Effect of echinalkamide identified from Echinacea purpurea (L.) Moench on the inhibition of osteoclastogenesis and bone resorption

Plant cell cultures have been exploited to provide stable production and new secondary metabolites for better pharmacological activity. Fractionation of adventitious root cultures of Echinacea purpurea resulted in the isolation of eleven constituents, including three new compounds. The structures of the three new compounds were determined to be an alkylamide (1), a polyacetylene (2) and a lignan (3) on the basis of combined spectroscopic analysis. To discover new types of antiresorptive agents, we screened for new compounds that regulate osteoclast differentiation, and survival. Among three new compounds, echinalkamide (compound 1) had considerably inhibitory effects on RANKL-induced osteoclast differentiation, and on proliferation of osteoclasts and efficiently attenuated osteoclastic bone resorption without toxicity. In addition, echinalamide treatment inhibited the osteoclast—specific gene expression level. Echinalkamide achieved this inhibitory effect by disturbing phosphorylation of MAPK and activation of osteoclast transcription factors c-Fos and NFATc1. Conclusionally, our study investigated that echinalkamide remarkably inhibited osteoclast differentiation and osteoclast specific gene expression through repression of the MAPK–c-Fos–NFATC1 cascade.

Echinacea purpurea (L.) Moench is a perennial herb of the Astraceae family. This plant is mainly cultivated in North America and Europe and widely used as an herbal medicine and dietary supplement worldwide 1 . It is used in most popular herbal medicines for the treatment of common cold and respiratory disorders. Several Echinacea species have been shown to have various biological activities including immunomodulatory, anti-inflammatory, antiviral and antibacterial properties [2][3][4] . Due to its beneficial effects, investigations for the sufficient production of E. purpurea have been established in many fields 5,6 . Recently, in vitro tissue culture techniques with plant cells have been exploited to provide stable production. Moreover, culture conditions for improved biomass and bioactive metabolites accumulation are developed by regulating nutrients, elicitors and culture environments 7,8 . Plant cell cultures have recently been used to find new secondary metabolites for better pharmacological activity 9,10 . In vitro cultures including adventitious and hairy root cultures have been successively developed for the production of E. purpurea with improved biomass and metabolite accumulation [11][12][13] . Therefore, our present study was conducted to find new secondary metabolites from adventitious cultures of E. purpurea.
Previous studies have reported that E. purpurea reduces monocyte and macrophage responses to the major antigenic components of endotoxin, lipopolysaccharide, by inhibiting tumor necrosis factor (TNF)-α and prostaglandin E2 production. The immune-regulatory effect of E. purpurea is mediated through modulation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways 14,15 .
Bone marrow-derived macrophages (BMM) isolation. The protocol for mouse use in this experiment was approved by the Institutional Animal Care and Use Committee at Wonkwang University (Approval number WK18-112). All methods were performed in accordance with relevant guidelines and regulations. Bone marrow cells (BMCs) were isolated from the long bones of 6-week-old C57BL6 mice by flushing with α-MEM containing antibiotics and red blood cells (RBCs) were removed using RBC lysis buffer.
Cell viability. RAW264 Anti-inflammation activity. Inhibitory effect of compounds on LPS-induced NO production and TNF-α secretion was assessed using RAW264.7cell 17 . RAW 264.7 cells were treated with 10 μg/ml lipopolysaccharide (LPS) in the presence or absence of compound 1-3. After 24 h incubation, the amount of Nitric oxide (NO) and Tumor necrosis factor-alpha (TNF-α) production was measured according to previous report 18 . Osteoclast differentiation and TRAP staining. BMCs were cultured for 4 days in the presence of M-CSF (30 ng/mL) to differentiate into BMM. To investigate the effect of differentiation of echinalkamide on osteoclast differentiation, BMMs with the various concentration of echinalkamide in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) in 96-well plates were processed. After 4 days, the cells were fixed with formalin, stained with TRAP, and then multinuclei (more than 3) were counted as osteoclasts.
Real-time PCR. Total RNA was extracted from harvested cells using Easy blue (Intron, Seongnam, Korea).
The amount of RNA was quantified with nanodrop (Thermofier, Massachusetts, USA). Each reaction mix (Applied Biosystems, California, USA) contained between 50 and 100 ng of RNA in a total reaction volume of 25 μL. Probes for the quantitative amplification of Complete information for FOS (c-FOS, Mm00487425, Applied Biosystems, California, USA), Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, Mm00479445, Applied Biosystems, California, USA), cathepsin K (Mm01255862, Applied Biosystems, California, USA), Matrix metallopeptidase 9 (MMP9, Mm00600164, Applied Biosystems, California, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm03302249, Applied Biosystems, California, USA) were validated using TaqMan Gene Expression Assay (Applied Biosystems, California, USA). Conditions for real-time quantitative RT-PCR were as follows: 30 min at 48 °C, 10 min at 95 °C (RT inactivation and initial activation), and then 40 cycles of amplification for 15 s at 95 °C (denaturation) and 1 min at 60 °C (annealing and extension). Data analysis was performed using SDS 2.1.1 software. To normalize expression to that of the GAPDH housekeeping gene, a mathematical model of partner expression ratio including PCR efficiency was applied to sample quantification.
Western blot analysis. Treated osteoclasts were harvest and lysed by direct addition of a lysis buffer (containing protease inhibitor and phosphatase inhibitor cocktails, Intron, Seongnam, Korea). The nuclear/cytosol fractionation kit (Bio Vision Technology Inc., New Minas, NS, Canada) was used to separate nuclear and cytoplasmic proteins according to the manufacturer's protocol. After the proteins were isolated, the concentration of the samples was determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific Inc., Massachusetts, USA). Sample (20 μg) per lane were electrophoresed on a 12% reducing SDS-PAGE gel and transferred onto a nitrocellulose membrane (Biorad, California, USA). The membrane was blocked with 5% skim milk and sequentially incubated with anti-c-FOS, anti-NFATc1, anti-MMP-9, anti-cathepsin K, and anti-actin antibodies at 4 °C overnight (all antibodies were used at a 1:1,000 dilution and were purchased from Cell Signaling Technology. Specific protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies (1:1,000 dilution, Enzo Life Sciences, Lausen, Switzerland) followed by Enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Protein images were captured using the FluorChem E image system (ProteinSimple, Santa Clara, CA, USA). Quantify images of western blot bands was measured using Image J software (NIH, Maryland, USA). Statistical analysis. Data are expressed as mean ± SD values. All the data were confirmed by technical replicated (n = 3). Significant differences were compared using repeated measures ANOVA followed by the Newman-Keuls multiple range test. Statistical significance was defined as P < 0.05. All statistical analyses were performed using GraphPad Software. Inc. (San Diego, CA).

