It inhibits key signaling pathways downstream of RANKL, including NF-κB, ERK, and calcium-NFATc1 signaling, which are critical for osteoclast differentiation and function. [1]

Cyanidin Chloride (IdB 1027) decreases the degradation of IκB-α, attenuates the phosphorylation of extracellular signal-regulated kinases (ERK), and inhibits receptor activator of NF-κB ligand (RANKL)-induced NF-κB activation. The expression of c-Fos, the activation of nuclear factor of activated T cells calcineurin-dependent 1 (NFATc1), and RANKL-induced calcium oscillations are all abolished by cyanidin chloride (IdB 1027)[1].

---

Cyanidin Chloride inhibited RANKL-induced osteoclast formation in a dose-dependent manner in bone marrow macrophage cultures, with an estimated half-maximal inhibitory concentration (IC50) of approximately 5 μM. At concentrations of 0.5, 1, 2.5, 5, 10, and 20 μM, a progressive reduction in TRAP-positive multinucleated cells was observed. [1]
Cyanidin Chloride (5 μM and 10 μM) significantly reduced the hydroxyapatite resorption area in osteoclast cultures, indicating impaired osteoclastic resorptive function. At 5 μM, osteoclast numbers were not significantly reduced, suggesting a direct effect on resorptive activity independent of cell number. [1]
Cyanidin Chloride (5 μM) significantly suppressed RANKL-induced mRNA expression of osteoclast marker genes, including calcitonin receptor (ctr), cathepsin K (ctsk), and tartrate-resistant acid phosphatase (trap), as determined by quantitative real-time PCR. [1]
Using a luciferase reporter gene assay, Cyanidin Chloride (2.5–10 μM) inhibited RANKL-induced NF-κB activation in a concentration-dependent manner. Similarly, it inhibited RANKL-induced NFAT activation (2.5–10 μM). [1]
Western blot analysis showed that Cyanidin Chloride (10 μM) suppressed RANKL-induced degradation of IκB-α, a key step in NF-κB activation. It also attenuated the RANKL-induced phosphorylation of ERK1/2, and reduced the protein expression levels of c-Fos and NFATc1 during osteoclast differentiation. [1]
Cyanidin Chloride (5 μM and 10 μM) significantly reduced the amplitude of RANKL-induced calcium oscillations in bone marrow macrophages, as measured by Fluo-4 AM staining and time-lapse fluorescence microscopy. [1]

The OVX-induced osteoporosis mouse model is protected against OVX-induced bone loss by cyclidine chloride (IdB 1027)[1].

---

In an ovariectomy-induced osteoporosis mouse model, Cyanidin Chloride (5 mg/kg, intraperitoneal injection every two days for 6 weeks) significantly protected against ovariectomy-induced bone loss. Micro-CT analysis revealed that Cyanidin Chloride treatment significantly increased bone volume/tissue volume ratio (BV/TV) and trabecular number (Tb.N) compared to the ovariectomy-vehicle control group. Trabecular separation (Tb.Sp) was also reduced. [1]
Histomorphometric analysis of decalcified tibial sections showed that Cyanidin Chloride treatment increased bone volume, and significantly decreased both osteoclast surface per bone surface (OcS/BS) and the number of osteoclasts per bone perimeter (N.Oc/BS) compared to the ovariectomy-vehicle control group. [1]

However, luciferase reporter gene assays were used to assess the activity of transcription factors. For the NF-κB reporter assay, RAW264.7 cells stably transfected with an NF-κB luciferase reporter construct were pre-treated with Cyanidin Chloride at indicated concentrations for 1 hour, then stimulated with RANKL for 6 hours, after which luciferase activity was measured. For the NFAT reporter assay, RAW264.7 cells stably transfected with an NFAT luciferase reporter construct were similarly pre-treated, stimulated with RANKL for 24 hours, and luciferase activity was measured. [1]

Bone marrow macrophages were isolated from the long bones of C57BL/J mice and cultured in α-MEM with M-CSF (10 ng/mL). For osteoclast differentiation, BMMs were seeded in 96-well plates (9 × 10³ cells/well) and treated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) in the presence or absence of various concentrations of Cyanidin Chloride (0, 0.5, 1, 2.5, 5, 10, 20 μM) for 5-7 days, with medium changed every 2 days. Cells were fixed with 2.5% glutaraldehyde and stained for TRAP. TRAP-positive multinucleated cells (≥3 nuclei) were counted. [1]
A cell apoptosis assay was performed using Annexin V/Propidium Iodide staining and flow cytometry. RAW264.7 cells (2 × 10⁶/well) were treated with Cyanidin Chloride (0, 1, 2.5, 5, 10, 20 μM) for 24 hours, harvested, stained, and analyzed. Cyanidin Chloride at concentrations ≤10 μM did not induce significant apoptosis. [1]
For the hydroxyapatite resorption assay, BMMs were differentiated into osteoclasts on collagen-coated plates, then dissociated and seeded onto hydroxyapatite-coated plates in the presence of RANKL (100 ng/mL) with or without Cyanidin Chloride. After 48 hours, cells were either stained for TRAP to count osteoclasts, or removed with bleach to visualize resorption pits. Resorption area was quantified. [1]
For quantitative real-time PCR, BMMs were cultured with M-CSF (10 ng/mL) and RANKL (100 ng/mL) with or without Cyanidin Chloride (5 μM) for 5 days. Total RNA was extracted, reverse-transcribed, and PCR was performed using specific primers for ctr, ctsk, trap, and 18S. [1]
For Western blot analysis, BMMs (0.5 × 10⁶/well) were pre-incubated with or without Cyanidin Chloride (10 μM) for 4 hours, then stimulated with RANKL (100 ng/mL) for indicated times. Cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against IκB-α, phosphorylated ERK, total ERK, c-Fos, NFATc1, and β-actin. [1]
For calcium oscillation measurement, BMMs were pre-treated with or without Cyanidin Chloride (5 or 10 μM) for 1 hour, then stimulated with RANKL (100 ng/mL) for 72 hours. Cells were loaded with Fluo-4 AM, and calcium oscillations were monitored by time-lapse fluorescence microscopy (488 nm excitation, images captured every 2 seconds for 3 minutes). [1]

