Method for preparing clitocine and applications in antineoplastic medicaments
A technology of critocin and drugs, applied in the field of medicine, can solve the problem of high mortality
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Embodiment 1
[0021] Example 1: Extraction, separation and purification of anti-tumor active compounds from fresh Pleurotus ostreatus fruiting bodies and their structure identification
[0022] experimental method
[0023] 1. Extraction and purification: The raw material fresh Pleurotus ostreatus fruiting bodies (2.7kg) are vacuum freeze-dried and crushed by a grinder. The resulting 340g of bacterial powder is immersed in 1.5L of 95% (v / v) alcohol, and the fresh solvent is replaced once in the meantime. The two extracts were combined and concentrated under reduced pressure to obtain an alcohol extract (25.7g). The extract was extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract (15.7g). Take 15g ethyl acetate extract on silica gel column, CH 2 Cl 2 / MeOH (10:0-0:10) gradient elution, collect cells to screen the active elution peak (6.5g), and then apply the reverse phase column RP-18 (ODS, 50μm, Φ25×420mm), MeOH / H 2 O(1:9) elution, and the acti...
Embodiment 2
[0030] Example 2: Test of the inhibitory activity of clitocine on the proliferation of human tumor cells
[0031] Experimental material method
[0032] 1. Human tumor cell lines and sources: A total of six tumor lines are breast cancer Bcap37, breast cancer MCF-7, cervical cancer Hela, liver cancer SMMC-7721, liver cancer HepG2, gastric cancer SGC-7901, all from American Type Culture Collection.
[0033] 2. Dissolve clitocine in DMSO to make 1mg / ml mother liquid, place it in the refrigerator at -20℃, dilute it with culture medium to the required concentration for use in the experiment, and make MTT (product of Sigma company) make 1mg / ml mother liquid with phosphate buffer. Place in a refrigerator at 4°C for later use.
[0034] 3. Cell culture: All cells were cultured in DMEM complete medium (Invitrogen), plus 5% newborn calf serum (Invitrogen), 2mmol / L L-glutamine. Choose logarithmic growth phase cells, with 1×10 4 The density was inoculated into 96-well plates, added with differe...
Embodiment 3
[0043] Example 3: Related mechanisms of clitocine inhibiting the proliferation of liver cancer HepG2 cells
[0044] 1. DNA ladder-like strip experiment method: tumor cells HepG2 were treated with different concentrations of clitocine for 48h and collected, and lysed at 45℃ for 1h (5mM Tris-HCl, 100mM EDTA, 1%(w / v)SDS, and Proteinase K), genomic DNA was extracted with phenol / chloroform / isoamyl alcohol (25:24:1), precipitated with 70% alcohol at -20°C, RNA was removed with RNase, and 40μg of the same amount of DNA was added to 1.2% agarose Electrophoresis on the gel and photographed by a gel imager.
[0045] The experimental results are attached Figure 5 .
[0046] One of the most typical characteristics of cell apoptosis is that after the cell receives the drug, the DNA will be cut at the nucleosome junctions by nucleases, resulting in DNA fragments of 180-200bp or an integer multiple of them. During agarose gel electrophoresis Presents regular strips of ladders, which is an impor...
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