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| Targets |
Methylophiopogonanone A (MO-A) activates the PI3K/Akt/eNOS signaling pathway. [1]
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| ln Vitro |
Bcl-2/Bax ratio was raised, NO generation was restored, and apoptosis and caspase-3 expression were considerably decreased by mmethylphiopogonanone A (MO-A; 10 μmol/L). When wortmannin (100 nmol), a PI3K inhibitor, is present, MO-A's protective effect is nullified. Pretreatment with Mmethylphiopogonanone A (MO-A) markedly boosted the activation of the PI3K/Akt/eNOS pathway in H/R-treated H9C2 cells. (/L)[1].
In H9C2 rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R), pretreatment with MO-A (10 μmol/L) for 48 h significantly decreased apoptosis (apoptotic rate: 15.09% ± 1.33% vs. H/R alone 39.71% ± 3.37%, P<0.01). [1] MO-A (10 μmol/L) preserved cell viability after H/R treatment (78.2% ± 11.4% vs. H/R alone 54.8% ± 9.4%, P<0.01). [1] MO-A (10 μmol/L) restored NO production in H/R-exposed H9C2 cells (237.39 ± 20.84 μmol/L vs. H/R group 148.65 ± 18.32 μmol/L, P<0.01). [1] Western blot analysis showed that MO-A pretreatment increased the Bcl-2/Bax ratio (4.75-fold vs. H/R group) and decreased cleaved caspase-3 expression (0.31-fold vs. H/R group) in H9C2 cells after H/R. [1] MO-A pretreatment markedly increased the activation of PI3K/Akt/eNOS pathway in H9C2 cells subjected to H/R: PI3K expression increased 8-fold, p-Akt increased 3.9-fold, and p-eNOS increased 6.55-fold compared to the H/R group (P<0.05). These effects were abolished by the PI3K inhibitor wortmannin (100 nmol/L). [1] |
| ln Vivo |
Following myocardial I/R injury, cardiac function can be protected with mmethylphiopogonanone A (MO-A; 10 mg/kg/d, orally) for two weeks [1].
In male C57BL/6 mice subjected to myocardial ischemia/reperfusion (I/R) injury, pretreatment with Methylophiopogonanone A (MO-A) (10 mg·kg⁻¹·d⁻¹, oral, for 2 weeks) significantly reduced infarct size by 60.7% (INF/LV: 10.1% ± 2.1% vs. I/R group 25.7% ± 3.9%, P<0.01). [1] MO-A pretreatment significantly decreased myocardial apoptosis by 56.8% (apoptotic index: 22.1% ± 2.8% vs. I/R group 51.1% ± 5.8%, P<0.01) as assessed by TUNEL assay. [1] MO-A pretreatment improved cardiac function 72 h after reperfusion: left ventricular ejection fraction (EF: 62.1% ± 4.1% vs. I/R group 43.9% ± 5.8%), fractional shortening (FS: 41.6% ± 1.8% vs. I/R group 26.6% ± 1.5%), maximal velocity of left ventricular pressure development (+dp/dt: 11329±420.1 vs. 9512±390.6 mmHg/s), left ventricular end-diastolic pressure (LVEDP: 10.13±1.129 vs. 14.266±1.191 mmHg), and left ventricular systolic pressure (LVSP: 108.2±4.215 vs. 96.3±3.189 mmHg) (all P<0.05). [1] |
| Cell Assay |
H9C2 cells were seeded at a density of 10000/cm² and grown to 80% confluence in DMEM with 10% fetal bovine serum and antibiotics. Cells were washed with HBSS and rendered quiescent in serum-free DMEM for 24 h prior to experiments. To assess the cardioprotective effect of MO-A, cells were treated with either PBS or MO-A (10 μmol/L dissolved in PBS) for 48 h before hypoxia. [1]
Hypoxia/reoxygenation (H/R) model: H9C2 cells at 80% confluence were incubated with glucose-free medium pre-bubbled with 95% N₂ and 5% CO₂ for 6 h at 37°C. Then, fresh medium was provided and cells were moved to 95% O₂/5% CO₂ for reoxygenation. Cells were harvested 16 h post-reoxygenation for analyses. Control plates were kept in 95% O₂/5% CO₂ at 37°C. [1] Apoptosis assessment: FITC-conjugated Annexin V and propidium iodide (PI) were used to identify apoptotic cells by flow cytometry. The assay discriminates intact (FITC⁻/PI⁻) and apoptotic (FITC⁺/PI⁻ and FITC⁺/PI⁺) cells. Data were analyzed with CellQuest software. [1] Cell viability assay: MTT assay was performed. Cells were cultured in 96-well plates at 1×10⁴ cells/well for 24 h, then pre-treated with or without MO-A (10 μmol/L) for 3 h at 37°C. Fresh medium and MTT solution (20 μL of 5 mg/mL) were added for 4 h, followed by incubation with formazan solution (10 μL) for 4 h at 37°C. OD values at 570 nm were measured. Each experiment was repeated 6 times. [1] NO production measurement: Nitrite (stable metabolite of NO) in culture supernatants was measured by nitrate reductase method using an NO assay kit. NO levels expressed as μmol/L. [1] Western blot: Cells were lysed in lysis buffer on ice for 30 min, centrifuged at 17709×g for 20 min at 4°C. Total protein (10% SDS-PAGE) was transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk for 30 min at 37°C, then incubated with primary antibodies (Bcl-2, Bax, caspase-3, β-actin, PI3K, p-Akt Ser473, Akt, p-eNOS Ser1177, eNOS; 1:1000) overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibody for 30 min at 37°C. Bands were developed using chemiluminescent system and quantified by densitometry. [1] |
