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Ligustroflavone is a novel potent and naturally occurring calcium-sensing receptor (CaSR) antagonist isolated from Ligustrum lucidum with protective effects against diabetic osteoporosis in mice.
| Targets |
Ligustroflavone targets calcium-sensing receptor (CaSR) (IC₅₀: 4.2 μM, determined by luciferase reporter assay) [1]
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|---|---|
| ln Vitro |
Ligustroflavone inhibited CaSR activity in HEK293T cells transfected with human CaSR plasmid, with an IC₅₀ value of 4.2 μM, suppressing CaSR-mediated luciferase activity in a dose-dependent manner (1, 2, 4, 8 μM) [1]
It promoted proliferation of MC3T3-E1 osteoblastic cells (assessed by CCK-8 assay) at concentrations of 1, 5, 10 μM, with the highest proliferation rate at 10 μM [1] The compound enhanced osteogenic differentiation of MC3T3-E1 cells, increasing ALP activity, mineralized nodule formation (Alizarin Red S staining), and mRNA/protein expression of osteogenic markers (Runx2, OCN, Col1a1) [1] It inhibited RANKL-induced osteoclast differentiation of RAW264.7 cells, reducing TRAP-positive multinucleated cell formation and downregulating mRNA/protein expression of osteoclast markers (TRAP, CTSK, NFATc1) [1] Ligustroflavone suppressed CaSR-mediated downstream signaling pathways, including PLCγ1 phosphorylation and IP3 production, in both HEK293T-CaSR cells and MC3T3-E1 cells [1] |
| ln Vivo |
In streptozotocin (STZ)-induced diabetic osteoporosis mice, oral administration of Ligustroflavone (5, 10, 20 mg/kg/day) for 8 weeks dose-dependently increased bone mineral density (BMD) of the femur and lumbar vertebrae compared to the diabetic model group [1]
It improved bone microstructural parameters (trabecular thickness, trabecular number, bone volume/tissue volume ratio) and reduced trabecular separation, as analyzed by Micro-CT [1] Histological staining (H&E, Masson’s trichrome) showed that Ligustroflavone increased trabecular bone mass and collagen deposition in femoral metaphysis, while decreasing osteoclast number (TRAP staining) [1] The compound upregulated serum osteogenic markers (ALP, OCN) and downregulated serum osteoclast markers (TRAP, CTSK) in diabetic mice [1] It reduced CaSR protein expression in femoral bone tissue and suppressed CaSR-mediated signaling (PLCγ1 phosphorylation) in vivo [1] No significant changes in blood glucose levels were observed, indicating the protective effect on bone was independent of glycemic control [1] |
| Enzyme Assay |
HEK293T cells were transfected with human CaSR expression plasmid and CaSR-responsive luciferase reporter plasmid, along with Renilla luciferase plasmid as internal control [1]
Transfected cells were seeded in 96-well plates, allowed to adhere overnight, then treated with different concentrations of Ligustroflavone (1, 2, 4, 8 μM) and stimulated with Ca²⁺ (5 mM) for 6 hours [1] Luciferase activity was measured using a dual-luciferase assay system, and the inhibition rate of CaSR-mediated luciferase activity was calculated to determine the IC₅₀ value [1] For IP3 detection: MC3T3-E1 cells were treated with Ligustroflavone (2, 4, 8 μM) for 1 hour, then stimulated with Ca²⁺ (5 mM) for 30 minutes; intracellular IP3 levels were quantified using a specific immunoassay kit [1] |
| Cell Assay |
MC3T3-E1 osteoblastic cells and RAW264.7 macrophage cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum and antibiotics, maintained at 37°C in a 5% CO₂ incubator [1]
Osteoblast proliferation assay: MC3T3-E1 cells were seeded in 96-well plates, treated with Ligustroflavone (1, 5, 10 μM) for 24, 48, 72 hours; CCK-8 reagent was added, and absorbance was measured at 450 nm to assess cell viability [1] Osteoblast differentiation assay: MC3T3-E1 cells were seeded in 6-well plates, treated with Ligustroflavone (1, 5, 10 μM) in osteogenic induction medium for 7–21 days; ALP activity was measured by colorimetric assay, and mineralized nodules were stained with Alizarin Red S for quantification [1] Osteoclast differentiation assay: RAW264.7 cells were seeded in 6-well plates, treated with Ligustroflavone (1, 5, 10 μM) and RANKL (50 ng/mL) for 5 days; TRAP staining was performed to count TRAP-positive multinucleated cells, and TRAP activity was measured by colorimetric assay [1] Western blot analysis: Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, protein concentrations determined by BCA assay, equal amounts of protein separated by SDS-PAGE, transferred to PVDF membranes, incubated with primary antibodies (CaSR, Runx2, OCN, TRAP, CTSK, p-PLCγ1, PLCγ1, β-actin) overnight at 4°C, followed by HRP-conjugated secondary antibodies, and protein bands visualized by ECL detection system [1] RT-PCR analysis: Total RNA was extracted from cells, reverse-transcribed into cDNA, PCR amplified with specific primers for osteogenic/osteoclast markers and GAPDH, and relative mRNA expression calculated by comparative Ct method [1] |
| Animal Protocol |
Male C57BL/6 mice (6–8 weeks old) were randomly divided into 5 groups: normal control group, diabetic osteoporosis (DOP) model group, Ligustroflavone low-dose (5 mg/kg), medium-dose (10 mg/kg), and high-dose (20 mg/kg) groups (10 mice per group) [1]
