Delayed rectifier K⁺ channel (I_K) in neuronal cells – IC₅₀ = 146.0 ± 5.8 μM (determined in rat hippocampal neurons). [1]

- Talatisamine (120 μM) suppressed the enhanced I_K density caused by 1 μM Aβ40 oligomers in cultured cortical neurons (12 h incubation). The current‑voltage (I/V) curves showed clear inhibition of I_K. [1]

- Talatisamine (120 μM) significantly restored cell viability after Aβ40 oligomer exposure. Aβ40 oligomers reduced cell viability to 45.24 ± 8.07% of control; talatisamine pre‑treatment (30 min before Aβ) increased viability to 76.83 ± 2.06% (P < 0.05). Talatisamine alone did not affect cell viability. [1]

- Talatisamine (120 μM) attenuated Aβ40 oligomer‑induced increase in cell membrane permeability. Aβ40 oligomers increased permeability to 156.1 ± 14.86% of control; talatisamine reduced it to 111.2 ± 4.95% (P < 0.05). [1]

- Talatisamine (120 μM) restored the impaired mitochondrial transmembrane potential (MMP) caused by Aβ40 oligomers. Aβ40 oligomers decreased MMP to 70.15 ± 6.96% of control; talatisamine increased it to 124.9 ± 1.86% (P < 0.01). [1]

- Western blot analysis showed that Aβ40 oligomers (1 μM, 24 h) decreased Bcl‑2 and increased Bax protein levels. Pre‑treatment with talatisamine (120 μM, 30 min before Aβ) significantly prevented these alterations. Talatisamine alone did not change Bcl‑2 or Bax expression. [1]

- Talatisamine (120 μM) significantly attenuated the activation of Caspase‑3 and Caspase‑9 induced by Aβ40 oligomers (1 μM, 24 h) as measured by a Caspase‑Glo assay. Talatisamine alone did not affect caspase activities. [1]

Whole‑cell patch clamp recording was used to measure voltage‑gated K⁺ currents (I_K) in cultured cortical neurons at 21–23 °C. The external solution contained 135 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 1 μM tetrodotoxin, 10 mM HEPES, and 10 mM glucose (pH 7.4). The internal solution consisted of 120 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 2 mM Na₂ATP, 10 mM EGTA, and 10 mM HEPES (pH 7.2). Recording pipettes had tip resistances of 5–7 MΩ when filled with internal solution. Series resistance was compensated by 75–85% using an EPC9 amplifier. Linear leak and residual capacitance currents were subtracted online using a P/6 protocol. Data were filtered at 3 kHz and digitized at 20 kHz. Current amplitude was measured at the end of each voltage step. To test the effect of talatisamine, neurons were incubated with Aβ40 oligomers for 12 h, then I_K was recorded in the presence of 120 μM talatisamine. [1]

- Primary cortical neurons were obtained from E16 Sprague‑Dawley rat embryos. Meninges were removed; tissues were minced and mechanically triturated. After centrifugation, cells were resuspended in Dulbecco’s minimum essential medium (high glucose) with 10% heat‑inactivated fetal bovine serum and 5% heat‑inactivated horse serum. Single‑cell suspensions were plated on poly‑lysine‑coated plates and incubated at 37 °C in 5% CO₂. The next day, medium was replaced with serum‑free Neurobasal medium supplemented with 2% B27 and 2 mM glutamine. On day 3 in vitro, 5 μM cytosine‑β‑D‑arabinofuranoside was added for 24 h to inhibit non‑neuronal cell growth, then changed back to fresh Neurobasal medium. Half of the medium was replaced every 3 days. Neurons were cultured for 7 days before treatments. [1]

- Cell viability was measured using a CCK‑8 kit. Cultured cortical neurons were pre‑incubated with talatisamine or TEA for 30 min before exposure to Aβ40 oligomers, then incubated for 24 h. Absorbance was read at 450 nm. [1]

- Multiparameter cytotoxicity assay (Cellomics kit) was used to assess membrane permeability and mitochondrial membrane potential (MMP). After treatments, cells were stained with fluorescent probes, scanned with a KineticScan reader, and analyzed using Cellular Health Profiling software. [1]

