppresses body weight gain induced by ovariectomy, without exerting estrogenic effects, as evidenced by the absence of an impact on uterine atrophy. While further

WEAG Inhibits Bone Loss
Given the observed inhibitory effect of WEAG on osteoclast differentiation, we examined the potential pharmacological impact of WEAG using an OVX mouse model. This model induces estrogen deficiency-induced osteoporosis, leading to an increase in RANKL expression that elevates bone resorption and bone turnover rate. Ultimately, it leads to a reduction in BMD and significant disruption of the trabecular microstructure [35]. µ-CT technology offers the capacity to provide both quantitative and visually informative data concerning bone microarchitecture. Three-dimensional µ-CT images provide a clear visualization of the trabecular bone and enable the acquisition of quantitative parameters, encompassing BMD, bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).
After 6 weeks of ovariectomy, µ-CT analysis of the femoral bone showed a notable reduction in trabecular bone in the OVX group when compared to the sham group. However, both WEAG-L (30 mg/kg/day) and WEAG-H (100 mg/kg/day) treatments effectively mitigated this bone loss. WEAG administration at both doses exhibited increased BMD, BV/TV, Tb.N, and Tb.Th, but decreased Tb.Sp compared to the OVX group ( Figure 3A,B). During the progression of osteoporosis induced by OVX surgery, uterine weight notably decreases while body weight increases [36]. In line with this, our study identified a substantial increase in body weight in the OVX group compared to the sham group. Remarkably, the elevated body weights observed in the OVX group were effectively restored to normal by administering two different doses of WEAG. However, in contrast to body weight changes, uterine atrophy induced by ovariectomy was not reversed in the WEAG-treated groups ( Figure 3C). implications for the potential use of WEAG as a therapeutic candidate for addressing two major health concerns prevalent in postmenopausal women-osteoporosis and obesity.

Phytochemical Profiling of WEAG
UHPLC-MS/MS analysis stands as a leading analytical method, providing accurate insight into the active components present within plant samples. This method facilitates detailed chemical profiling even before proceeding to the isolation and purification stages [37]. To uncover the molecular foundation behind WEAG's biological activity, we delved into its phytochemical composition. Through UHPLC-MS/MS, we discerned six phenolics (quinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, feruloylquinic acid, malonyl-tri-O-caffeoylquinic acid) and five flavonoids (schaftoside, quercetin-3-Orutinoside, quercetin-O-glucuronide, kaempferol-3-O-glucuronide, iso-rhamnetin-3-Oglucuronide) present in WEAG, as detailed in Table 1. Figure 4 displays both the base peak Our animal study collectively demonstrates that WEAG effectively mitigates bone loss and suppresses body weight gain induced by ovariectomy, without exerting estrogenic effects, as evidenced by the absence of an impact on uterine atrophy. While further investigation is warranted, including an in-depth exploration of WEAG's influence on osteoclasts and osteoblasts in vivo, to fully uncover the underlying mechanisms responsible for the beneficial effects of WEAG observed in OVX mice, our findings hold significant implications for the potential use of WEAG as a therapeutic candidate for addressing two major health concerns prevalent in postmenopausal women-osteoporosis and obesity.

Phytochemical Profiling of WEAG
UHPLC-MS/MS analysis stands as a leading analytical method, providing accurate insight into the active components present within plant samples. This method facilitates detailed chemical profiling even before proceeding to the isolation and purification stages [37]. To uncover the molecular foundation behind WEAG's biological activity, we delved into its phytochemical composition. Through UHPLC-MS/MS, we discerned six phenolics  Table 1. Figure 4 displays both the base peak chromatograms and the extracted ion chromatogram of the pinpointed phytochemicals in WEAG. chromatograms and the extracted ion chromatogram of the pinpointed phytochemicals in WEAG.

