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Aristolochic acid I (AA-I) is major metabolite of aristolochic acid I (AA-I), and is able to form DNA adducts, thus causing renal damage. The cytotoxic potency of Aristololactam I (AL-I) is higher than that of Aristololactam I (AA-I), and the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase-3-dependent pathway. AL-I directly damages renal proximal tubule cells.
| ln Vitro |
Aristolactam I (AL-I) exhibits cytotoxic effects on human proximal tubular epithelial cells (HK-2). Its potency is greater than that of its parent compound, Aristolochic Acid I (AA-I), as indicated by lower IC₅₀ values.
AL-I inhibits HK-2 cell proliferation in a concentration- and time-dependent manner, as determined by MTT assay. Treatment with AL-I (e.g., 80 µM for 24 hours) induces morphological changes in HK-2 cells, including cell shrinkage and rounding, observed under a contrast microscope. Nuclear condensation and chromatin margination, characteristic of apoptosis, are visible after Hoechst 33258 staining. Flow cytometric analysis (PI staining) shows that AL-I induces a sub-G1 peak (indicative of apoptotic cells with hypodiploid DNA) and causes cell cycle arrest in the S-phase. Annexin V/PI staining confirms that AL-I induces early apoptosis (Annexin V⁺/PI⁻) in a dose-dependent manner, with minimal necrosis observed. AL-I increases caspase-3-like activity in HK-2 cells in a concentration-dependent manner, as measured by the cleavage of the colorimetric substrate Ac-DEVD-pNA, indicating apoptosis proceeds through a caspase-3-dependent pathway. [1] |
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| Cell Assay |
Cell Viability (MTT Assay): HK-2 cells are seeded at a density of 5 × 10⁴ cells/mL in culture plates and treated with various concentrations of AL-I for 24, 48, or 72 hours. After treatment, MTT reagent is added to each well to a final concentration of 0.5 mg/mL and incubated for 4 hours at 37°C. The resulting formazan crystals are dissolved, and the absorbance is measured at 570 nm to determine cell viability and calculate inhibition rates.
Morphological Assessment: HK-2 cells are treated with AL-I (e.g., 80 µM) for 24 hours. Cellular morphology is observed using a phase-contrast microscope. For nuclear morphology assessment, treated cells fixed on coverslips are stained with Hoechst 33258 and visualized under a fluorescence microscope to detect apoptotic features like chromatin condensation. Cell Cycle and Apoptosis Analysis (Flow Cytometry): For cell cycle analysis, HK-2 cells treated with AL-I are collected, fixed in ethanol, treated with RNase A and propidium iodide (PI), and analyzed by flow cytometry to determine DNA content and cell cycle phase distribution. The sub-G1 peak is used to quantify apoptotic cells. For apoptosis detection via Annexin V/PI staining, treated HK-2 cells are harvested, washed, and resuspended in a binding buffer containing FITC-conjugated Annexin V and PI. After incubation in the dark, cells are analyzed by flow cytometry to distinguish viable, early apoptotic, late apoptotic, and necrotic cell populations. Caspase-3 Activity Assay: HK-2 cells are treated with AL-I, harvested, and lysed. Cell lysates containing 50 µg of protein are incubated with 100 µM of the caspase-3 colorimetric substrate Ac-DEVD-pNA at 37°C for 2 hours. The release of p-nitroaniline is measured by absorbance at 405 nm to determine caspase-3-like protease activity. [1] |
| Toxicity/Toxicokinetics |
Aristolochic acid I (AL-I) exhibited significant in vitro cytotoxicity against human renal tubular epithelial cells (HK-2), inhibiting cell proliferation and inducing apoptosis. Comparison of IC₅₀ values in the MTT assay showed that AL-I was more cytotoxic than its precursor, aristolochic acid I (AA-I). The main mechanism of this renal cytotoxicity involves the induction of caspase-3-dependent apoptosis and S-phase cell cycle arrest. [1]
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| References | |
| Additional Infomation |
Aristololactam kankauensis, Aristololactam debilis, and other organisms with relevant data have been reported. Aristololactam I (AL-I) is the major metabolite of aristolochic acid I (AA-I). AA-I and AL-I are active ingredients in plants of the genus Aristololactam, which are widely used in traditional medicine but are associated with severe nephrotoxicity, including a disease called aristolochic acid nephropathy. Studies have shown that AL-I can rapidly enter and accumulate in the proximal tubular epithelial cells of the kidney, which may be one of the reasons for its direct cytotoxic effects and the persistent nephrotoxicity observed in the development of aristolochic acid nephropathy. [1]
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| Molecular Formula |
C17H11NO4
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|---|---|
| Molecular Weight |
293.2735
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| Exact Mass |
293.068
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| Elemental Analysis |
C, 69.62; H, 3.78; N, 4.78; O, 21.82
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| CAS # |
13395-02-3
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| Related CAS # |
13395-02-3
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| PubChem CID |
96710
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
463.9±34.0 °C at 760 mmHg
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| Melting Point |
319 °C
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| Flash Point |
234.4±25.7 °C
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| Vapour Pressure |
0.0±1.1 mmHg at 25°C
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| Index of Refraction |
1.763
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| LogP |
2.97
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
22
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| Complexity |
483
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C([H])([H])OC2C([H])=C3C(N([H])C4C([H])=C5C(=C([H])C([H])=C([H])C5=C(C1=2)C=43)OC([H])([H])[H])=O
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| InChi Key |
MXOKGWUJNGEKBH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H11NO4/c1-20-12-4-2-3-8-9(12)5-11-14-10(17(19)18-11)6-13-16(15(8)14)22-7-21-13/h2-6H,7H2,1H3,(H,18,19)
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| Chemical Name |
14-methoxy-3,5-dioxa-10-azapentacyclo[9.7.1.02,6.08,19.013,18]nonadeca-1(18),2(6),7,11(19),12,14,16-heptaen-9-one
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| Synonyms |
Aristolactam I; Aristololactam
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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 : ~12.5 mg/mL (~42.62 mM)
H2O : < 0.1 mg/mL |
|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4098 mL | 17.0491 mL | 34.0983 mL | |
| 5 mM | 0.6820 mL | 3.4098 mL | 6.8197 mL | |
| 10 mM | 0.3410 mL | 1.7049 mL | 3.4098 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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