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| Targets |
Ilexaponin A modulates the apoptotic pathway. It reduces pro‑apoptotic proteins (caspase‑3, cleaved caspase‑3, Bax) and increases anti‑apoptotic protein Bcl‑2. It also activates the PI3K/Akt pathway by increasing the expression of p‑Akt (without affecting total Akt) [1].
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| ln Vitro |
In hypoxic/reoxygenated cardiomyocytes, ilexsaponin A markedly decreased pro-apoptotic proteins, Bax, cleaved caspase-3, and caspase-3, and elevated anti-apoptotic protein bcl-2. Treatment with ilex saponin A has been shown to upregulate p-Akt expression in animal models of cardiac ischemia/reperfusion and hypoxia/reoxygenation [1].
- Ilexaponin A pretreatment (10, 50, 250 μg/ml for 24 h) increased cell viability in neonatal rat cardiomyocytes subjected to hypoxia (4 h) / reoxygenation (4 h) (H/R). Cell viability in H/R‑only group was 45.10 ± 3.10 % of control; after Ilexaponin A at 10, 50, 250 μg/ml, viability increased to 56.09 ± 3.95 %, 64.60 ± 4.16 %, and 78.03 ± 2.56 %, respectively, in a dose‑dependent manner [1]. - Ilexaponin A pretreatment reduced apoptosis in H/R‑treated cardiomyocytes as determined by TUNEL assay. The apoptotic index (percentage of TUNEL‑positive nuclei) was significantly decreased by Ilexaponin A at all tested concentrations compared to H/R‑only group [1]. - Western blot analysis showed that in H/R‑treated cardiomyocytes, Ilexaponin A pretreatment decreased the expression levels of caspase‑3, cleaved caspase‑3, and Bax, while increasing Bcl‑2 expression. These effects were dose‑dependent. Additionally, Ilexaponin A increased the expression level of p‑Akt (without changing total Akt) in H/R‑treated cells [1]. |
| ln Vivo |
Male Sprague-Dawley rats weighing 280–320 g show dose-dependent reductions in myocardial infarct size when exposed to ilexsaponin A (10 or 40 mg/kg) [1].
- In male Sprague‑Dawley rats subjected to myocardial ischemia (30 min) followed by reperfusion (4 h), Ilexaponin A (10 mg/kg or 40 mg/kg, given 10 min before ligation) significantly reduced myocardial infarct size. The IS/AAR ratio was 41.55 ± 8.99 % in I/R group, 25.89 ± 9.33 % in low‑dose Ilexaponin A group, and 20.49 ± 6.55 % in high‑dose Ilexaponin A group, showing a dose‑dependent protective effect [1]. - Ilexaponin A treatment significantly lowered serum levels of myocardial injury markers: CK‑MB was reduced in both low‑ and high‑dose groups; LDH and AST were significantly reduced only in the high‑dose (40 mg/kg) group compared to I/R group [1]. - Western blot analysis of heart tissues from I/R rats showed that Ilexaponin A decreased the expression of caspase‑3 and Bax, increased Bcl‑2 expression, and increased p‑Akt expression (without changing total Akt) [1]. |
| Cell Assay |
- Primary neonatal rat cardiomyocyte culture: Hearts from 1‑3‑day‑old rats were harvested, and isolated cardiomyocytes were seeded and cultured at 37 °C in humidified 5 % CO₂. After 48 h, medium was replaced with fresh DMEM and cells were cultured for another 24 h [1].
- Hypoxia/reoxygenation (H/R) model: Cells were placed in a hypoxic/ischemic chamber (Modular Incubator Chamber) at 37 °C for 4 h in a humidified atmosphere of 5 % CO₂ and 95 % nitrogen. Reoxygenation was performed by incubating cells in DMEM supplemented with 10 % fetal calf serum at 37 °C under 5 % CO₂ for 4 h [1]. - Cell viability (MTT assay): After H/R treatment, MTT was added and the optical density was measured. Viability was calculated by dividing the optical density of samples by that of control [1]. - Apoptosis detection (TUNEL assay): An in situ cell death detection kit was used following the manufacturer’s instructions. Apoptotic index was expressed as the number of positively stained apoptotic cardiomyocytes divided by the total number of cardiomyocytes [1]. - Western blot analysis: Cardiomyocyte samples were lysed in lysis buffer on ice for 20 min, then centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentration was quantified. 30 μg of total protein was separated by SDS‑PAGE and transferred to a PVDF membrane. Membranes were incubated with primary antibodies against caspase‑3, cleaved caspase‑3, Bcl‑2, Bax, p‑Akt, Akt, and actin, followed by secondary antibodies. Immunodetection was performed using enhanced chemiluminescence and a quantitative digital imaging system [1]. |
| Animal Protocol |
- Myocardial ischemia/reperfusion (I/R) model in rats: Male Sprague‑Dawley rats (280‑320 g) were anesthetized with 3 % pentobarbital sodium (50 mg/kg). A left thoracic incision was made to exteriorize the heart, and a 6.0 silk suture with a silicone rubber venous cannula was placed around the left anterior descending coronary artery. Ischemia was maintained for 30 min (confirmed by myocardial blanching and ST‑segment elevation), then the cannula was removed to allow reperfusion for 4 h. Sham‑operated animals underwent the same surgical procedure without tying the suture [1].