The Effects of Phytochemicals in WEAG on Osteoclast Differentiation
We proceeded to examine the influence of the phytochemicals identified in WEAG on osteoclast differentiation, aiming to determine if the inhibitory effect of WEAG on osteoclas-togenesis could be ascribed to the bioactive properties of these constituent phytochemicals. Out of the eleven identified compounds, our investigation centered on nine readily available phytochemicals. Significantly, we discovered that one particular compound, isorhamnetin-3-glucuronide, potently suppressed RANKL-induced osteoclast differentiation without any associated cytotoxicity ( Figure 5A,B). While the effects of the phytochemicals in WEAG on bone metabolism remain underexplored, it is important to note that caffeic acid has displayed varied impacts on bone-both beneficial and harmful-depending on the specific animal model used [38]. Isorhamnetin-3-O-glucuronide, which showed anti-osteoclastogenic effects in our study, has previously demonstrated anti-inflammatory properties by inhibiting LPS-stimulated JNK and p38 activation in RAW264.7 macrophage cells [39]. In alignment with our observations on isorhamnetin-3-O-glucuronide, its aglycone variant, isorhamnetin, has been found to counteract RANKL-induced osteoclastogenesis in BMMs. Moreover, in a surgically induced osteoarthritis mouse model, the administration of isorhamnetin resulted in a reduction in osteoclast numbers and a decrease in bone loss within the subchondral region [40].
phytochemicals. Out of the eleven identified compounds, our investigation centered on nine readily available phytochemicals. Significantly, we discovered that one particular compound, isorhamnetin-3-glucuronide, potently suppressed RANKL-induced osteoclast differentiation without any associated cytotoxicity ( Figure 5A,B). While the effects of the phytochemicals in WEAG on bone metabolism remain underexplored, it is important to note that caffeic acid has displayed varied impacts on bone-both beneficial and harmful-depending on the specific animal model used [38]. Isorhamnetin-3-O-glucuronide, which showed anti-osteoclastogenic effects in our study, has previously demonstrated anti-inflammatory properties by inhibiting LPS-stimulated JNK and p38 activation in RAW264.7 macrophage cells [39]. In alignment with our observations on isorhamnetin-3-O-glucuronide, its aglycone variant, isorhamnetin, has been found to counteract RANKLinduced osteoclastogenesis in BMMs. Moreover, in a surgically induced osteoarthritis mouse model, the administration of isorhamnetin resulted in a reduction in osteoclast numbers and a decrease in bone loss within the subchondral region [40].
The unearthing of isorhamnetin-3-O-glucuronide's anti-osteoclastogenic ability, combined with the detection of phytochemicals with anti-obesity properties within WEAG, offers profound insights into the underlying mechanisms of WEAG's dual action against osteoporosis and obesity. This suggests the potential of WEAG as a comprehensive alternative treatment, addressing both bone health and weight concerns, which are critical health challenges often faced by postmenopausal women.  Regarding WEAG's potential anti-obesity activity, several of its phytochemicals, including quinic acid [41], 5-O-caffeoylquinic acid [42], caffeic acid [43], and kaempferol-3-Oglucuronide [44], have exhibited anti-obesity effects in various animal studies.
The unearthing of isorhamnetin-3-O-glucuronide's anti-osteoclastogenic ability, combined with the detection of phytochemicals with anti-obesity properties within WEAG, offers profound insights into the underlying mechanisms of WEAG's dual action against osteoporosis and obesity. This suggests the potential of WEAG as a comprehensive alternative treatment, addressing both bone health and weight concerns, which are critical health challenges often faced by postmenopausal women.

Conclusions
This research illuminates the previously uncharted potential of WEAG for bone protection. We have shown that WEAG curbs osteoclast differentiation by dampening RANKL expression in osteoclast-supportive cells and directly intervening in RANKL-induced osteoclastogenesis. Additionally, administering WEAG to OVX mice markedly reduced both bone loss and weight gain. Through our phytochemical profiling of WEAG, we pinpointed several constituents recognized for their influence on bone health and obesity management. Notably, we discovered a particular compound, isorhamnetin-3-O-glucuronide, that exhibits an anti-osteoclastogenic effect. Collectively, these revelations indicate that WEAG could be a promising pharmacological solution to counter bone loss stemming from heightened osteoclast formation. This potential is especially pertinent when considering conditions like postmenopausal osteoporosis, which often coexists with weight management challenges.