Application of brazilin in preparation of drugs
A Brazilian lignin and compound technology, applied in the field of medicine, can solve the problem of no discovery
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Embodiment 1
[0030] Example 1: Evaluation of the inhibitory effect of brazilnin on PTKs activity.
[0031] In this example, 10 μL of the sample and kinase buffer (50mM HEPES buffer, pH7.4, 20mM MgCl 2 , 0.1 mM MnCl 2 , 1 mM Na 3 VO 4 ) 50 μL of the diluted PTKs crude extract was added to a 96-well microtiter plate coated with the substrate polyglutamic acid-tyrosine (Poly-Glu-Ty, PGT), and incubated at 37°C for 10 minutes. Afterwards, 80 μL of ATP (0.4 mM) diluted in kinase buffer was added, and after incubation at 37°C for 40 min, the reaction solution in the plate was removed, and the microwell plate was washed 5 times with PBS containing 0.1% Tween-20. Add 100 μL / well of horseradish peroxidase-labeled phosphorylated tyrosine-antibody complex and incubate at 37°C for 40 min. Wash the microplate 5 times, add 100 μL / well of TMB chromogenic solution, and react for 10 min at room temperature in the dark. Add 100 μL / well of stop solution, and immediately measure the absorbance value at 4...
Embodiment 2
[0035] Example 2: Effects of brazilianin on PDGF-BB-induced VSMC proliferation.
[0036] In this example, primary cultured VSMCs were used to carry out the anti-PDGF-BB-induced VSMC proliferation experiment of brazilin.
[0037] Detection of the effect of Brazilin on the viability of VSMC cells:
[0038] Take the VSMC (3-6 generations) in the logarithmic growth phase, and use 4×10 4 cells / mL, inoculated into a 96-well plate, and added 100 μL to each well. 37°C, 5% CO 2 After culturing in the incubator for 24 hours, the medium was replaced with serum-free DMEM medium, and the cells were starved for 24 hours to synchronize the cells. Drugs of different concentrations (0, 0.1, 1, 3, 10 μM) were added, and the culture was continued. After 12, 24, and 48 hours respectively, the cells were washed twice with PBS, and the cell viability was detected by the MTT method.
[0039] Detection of the effect of Brazilin on the proliferation of VSMC:
[0040]VSMC cells were treated the sa...
Embodiment 3
[0042] Example 3: Effects of brazilianin on PDGF-BB-induced VSMC migration
[0043] In this example, the in vitro wound healing experiment and the cell transmembrane migration experiment were respectively used to conduct the anti-VSMC migration experiment of brazilianin.
[0044] In vitro wound healing experiments:
[0045] Get the VSMC (3-6 generation) of logarithmic growth phase, with 10 5 cells / mL were inoculated into a 24-well plate, and 400 μL was added to each well, at 37°C, 5% CO 2 After incubating in a humidity incubator for 24 hours, use a 200 μL pipette tip to scratch vertically in the 24-well plate in the shape of a "ten", marking four quadrants A, B, C, and D respectively. After scratching, wash the cells twice with PBS, remove the scratched cells, and take pictures at the position of "cross". That is the T0 period. Afterwards, brazilin at a final concentration of 1, 3, 10 and 30 μM diluted in serum-free medium was added, and 20 ng / mL of PDGF-BB was added after...
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