Oleonuezhenide targets casein kinase 2 alpha (CK2α). It increases CK2α expression and directly binds to CK2α, acting as an agonist. Docking studies showed binding cavity on CK2α with residue Arg43 involved. No IC50/Ki/EC50 values reported for this interaction. [1]
Also involved in non-canonical Wnt pathway via upregulation of Wnt5a expression. [1]

Oleonuezhenide (0.9, 4.5, 9 μM) increased proliferation of BMSCs in a dose-dependent manner. The combination of Oleonuezhenide (9 μM) with wedelolactone (6 μM) increased BMSC proliferation but showed no significant change compared to Oleonuezhenide treatment alone. [1]
When BMSCs were cultured in osteogenic medium for 9 days, Oleonuezhenide treatment increased alkaline phosphatase (ALP) activity. The combination of 9 μM Oleonuezhenide with 6 μM wedelolactone induced a more significant increase in ALP activity compared to either compound alone. Morphological observations confirmed increased ALP-positive cells with the combination. [1]
After 21 days of treatment with 9 μM Oleonuezhenide plus 6 μM wedelolactone, calcium deposits (mineralization) were increased based on alizarin red S staining. [1]
Oleonuezhenide (9 μM) increased mRNA expression of osteoblastogenesis marker genes including Osterix (SP7), osteocalcin (Bglap), and Runx2 after 9 days of treatment (alone or in combination with wedelolactone). [1]
Oleonuezhenide (9 μM) increased CK2α expression and promoted nuclear accumulation of β-catenin and Runx2. It did not affect GSK3β phosphorylation. [1]
Addition of DMAT (a CK2α inhibitor, 40 μM for 1 h pretreatment) inhibited Oleonuezhenide-induced ALP activity and suppressed Oleonuezhenide/wedelolactone-induced nuclear accumulation of β-catenin. [1]
Oleonuezhenide at 0.9, 4.5, and 9 μM dose-dependently reversed wedelolactone-induced cell death (30 μM wedelolactone reduced cell survival to 68%; addition of Oleonuezhenide increased survival to 89%). Morphological observations confirmed that Oleonuezhenide inhibited high-dose wedelolactone-induced cytotoxicity. [1]
Oleonuezhenide (9 μM) reversed wedelolactone-reduced ALP activity in BMSCs treated with 30 μM wedelolactone for 9 days, and increased the number of ALP-positive cells. [1]
Oleonuezhenide (9 μM) increased Wnt5a and CK2α expression, and reversed wedelolactone-induced reduction in ERK1/2 phosphorylation. DMAT (CK2α inhibitor) blocked Oleonuezhenide-induced increase in BMSC survival rate and reduced CK2α expression and ERK1/2 phosphorylation induced by Oleonuezhenide. [1]

In ovariectomized (OVX) mice, intraperitoneal injection of Oleonuezhenide (50 mg/kg) every two days for one month, alone or in combination with wedelolactone (10 mg/kg), prevented OVX-induced bone loss. The combination significantly alleviated OVX-induced decrease in bone volume and trabecular number at the femur compared to either compound alone. [1]
Bone formation rate per bone surface was increased by combined treatment with Oleonuezhenide and wedelolactone compared to either alone. Eroded surface/bone surface (osteoclastic parameter) was decreased by Oleonuezhenide but no further decrease with combination. [1]
H&E staining showed increased trabecular bone volume and abundant osteoblast lineage cells on trabecular bone surface in Oleonuezhenide-treated mice. Calcein double staining showed increased distance between labels (new bone formation) after treatment with Oleonuezhenide plus wedelolactone. [1]

Isolation and culture of mouse bone marrow mesenchymal stem cells (BMSCs): BMSCs were isolated from BALB/c mice (female, 8-week old, body weight 20-25 g). Cells were collected using gradient centrifugation of mesenchymal stem cell-specific gradient solutions. A layer of bone marrow cell fraction in PBS was placed on top of the gradient solution and centrifuged at 340g for 20 min. The cell fraction was collected and washed with PBS. Cells were resuspended in minimum essential medium alpha medium (α-MEM) supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin, and maintained in 5% CO2 at 37°C in a humidified atmosphere. Three days later, cell suspension was decanted and replaced with fresh medium. BMSCs were further separated by differential adhesion. Medium was replaced every 3 days until confluence. [1]
MTT assay: BMSCs were seeded into 96-well plates at density of 1×10^5/mL, grown overnight in α-MEM with 10% FBS, then treated with various concentrations of compounds. MTT solution was added at different time points. Cell yield measured with plate reader. Inhibition percentage calculated as [(A492(control)-A492(sample))/A492(control)]×100%. [1]
ALP activity assay: BMSCs were reseeded (1×10^4/cm^2) and cultured with osteogenic medium (0.1 μM dexamethasone, 5 μM L-ascorbic acid 2-phosphate, 1 mM β-glycerophosphate). Medium replaced every 3 days. After 9 days, cells were fixed with 60% citrate buffered acetone for 30 s, washed with water, and incubated with 0.1 mL phosphatase substrate solution (10 mM pNPP and 10 mM sodium tartrate in 50 mM citrate buffer, pH 9.5) at 37°C for 1 h. The reaction mixture was transferred and stopped with 0.1 mL 0.1 N NaOH. Absorbance measured at 405 nm. ALP activity represented as OD value normalized with protein content. ALP staining was also performed per manufacturer's instructions. [1]
Alizarin red S staining: BMSCs were reseeded (1×10^4/cm^2) and incubated with osteogenic medium. After 21 days, cells were fixed with 4% paraformaldehyde for 10 min, washed twice with deionized water, stained with 2% ARS (pH 4.1) for 10 min at room temperature, then washed three times. Intensity of calcium deposits indicated by orange red staining. Quantification: absorbance of ARS released by cetylpyridinium measured and normalized to mg total protein. [1]
Quantitative real-time PCR: TRIZOL reagent used to extract total RNA. Runx2, Osteocalcin, Osterix transcripts quantified on Mx3005P system using SYBR green dye and normalized with β-actin. Primers sequences provided. SuperScript III first-strand synthesis kit used to synthesize cDNA. Amplification conditions: 15 min at 95°C; then 55 cycles at 95°C for 10 s; 58°C for 45 s; 72°C for 45 s. β-actin levels used to normalize gene expression. Fold changes calculated relative to untreated control. [1]
Western blot analysis: Cells resuspended in buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 mM NaF, 2 mM Na3VO4, 1 mM pyrophosphoric acid, and protease inhibitors) and incubated on ice for 10 min, then centrifuged at 700g for 10 min at 4°C. Supernatant (cytosolic fraction) collected. For nuclear protein extraction, pellet washed with buffer A, resuspended in buffer B (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM pyrophosphoric acid, and protease inhibitors), incubated on ice for 5 min. Protein concentration determined by Bradford assay. Equal amounts run on SDS-PAGE and transferred to PVDF membrane. Blocked with 5% non-fat dry milk, probed with primary antibodies overnight at 4°C, secondary antibodies for 2 h at room temperature. Antibodies used: β-catenin, Runx2, Dvl2, Wnt5a, CK2α, GSK3β, P-GSK3β, ERK, P-ERK. Bands visualized using DAB or ECL substrate. [1]