Effect of newly isolated compounds on anti-inflammatory activity.
We investigated the antiinflammatory effects of newly isolated compounds by measuring the production of NO and TNF-α in LPSstimulated RAW 264.7 macrophages. The cytotoxic effects of each compound were also tested to ensure that the inhibitory effect on NO production was whether it was due to cell death. Compounds 1 and 2 dose-dependently reduced NO and TNF-α production stimulated by LPS without any significant cytotoxic effects at the concentration ranging from 1 to 100 μM. However, compound 1 reduced NO production and displayed cytotoxic effects at 100 μM. Compounds 1-3 conclusively showed anti-inflammatory activity probably by interfering with NO and TNF-α production (Fig. 4).
Effect of echinalkamide on cell viability. BMMs were treated of echinalkamide for 4 days, and viability was evaluated using the MTT assay. Compared to the control group, echinalkamide had any cytotoxic effects on the cells at concentrations less than 30 µM. To exclude the cytotoxic effects, in the following study, echinalkamide concentrations below 30 µM were used for further analysis. (Fig. 5).

Effect of echinalkamide on osteoclast differentiation in RANKL-stimulated BMMs. To exam-
ine the effect of echinalkamide on RANKL-induced osteoclast differentiation, BMMs were treated with various concentrations (1-30 µM) of echinalkamide in the presence of M-CSF or/and RANKL. BMMs without treated to echinalkamide differentiated into TRAP-positive multinucleated cells. Whereas, treated to echinalkamide observed a dose-dependently inhibits in the number of TRAP-positive multinucleated cells (MNCs) (Fig. 6C). The number of TRAP-positive MNCs was significantly decreased when echinalkamide was treated with BMM cells at a concentration of 1-30 μM. The quantification of TRAP-positive MNCs during echinalkamide treatment was from 411 ± 10.1 (0 μM) to 54 ± 9.8 (30 μM) per well. (Fig. 6B).

