Background
Daurisoline is an alkaloid that has been found in M. dauricum and has diverse biological activities.1,2,3 It enhances cytotoxicity induced by camptothecin in HeLa cells when used at a concentration of 10 ?M.1 Daurisoline inhibits camptothecin-induced autophagy in HeLa, A549, and HCT116 cells (IC50s = 74.75, 50.54, and 80.81 μM, respectively). It inhibits ADP-induced aggregation of platelets in isolated rabbit whole blood (IC50 = 100 ?M).2 Daurisoline prolongs action potential duration (APD) and reduces early afterdepolarizations (EADs) in papillary muscle preparations isolated from hypertrophied rabbit hearts when used at a concentration of 15 ?M.3
1.Wu, M.-Y., Wang, S.-F., Cai, C.-Z., et al.Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicityOncotarget8(44)77673-77684(2017)
2.Hu, S.-M., Xu, S.-X., Yao, X.-S., et al.Dauricoside, a new glycosidal alkaloid having an inhibitory activity against blood-platelet aggregationChem. Pharm. Bull. (Tokyo)41(10)1866-1868(1993)
3.Liu, Q.-N., Zhang, L., Gong, P.-L., et al.Daurisoline suppressed early afterdepolarizations and inhibited L-type calcium currentAm. J. Chin. Med.38(1)37-49(2010)
Protocol
Kinase experiment: | HEK293 cells are incubated overnight with 35 μg/mL Dx-OG514. Cells are washed and incubated with serum-free Dulbecco's modified Eagle's medium (DMEM) for 2 h. 15 minutes prior to lysis, FCCP is added into the medium to a final concentration of 1 μM. Cells are scraped in fraction buffer (50 mM KCl, 90 mM K-Gluconate, 1 mM EGTA, 50 mM Glucose, 20 mM HEPES, protease inhibitor cocktail, pH=7.4) supplemented with 1 μM FCCP. After spraying with needle, cells are spun down at 10,000 rpm for 15 sec. at 4°C. Then, re-centrifuge the supernatant at max speed for another 20 minutes. The pellet is resuspended in pre-warmed fractionation buffer supplemented with 1% BSA, and split into several aliquots with DAC, Daurisoline (DAS) or BAF treatment for 30 min. Baseline fluorescence is measured at 530 nm upon 511 nm excitation in 96-well plate at 30 s intervals for 5 min[2]. |
Cell experiment: | Cell proliferation is determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HeLa cells are seeded at 7000 cells per well in 96-well plates in DMEM (1% serum). After cells are treated with different compounds (including Daurisoline) for indicated times, 20 μL of MTT (2.5 mg/mL in PBS) is added to each well. The plates are incubated for an additional 4 h at 37°C. Then the purple-blue MTT formazan precipitate is dissolved in 100 μL DMSO. The cell viability of HeLa cell is evaluated by measuring optical density at 572 nm with a microplate reader[2]. |
Animal experiment: | After the beagle dogs are anesthetized with sodium pentobarbital (30 mg/kg, iv) a canula is advanced into the left ventricle through the right common carotid artery. And the canula is connected to a pressure transducer which is connected to an amplifier and polygraph. The right femoral artery is canulated to measure the blood pressure wave. ECG (lead II) is observed simultaneously. After iv injection of Daurisoline (DS) (n=4) or Dau (n=4) to beagle dogs, the ECG, BP, and LVP signals are recorded. Blood samples are taken before dosing and at 2, 5, 10, 15, 20, 30, 45 min, and 1, 1.5, 2, 3, 4, 6, 8 h after dosing[3]. |
参考文献: [1]. Liu Q, et al. Effect of daurisoline on HERG channel electrophysiological function and protein expression. J Nat Prod. 2012 Sep 28;75(9):1539-45.
[2]. Wu MY, et al. Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells tocamptothecin-induced toxicity. Oncotarget. 2017 Sep 8;8(44):77673-77684.
[3]. Shi SJ, et al. Pharmacokinetic-pharmacodynamic modeling of daurisoline and dauricine in beagle dogs. Acta Pharmacol Sin. 2003 Oct;24(10):1011-5. |
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