Ovariectomized mouse model: Nine-week-old C57BL/6 female mice were ovariectomized or sham operated. Two days after ovariectomy, mice were divided into five groups of eight mice each: sham, OVX vehicle, OVX treated with Oleonuezhenide (50 mg/kg), OVX treated with wedelolactone (10 mg/kg), and OVX treated with combination. Oleonuezhenide was injected intraperitoneally every two days for one month. After treatment, sections of femurs and lumbar vertebrae were obtained for histomorphometric analysis using the OsteoMeasure Analysis System. Three-dimensional microcomputed tomography analyses and bone morphometric analyses were performed. For H&E staining and calcein staining, proximal tibiae were isolated and fixed in 10% paraformaldehyde, decalcified with 10% EDTA for 2 weeks, then embedded in paraffin for staining. Calcein double labeling was used to assess new bone formation (arrows mark distance between calcein-labeled layers). [1]

[1]. The combined effect of oleonuezhenide and wedelolactone on proliferation and osteoblastogenesis of bone marrow mesenchymal stem cells. Phytomedicine. 2019 Dec;65:153103.

See also: Oleonuezhenide (Note moved to).

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Oleonuezhenide is isolated from Fructus Ligustri Lucidi, a component of Er-Zhi-Wan, a traditional Chinese formulation first recorded in "Yi Bian" during the Ming Dynasty. Er-Zhi-Wan is used to treat kidney diseases and strengthen bone. Oleonuezhenide combined with wedelolactone enhanced osteoblast differentiation and bone mineralization in vitro and in vivo. The combination promoted GSK3β phosphorylation (by wedelolactone) and increased CK2α expression (by Oleonuezhenide), leading to β-catenin nuclear translocation and enhanced osteoblastogenesis. Simultaneously, Oleonuezhenide protected BMSCs from wedelolactone-induced cytotoxicity by activating Wnt5a/CK2α/ERK pathway. The authors suggest these two compounds could be developed as a combined therapeutic agent for osteoporosis. [1]

C48H64O27

1073.00556

1072.363

112693-21-7

23843954

White to off-white solid

1.6±0.1 g/cm3

1149.3±65.0 °C at 760 mmHg

325.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.642

-2.31

11

27

23

75

2050

15

OC1C=CC(CCO[C@@H]2O[C@H](COC(CC3C(C(OC)=O)=CO[C@@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)/C/3=C/C)=O)[C@@H](O)[C@H](O)[C@H]2OC(CC2C(C(OC)=O)=CO[C@@H](O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)/C/2=C/C)=O)=CC=1

MFZDFMOKBMJUGB-CWERYYTKSA-N

InChI=1S/C48H64O27/c1-5-22-24(26(42(62)64-3)17-68-44(22)74-46-39(60)36(57)33(54)28(15-49)70-46)13-31(52)67-19-30-35(56)38(59)41(48(72-30)66-12-11-20-7-9-21(51)10-8-20)73-32(53)14-25-23(6-2)45(69-18-27(25)43(63)65-4)75-47-40(61)37(58)34(55)29(16-50)71-47/h5-10,17-18,24-25,28-30,33-41,44-51,54-61H,11-16,19H2,1-4H3/b22-5+,23-6+/t24?,25?,28-,29-,30-,33-,34-,35-,36+,37+,38+,39-,40-,41-,44+,45+,46+,47+,48-/m1/s1

methyl (5E,6S)-5-ethylidene-4-[2-[[(2R,3S,4S,5R,6R)-5-[2-[(2S,3E)-3-ethylidene-5-methoxycarbonyl-2-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-4-yl]acetyl]oxy-3,4-dihydroxy-6-[2-(4-hydroxyphenyl)ethoxy]oxan-2-yl]methoxy]-2-oxoethyl]-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylate

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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