Seven-week-old female C57BL/6 mice (body weight 23 ± 1.5 g) were used. After one week of acclimatization, mice underwent either ovariectomy or sham surgery. Mice were divided into three groups (n = 6 per group): Sham + vehicle (PBS), OVX + vehicle (PBS), and OVX + Cyanidin Chloride (5 mg/kg). Cyanidin Chloride (dissolved in PBS) or vehicle was administered via intraperitoneal injection every two days for 6 weeks. [1]
At the end of the treatment period, mice were sacrificed. Left tibias were collected and scanned using high-resolution micro-CT. Bone parameters including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were analyzed. Right tibias were fixed, decalcified, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) and for TRAP. Histomorphometric analysis was performed to quantify bone volume, osteoclast surface per bone surface (OcS/BS), and number of osteoclasts per bone perimeter (N.Oc/BS). [1]

---

Seven-week-old female C57BL/6 mice (body weight 23 ± 1.5 g) were used. After one week of acclimatization, mice underwent either ovariectomy or sham surgery. Mice were divided into three groups (n = 6 per group): Sham + vehicle (PBS), OVX + vehicle (PBS), and OVX + Cyanidin Chloride (5 mg/kg). Cyanidin Chloride (dissolved in PBS) or vehicle was administered via intraperitoneal injection every two days for 6 weeks. [1]
At the end of the treatment period, mice were sacrificed. Left tibias were collected and scanned using high-resolution micro-CT. Bone parameters including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were analyzed. Right tibias were fixed, decalcified, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) and for TRAP. Histomorphometric analysis was performed to quantify bone volume, osteoclast surface per bone surface (OcS/BS), and number of osteoclasts per bone perimeter (N.Oc/BS). [1]

In an Annexin V/Propidium Iodide apoptosis assay using RAW264.7 cells, Cyanidin Chloride at concentrations of 10 μM and below did not induce significant cellular apoptosis. At 20 μM, a slight increase in apoptotic cells was observed. This indicates that the inhibitory effects of Cyanidin Chloride on osteoclast formation at concentrations ≤10 μM are not due to general cytotoxicity. [1]

[1]. Cyanidin Chloride inhibits ovariectomy-induced osteoporosis by suppressing RANKL-mediated osteoclastogenesis and associated signaling pathways. J Cell Physiol. 2018 Mar;233(3):2502-2512.

See also: Blueberries (partial).

---

Cyanidin Chloride is a naturally occurring anthocyanidin, a subclass of anthocyanins, found in various colorful fruits and plants. It possesses antioxidant and anti-carcinogenesis properties. [1]
This study demonstrates that Cyanidin Chloride inhibits osteoclast formation, hydroxyapatite resorption, and RANKL-induced expression of osteoclast marker genes. Mechanistically, it suppresses RANKL-induced NF-κB activation, ERK phosphorylation, calcium oscillations, and subsequent NFATc1 and c-Fos activation, which are critical pathways for osteoclast differentiation and function. In an OVX-induced osteoporosis mouse model, Cyanidin Chloride treatment protected against bone loss by reducing osteoclast numbers and bone resorption. These findings suggest that Cyanidin Chloride may have therapeutic potential for osteolytic diseases such as osteoporosis. [1]

C15H11CLO6

322.6972

322.024

528-58-5

13306-05-3 (Parent)

68247

Brown to black solid powder

5

6

1

22

364

0

[Cl-].[O+]1=C(C(=C([H])C2=C(C([H])=C(C([H])=C12)O[H])O[H])O[H])C1C([H])=C([H])C(=C(C=1[H])O[H])O[H]

COAWNPJQKJEHPG-UHFFFAOYSA-N

InChI=1S/C15H10O6.ClH/c16-8-4-11(18)9-6-13(20)15(21-14(9)5-8)7-1-2-10(17)12(19)3-7;/h1-6H,(H4-,16,17,18,19,20);1H

2-(3,4-dihydroxyphenyl)chromenylium-3,5,7-triol;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~77.47 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.75 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)