- Drug administration: Ilexaponin A was dissolved in DMSO and then diluted with saline to the required concentrations. It was given 10 min before ligation of the left anterior descending coronary artery. Doses used were 10 mg/kg (low dose) and 40 mg/kg (high dose). The sham and I/R groups received vehicle only [1]. - Infarct size determination: At the end of reperfusion, hearts were perfused with Evan’s blue at constant pressure (80 mmHg). The left ventricle was cut into 2‑3 mm slices. The unstained area at risk (AAR) was incubated in 1 % TTC (in 0.1 mol/L phosphate buffer, pH 7.4) at 37 °C for 30 min. Infarcted myocardium appeared pale white, non‑infarcted myocardium deep red. Infarct size was expressed as percentage of AAR (IS/AAR) [1]. - Serum marker measurement: Blood was collected from the trunk, kept at room temperature for 30 min, then centrifuged at 4,500 g for 15 min at 4 °C. Serum levels of CK‑MB, LDH, and AST were assayed using commercial kits [1]. |
| References | |
| Additional Infomation |
It has been reported that holly saponin A1 has been found in both Ilex pubescens and Mussaenda pubescens, and relevant data are available for reference.
- Background: Ilexaponin A is one of the representative saponins isolated from the roots of Ilex pubescens (Mao Dong Qing). Its purity was >95 % as detected by HPLC. Its structure was established by HR‑ESI‑MS, IR, UV, ¹H NMR, and ¹³C NMR spectroscopy [1]. - Mechanism: Ilexaponin A exerts cardioprotective effects against ischemia‑reperfusion injury by inhibiting apoptosis (reducing caspase‑3, cleaved caspase‑3, and Bax, and increasing Bcl‑2) and by activating the PI3K/Akt pathway (increasing p‑Akt expression). Both in vivo and in vitro results support an anti‑apoptotic mechanism [1]. - Potential indication: Ilexaponin A may have potential as a novel cardioprotective agent for treating myocardial ischemia/reperfusion injury [1]. |
| Molecular Formula |
C36H56O11
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|---|---|
| Molecular Weight |
664.8233
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| Exact Mass |
664.382
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| CAS # |
108524-93-2
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| PubChem CID |
72163175
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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 |
788.1±60.0 °C at 760 mmHg
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| Flash Point |
240.1±26.4 °C
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| Vapour Pressure |
0.0±6.2 mmHg at 25°C
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| Index of Refraction |
1.611
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| LogP |
5.11
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| Hydrogen Bond Donor Count |
7
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
47
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| Complexity |
1320
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| Defined Atom Stereocenter Count |
16
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| SMILES |
O([H])[C@]1(C([H])([H])[H])[C@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[C@]2(C(=O)O[C@@]3([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O3)O[H])O[H])O[H])C([H])([H])C([H])([H])[C@]3(C([H])([H])[H])C(=C([H])C([H])([H])[C@]4([H])[C@@]5(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])([C@@](C(=O)O[H])(C([H])([H])[H])C5([H])C([H])([H])C([H])([H])[C@]43C([H])([H])[H])O[H])[C@@]21[H]
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| InChi Key |
AWFZJSUJFSUBQU-MOJLLMCOSA-N
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| InChi Code |
InChI=1S/C36H56O11/c1-18-9-14-36(30(44)47-28-26(41)25(40)24(39)20(17-37)46-28)16-15-32(3)19(27(36)35(18,6)45)7-8-21-31(2)12-11-23(38)34(5,29(42)43)22(31)10-13-33(21,32)4/h7,18,20-28,37-41,45H,8-17H2,1-6H3,(H,42,43)/t18-,20-,21-,22-,23+,24-,25+,26-,27-,28+,31-,32-,33-,34-,35-,36+/m1/s1
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| Chemical Name |
(3S,4R,4aR,6aR,6bS,8aS,11R,12R,12aS,14aR,14bR)-3,12-dihydroxy-4,6a,6b,11,12,14b-hexamethyl-8a-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxycarbonyl-1,2,3,4a,5,6,7,8,9,10,11,12a,14,14a-tetradecahydropicene-4-carboxylic acid
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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 |
| 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 (~150.42 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.76 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 (3.76 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5042 mL | 7.5208 mL | 15.0417 mL | |
| 5 mM | 0.3008 mL | 1.5042 mL | 3.0083 mL | |
| 10 mM | 0.1504 mL | 0.7521 mL | 1.5042 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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