Effect of echinalkamide on regulators of osteoclastogenesis.
We investigated RT-PCR and western blot to measure whether echinalkamide is related to modulator of osteoclastogenesis. Osteoclast precursors were pretreated with echinalkamide and then stimulated with RANKL for different time intervals (6, 12, 24, and 48 h). We found that c-Fos and NFATc1 mRNA and protein levels increased upon treated to RANKL. However, c-Fos and NFATc1 expression was significantly supressed by echinalkamide (Fig. 7A). These results indicated that the inhibitory effects of echinalkamide includes the inhibition of transcription factors such as c-Fos and NFATc1.
Effect of echinalkamide on bone resorption. BMMs were seeded onto osteoclastic resorption assay plates in induction medium and treated to 1, 10, and 30 μM echinalkamide. As a result, bone resorption was www.nature.com/scientificreports/ activated by RANKL stimulation. Whereas that the percentage of resorption area decreased after treated to 1 μM echinalkamide, and the resorption was thoroughly suppressed at 30 μM (Fig. 7A, B). Ultimately, the results suggested that treatment with echinalkamide reduced bone resorption in vitro. The RT-PCR and western blot data proved that echinalkamide supressed mRNA and the protein expression of some transcription factors (MMP9 and cathepsin K) associated with cell fusion and osteoclastic bone resorption (Fig. 7C-E).

Effect of echinalkamide on RANKL-stimulated MAPK and NFkB signaling. RANKL-stimulated
signaling pathways were investigated to demonstrate the underlying molecular mechanisms of echinalkamide inhibitory effects on osteoclast differentiation. JNK, p38, and ERK are members of the MAPKinase and can be activated by RANKL. In the control group, the phosphorylation of ERK, JNK, and p38 peaked within 15 min after RANKL stimulation. However, JNK and ERK phosphorylation was remarkably inhibited after treat with echinalkamide. The quantitative analysis confirmed these observations (Fig. 8).