- Immunofluorescence staining: Cells on poly‑lysine‑coated coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature, washed with PBS, blocked with 10% normal goat serum in 0.3% Triton X‑100/PBS for 1 h, then incubated with monoclonal mouse anti‑β‑III tubulin antibody (1:800) overnight at 4 °C. After washing, cells were incubated with FITC‑conjugated goat anti‑mouse IgG (1:100) for 1 h at 37 °C, rinsed, and mounted with medium containing Hoechst 33342. [1]

- Western blot: Cells were lysed in buffer (50 mM Tris‑HCl pH 7.4, 150 mM NaCl, 0.2 mM PMSF, 1% NP‑40, 1.0 mM Na₃VO₄, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 4 μg/ml leupeptin, 2 μg/ml aprotinin). Lysates were centrifuged at 12,000×g for 20 min at 4 °C. Supernatants were mixed with sample buffer (62.5 mM Tris‑HCl pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue) and boiled for 5 min. Proteins were separated by 12% SDS‑PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST for 2 h, then incubated with primary antibodies (anti‑Bcl‑2 1:500, anti‑Bax 1:500, anti‑β‑actin 1:1000) overnight at 4 °C. After washing, membranes were incubated with peroxidase‑conjugated secondary antibody (1:1000) for 1 h at room temperature. Signals were visualized by enhanced chemiluminescence and densitometry was performed. [1]

- Caspase‑3 and Caspase‑9 activities were measured using a Caspase‑Glo assay kit. Cells were plated in white‑walled 96‑well plates at 2×10⁶ cells/well. The proluminescent substrates containing DEVD (for Caspase‑3) or LEHD (for Caspase‑9) were cleaved by the respective caspases. After 1 h incubation at room temperature, luminescence was read with a plate‑reading luminometer. [1]

- Talatisamine (120 μM) alone did not affect cell viability of cultured cortical neurons. [1]

- Talatisamine alone did not alter cell membrane permeability or mitochondrial transmembrane potential in cortical neurons. [1]

- Talatisamine alone (24 h incubation) did not change the expression levels of Bcl‑2 or Bax protein. [1]

- Talatisamine alone did not affect Caspase‑3 or Caspase‑9 activity. [1]

[1]. The newly identified K+ channel blocker talatisamine attenuates beta-amyloid oligomers induced neurotoxicity in cultured cortical neurons. Neurosci Lett. 2012 Jun 19;518(2):122-7.

Tarlatizamin is a diterpenoid compound.

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- Talatisamine is an aconitum alkaloid identified from the China Natural Products Database (CNPD) through computational virtual screening and electrophysiological characterization. [1]

- The neuroprotective effect of talatisamine against Aβ oligomer‑induced cytotoxicity is mediated by its I_K channel blocking action, which suppresses K⁺ loss‑related apoptotic responses (e.g., decreased Bcl‑2, increased Bax, activation of Caspase‑3 and Caspase‑9). [1]

- Talatisamine is suggested as a potential leading compound for I_K channel blocker‑based neuroprotection, possibly for Alzheimer’s disease treatment, but requires evaluation in AD animal models. [1]

C24H39NO5

421.5702

421.282

C, 68.38; H, 9.32; N, 3.32; O, 18.98

20501-56-8

441761

White to off-white solid powder

1.3±0.1 g/cm3

533.9±50.0 °C at 760 mmHg

151-152℃

276.7±30.1 °C

0.0±3.2 mmHg at 25°C

1.588

-0.5

2

6

5

30

720

11

O([H])[C@]12C([H])([H])[C@@]([H])(C3([H])C([H])([C@@]1([H])[C@@]([H])(C3([H])[H])C13C([H])(C([H])([H])C([H])([H])[C@@]4(C([H])([H])OC([H])([H])[H])C([H])([H])N(C([H])([H])C([H])([H])[H])[C@@]1([H])[C@]2([H])C([H])([H])[C@@]34[H])OC([H])([H])[H])O[H])OC([H])([H])[H]

BDCURAWBZJMFIK-UHFFFAOYSA-N

InChI=1S/C24H39NO5/c1-5-25-11-22(12-28-2)7-6-18(30-4)24-14-8-13-16(29-3)10-23(27,19(14)20(13)26)15(21(24)25)9-17(22)24/h13-21,26-27H,5-12H2,1-4H3

11-ethyl-6,16-dimethoxy-13-(methoxymethyl)-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecane-4,8-diol

Talatisamine

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)

DMSO : ~50 mg/mL (~118.60 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.93 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.93 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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