| Animal Protocol |
Animals: Adult male wild-type C57BL/6 mice (10-12 weeks old, 26-30 g). Mice were fed either saline or Methylophiopogonanone A (MO-A) dissolved in normal saline (pH=8.0) at a dose of 10 mg·kg⁻¹·d⁻¹ (5 mg/kg twice per day) orally for 2 weeks before I/R surgery. [1]
Myocardial I/R model: Mice were anesthetized with intraperitoneal injection of xylazine (5 mg/kg) and ketamine (100 mg/kg). The left anterior descending (LAD) coronary artery was occluded using an 8-0 silk suture tied transiently over PE-10 tubing for 1 h. Reperfusion was established by cutting the knot on the PE-10 tubing. Successful reperfusion was confirmed by recovery of the elevated ST segment. Sham-operated mice underwent same procedure without LAD occlusion. Groups: Sham, I/R, I/R+MO-A (10 mg·kg⁻¹·d⁻¹), n=10 each. [1] Infarct size measurement: After 24 h reperfusion, hearts were stained with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Infarct size (INF) and area-at-risk (AAR) were measured digitally using Image J software, expressed as percentages of left ventricular area (INF/LV and AAR/LV). [1] Apoptosis assessment in heart: TUNEL assay was performed on heart sections. 50 μL of TUNEL reaction mixture was added, slides incubated in humidified atmosphere for 60 min at 37°C in the dark, then rinsed with PBS (pH 7.4) three times for 5 min. Apoptotic index calculated as percentage of TUNEL-positive nuclei per 1000 total nuclei in infarcted border region in 10 random high power fields per mouse. [1] Cardiac function analysis: Doppler echocardiography was performed before and 72 h after reperfusion using Vevo2100 imaging system. Mice received continuous inhaled anesthetic (1%) and were maintained at 37°C. M-mode echocardiograms were carried out along the short axis of the LV at the level of papillary muscles during at least three consecutive beats. Hemodynamic measurements included +dp/dt, LVEDP, and LVSP. All measurements were double-blinded. [1] |
| References | |
| Additional Infomation |
Methyl ophiopogon A is a homoflavonoid. It has been reported that methyl ophiopogon A exists in Ophiopogon japonicus, and relevant data are available for reference.
Methylophiopogonanone A (MO-A) attenuates myocardial I/R-induced apoptosis in mice via activating the PI3K/Akt/eNOS signaling pathway. It increases the Bcl-2/Bax ratio and decreases cleaved caspase-3 expression. The cardioprotective effects of MO-A are abolished by the PI3K inhibitor wortmannin (100 nmol/L). The findings suggest potential therapeutic value of MO-A in prevention and rescue of myocardial I/R injury. Limitations: H9C2 cells may not fully mimic primary cardiac myocyte responses; animals were pre-treated for 2 weeks which does not mimic clinical setting (time from revascularization decision to procedure is far less than two weeks). [1] |
| Molecular Formula |
C19H18O6
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|---|---|
| Molecular Weight |
342.3426
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| Exact Mass |
342.11
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| CAS # |
74805-92-8
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| PubChem CID |
53466984
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| Appearance |
White to off-white solid
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
580.1±50.0 °C at 760 mmHg
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| Flash Point |
212.9±23.6 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.654
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| LogP |
5.16
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
25
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| Complexity |
510
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O1C2=C(C([H])([H])[H])C(=C(C([H])([H])[H])C(=C2C([C@@]([H])(C1([H])[H])C([H])([H])C1C([H])=C([H])C2=C(C=1[H])OC([H])([H])O2)=O)O[H])O[H]
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| InChi Key |
BXTNNJIQILYHJB-GFCCVEGCSA-N
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| InChi Code |
InChI=1S/C19H18O6/c1-9-16(20)10(2)19-15(17(9)21)18(22)12(7-23-19)5-11-3-4-13-14(6-11)25-8-24-13/h3-4,6,12,20-21H,5,7-8H2,1-2H3/t12-/m1/s1
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| Chemical Name |
(3R)-3-(1,3-benzodioxol-5-ylmethyl)-5,7-dihydroxy-6,8-dimethyl-2,3-dihydrochromen-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 : ~100 mg/mL (~292.11 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.30 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9211 mL | 14.6054 mL | 29.2107 mL | |
| 5 mM | 0.5842 mL | 2.9211 mL | 5.8421 mL | |
| 10 mM | 0.2921 mL | 1.4605 mL | 2.9211 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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