Discussion
Bone loss occurs when cycle of removal of old bone faster than the deposition of new bone 27,28 . The loss of bone occurs with age, poor diet, excessive vitamin A levels, low levels of sex hormones, bed rest or inactivity, smoking, and excessive consumption of alcohol and caffeine [29][30][31] . Bone loss can contribute to decrease of bone density, bone weakness, and ultimately osteoporosis. Osteoporosis is the result of bone loss and accumulative bone structure damage that can break the bone with minimal trauma. These bone loss or osteoporosis can be prevented through appropriate nutrition, physical activity and, if necessary, appropriate treatment 32,33 . www.nature.com/scientificreports/ The existing approved drugs for osteoporosis include bisphosphonates, ecombinant human parathyroid hormone (PTH), hormone replacement therapy (HRT), selective estrogen receptor modulators (SERMS), and denosumab 29,31 . However, these osteoporosis treatments can cause serious side effects such as hypocalcemia, stroke, heart attack, thrombosis and osteonecrosis, and alternative therapies are needed to prevent this. Natural compounds derived from medicinal plants and foods are interested in developing effective and safe treatments for bone diseases [34][35][36]  Many osteoclast suppressors are associated with anti-inflammatory and antioxidant 52,53 . According to Aarland et al., E. purpurea has already been studied for its effect on anti-inflammatory and anti-oxidant activity 54 . Therefore it was hypothesized that it may also be effective on inflammation-related osteoclastogenesis. Fractionation of adventitious roots of E. purpurea using various chromatographic techniques yielded three new compounds together with eight known compounds. We first performed an antioxidant and anti-inflammatory activity, in order to select a new compound effective in inhibiting osteoclasts. Echinalkamide (compound 1, undeca-2Z-4E-diene-8,10-diynoic acid isobutylamide), which has the highest antioxidant and anti-inflammatory effects among the new compounds was selected. In the present study, we investigated the potential of echinalkamide as an osteoporosis treatment and its underlying mechanism.
Most disorders of bone metabolism induce activation of osteoclasts 28,55 . As a result, bone resorption goes beyond bone formation, leading to pathological bone resorption activity and causing osteopenia, which increases the risk of fracture. Osteoclasts are important target cells for osteoporosis treatment 29,56 . www.nature.com/scientificreports/ Osteoclasts play an important role in the pathological destruction of bone. The differentiation of osteoclasts differentiates into osteoclasts that resorpsion bone through several stages. When RANKL is bound to RANK expressed in monocytes or macrophages, it differentiates into tartrate-resistant acid phosphatase (TRAP) positive cells. The binding of RANKL to RANK on the surface of osteoclasts induces intracellular signal transduction pathways such as NF-κB, MAPK and calcium rash to increase expression of NFATc1, an essential transcription factor involved in the formation of osteoclasts. 55,57,58 .
In the present study, echinalkamide has been demonstrated to be an effective inhibitor of osteoclastogenesis in vitro in terms of number and area reduction of TRAP-positive multinucleated cells. It has been reported that embryonic stem cells deficient in NFATc1 do not differentiate into osteoclasts in response to RANKL stimulation 55,58,59 . Our study has demonstrated that echinalkamide supresses RANKL-induced NFATc1 activation. We further investigated its effect on RANKL activation of MAPKs.
The binding of RANKL and RANK results in the congestion of the cell RANK domain with TNT receptorassociated factor 6 (TRAF6). This activates transcription factors such as nuclear factor kappa B (NF-κB), activator protein-1 (AP1) and NFATc1, which are the subsequent genes of TRAF6, resulting in activation of p38, JNK and extracellular-signal regulated kinases (ERK). Activation is carried out at the protein level through phosphorylation processes including mitogen-activated protein kinases (MAPKs) and Phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway 60,61 . Many studies have revealed that MAPKs can be stimulated by RANKL stimulation and are associated with osteoclastogenesis [62][63][64][65] . RANKL stimulated ERK, JNK, and p38 through activation of MEK1/2, MKK7, and MKK6 to induce activation of their downsignal targets such as c-Fos, AP-1 transcription  Supplementary  Fig. S01. (B, C) Total RNA was then isolated using easy blue kit, and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. All the data were confirmed by technical replicated (n = 3). The results are presented as the mean ± SD. Values with different letters (a, b, c, d, e) are significantly different one from another (one-way ANOVA followed by Newman-Keuls multiple range test, p < 0.05).    67,68 . Dominant-negative JNK prevents RANKL-induced osteoclastogenesis. JNK plays an important role in osteoclast generation, according to studies using knockout models 69 . We observed that echinalkamide inhibits the MAPK signaling pathway by suppressing phosphorylation of p-38, JNK and ERK. Several of evidence surggest that both NF-κB and c-Fos serve as downstream targets of TRAF6 and play important roles in NFATc1 activation. NF-κB is important for RANKL-mediated induction of NFATc1 in the early stages of osteoclastogenesis. After RANKL stimulation for 24 h, AP-1 containing c-FOS is recruited to the NFATc1 promoter and contributes to the automatic amplification of NFATc1. NFATc1 increases expression of osteoclast-specific genes TRAP, DC-STAMP, and cathepsin K 55,70 . Our results showed that echinalkamide inhibited the RANKL-induced expression of NFATc1 and c-FOS. NFATc1 is a master transcription factor be demanded for osteoclastogenesis and controlled of marker genes, including TRAP, cathepsin K, DC-STAMP, and MMP-9, which are indispensable for osteoclastic fusion and resorption 55,71 . The presence of echinalkamide attenuated the MAPKinase pathway, protein induction and transcriptional activity of NFATc1. The marker gene, cathepsin K and MMP9, was also significantly reduced. These results suggest that the effect of echinalkamide on osteoclast formation may be partly due to mitigation of the MAPK signaling pathway and subsequent mitigation of NFATc1 induction.  Supplementary Fig. S03. All the data were confirmed by technical replicated (n = 3). The results are presented as the mean ± SD. Values with different letters (a, b, c, d, e) are significantly different one from another (one-way ANOVA followed by Newman-Keuls multiple range test, p < 0.05).
Taken together, our present study demonstrated that adventitious root cultures of E. purpurea can be used not only for securing materials but also for the discovery of new compounds. In addition, the newly isolated compounds might contribute to the anti-inflammatory effect of E. purpurea. Among them, studies have shown that echinalkamide inhibits osteoclast formation and osteoclast function in vitro through inhibition of MAPK signaling pathway and NFATc1. Echinalkamide can be used as a treatment for osteoclast-related diseases as well as anti-inflammatory agents (Fig. 9).