Application of pterostilbene in preparation of TOP1 enzyme inhibitor
A technology of pterostilbene and an inhibitor is applied in the application field of pterostilbene in the preparation of TOP1 enzyme inhibitors, which can solve problems such as no reports on pterostilbene, and achieve the effect of high safety.
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Embodiment 1T
[0027] Embodiment 1 TOP1 enzyme activity inhibition test
[0028] Under the condition of incubation at 37°C for 30 minutes, one unit of TOP1 enzyme activity can unwind 1 ng of supercoiled DNA. The supercoiled DNA used in this experiment is pHOT1 DNA plasmid, the concentration is 0.25 μg / mL, and the topoisomerase I used is a high-purity, high-catalytic activity enzyme overexpressed and purified in baculovirus, with an enzyme activity of 330 units / μL . The preparation of the experimental system is shown in Table 1.
[0029] Table 1: Sample preparation for TOP1 enzyme activity assay
[0030]
[0031] After the above system configuration is completed, put it in a water bath at 37°C for 30 minutes, take it out, add 2 μL of 10% SDS solution to each tube, add 2.5 μL of proteinase K (0.5 mg / mL), stop the reaction and degrade the TOP1 enzyme, and continue to bathe in 37°C for 15 minutes , add 4μL 6X Loading Buffer to each tube to stop the reaction.
[0032] A 2% agarose gel was ...
Embodiment 2
[0034] Example 2 In vitro tumor cell proliferation inhibition test
[0035] In this experiment, CCK-8 kit was used to detect cell proliferation. Cell Counting Kit-8, referred to as CCK-8 kit or CCK8, is a rapid and highly sensitive detection kit based on WST-8 and widely used in cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of electron coupling reagents, it can be reduced by some dehydrogenases in mitochondria to produce orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cell, there is a linear relationship between the depth of the color and the number of cells.
[0036] A blank control group, a positive control group and a test group were set up in the experiment, and 6 replicate holes were set up in each group. Tumor cells in the logarithmic growth phase were taken, and the cell density was adjusted to 5×10 4 cells / mL, seeded...
Embodiment 3
[0044] Example 3 Intracellular TOP1 protein content detection test
[0045] The experiment set up blank control group, positive control group and test group. Tumor cells in the logarithmic growth phase were taken, and the cell density was adjusted to 5×10 5 cells / mL, seeded in a 12-well plate, 1 mL per well, and incubated in a cell culture incubator for 24 hours. Prepare media containing different concentrations of pterostilbene, add 1 mL to each well, so that the final concentration of pterostilbene acting on the cells is 1, 10, 20, 50, 80, 100 μM. The positive control group was camptothecin at a final concentration of 1 μM. Continue to incubate in the incubator for 12h / 24h / 36h / 48h.
[0046] After the incubation, discard the supernatant, wash the cells twice with sterile PBS, and lyse them on ice with RIPA lysate containing 1% PMSF for 5-15 minutes.
[0047] The protein content was determined by the BCA method, and the total protein concentration of each sample was adjust...
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