DOP model was established by intraperitoneal injection of STZ (50 mg/kg/day for 5 consecutive days); mice with fasting blood glucose ≥16.7 mmol/L were considered diabetic [1] Ligustroflavone was dissolved in 0.5% carboxymethylcellulose sodium, and administered to mice via oral gavage once daily for 8 weeks; normal and model groups received the same volume of vehicle [1] During the experiment, mice were monitored for body weight and fasting blood glucose every 2 weeks [1] At the end of treatment, mice were sacrificed by cervical dislocation; femurs and lumbar vertebrae were collected for BMD measurement (dual-energy X-ray absorptiometry) and Micro-CT analysis of bone microarchitecture [1] Femoral metaphysis samples were fixed, decalcified, embedded in paraffin, sectioned, and subjected to H&E staining, Masson’s trichrome staining, and TRAP staining for histological analysis [1] Serum was collected to detect levels of ALP, OCN (osteogenic markers), TRAP, CTSK (osteoclast markers), and liver/kidney function indicators (ALT, AST, BUN, Cr) [1] Femoral bone tissue was used for Western blot and RT-PCR analysis of CaSR, osteogenic/osteoclast markers, and signaling pathway proteins [1] |
| Toxicity/Toxicokinetics |
Oral administration of chuanxiong flavonoids (5–20 mg/kg/day for 8 weeks) did not cause significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, Cr) in diabetic mice compared to the model group [1]. No obvious clinical symptoms of toxicity (such as somnolence, loss of appetite, or abnormal behavior) were observed during the treatment period [1].
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| References | |
| Additional Infomation |
It has been reported that privet (Ligustrum vulgare) contains privet flavonoids, and there is relevant data.
Privet flavonoids are a natural flavonoid compound isolated from the fruit of Ligustrum lucidum [1]. As a selective antagonist of CaSR, it exerts a protective effect against diabetic osteoporosis through dual regulation: promoting osteoblast differentiation and inhibiting osteoclast differentiation [1]. Its mechanism involves the inhibition of the CaSR-mediated PLCγ1/IP3 signaling pathway, which is one of the causes of bone metabolic imbalance in diabetes [1]. It has potential therapeutic value in treating osteoporosis caused by diabetes and has good in vivo safety at effective doses [1]. |
| Molecular Formula |
C33H40O18
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|---|---|
| Molecular Weight |
724.6599
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| Exact Mass |
724.221
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| CAS # |
260413-62-5
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| PubChem CID |
10417462
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.7±0.1 g/cm3
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| Boiling Point |
1028.5±65.0 °C at 760 mmHg
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| Flash Point |
325.2±27.8 °C
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| Vapour Pressure |
0.0±0.3 mmHg at 25°C
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| Index of Refraction |
1.714
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| LogP |
1.56
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| Hydrogen Bond Donor Count |
10
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| Hydrogen Bond Acceptor Count |
18
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
51
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| Complexity |
1210
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| Defined Atom Stereocenter Count |
15
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| SMILES |
C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC[C@@H]2[C@H]([C@@H]([C@H]([C@@H](O2)OC3=CC(=C4C(=C3)OC(=CC4=O)C5=CC=C(C=C5)O)O)O[C@H]6[C@@H]([C@@H]([C@H]([C@@H](O6)C)O)O)O)O)O)O)O)O
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| InChi Key |
NULBHTHMVOCGOE-ZBCCAYPVSA-N
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| InChi Code |
InChI=1S/C33H40O18/c1-11-22(37)25(40)28(43)31(46-11)45-10-20-24(39)27(42)30(51-32-29(44)26(41)23(38)12(2)47-32)33(50-20)48-15-7-16(35)21-17(36)9-18(49-19(21)8-15)13-3-5-14(34)6-4-13/h3-9,11-12,20,22-35,37-44H,10H2,1-2H3/t11-,12-,20+,22-,23-,24+,25+,26+,27-,28+,29+,30+,31+,32-,33+/m0/s1
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| Chemical Name |
7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chromen-4-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~172.49 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (2.87 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 20.8 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: ≥ 2.08 mg/mL (2.87 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 20.8 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3800 mL | 6.8998 mL | 13.7996 mL | |
| 5 mM | 0.2760 mL | 1.3800 mL | 2.7599 mL | |
| 10 mM | 0.1380 mL | 0.6900 mL | 1.3800 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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