Pharmaceutical composition of functional red yeast rice active ingredients and docosahexaenoic acid and its application in the preparation of antitumor drugs
A technology of docosahexaenoic acid and functional red yeast rice, applied in the field of medicine, can solve the problems of tumor cell drug resistance and treatment failure, achieve enhanced cytotoxicity, obvious inhibitory effect, and enhanced anti-tumor effect Effect
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Embodiment 1
[0025] Embodiment 1: cell proliferation experiment
[0026] The SRB colorimetric method was used to investigate the effect of the combination of functional red yeast rice extract and DHA in inhibiting the proliferation of breast cancer cells in vitro, and the inhibition rate was calculated.
[0027] experimental method:
[0028] 1) Seed tumor cells in a 96-well plate at 3000-5000 cells / well.
[0029] 2) After 24 hours, add different concentrations of functional red yeast rice extract and DHA (half-dilution, set 6 concentrations), and the corresponding mixed solution, and repeat 3 wells for each concentration.
[0030] 3) After 72 hours, add 50 μL 4°C pre-cooled TCA solution (30%, w / v) to each well to fix the cells, and the final concentration of TCA solution is 10%. Let it stand for 5 minutes, move it into a refrigerator at 4°C for 1 hour, take it out, rinse it with deionized water 5 times, and dry it at room temperature. After the 96-well plate was dried at room temperatur...
Embodiment 2
[0034] Embodiment 2: Apoptosis experiment
[0035] PI single staining and Annexin-V / PI double staining were used to investigate the ability of DHA and functional red yeast extract to promote tumor cell apoptosis in vitro.
[0036] experimental method:
[0037] 1) Inoculate tumor cells in a 6-well plate at 300,000-500,000 cells / well;
[0038] 2) After 24 hours, add the corresponding concentration of DHA and functional red yeast extract and the mixture;
[0039] 3) After 24 hours, collect the cells and stain with Annexin-V for 15 minutes at room temperature in the dark;
[0040] 4) Then stain with PI at room temperature in the dark for 5 minutes;
[0041] 5) 10,000 cells were collected by flow cytometry for analysis, and early apoptosis (PI - AV + ) and late apoptosis (PI + AV + ) percentage, the total apoptotic rate was obtained by adding the two together.
[0042] Experimental results:
[0043] Such as image 3 , Figure 4As shown, the combined use of DHA and functi...
Embodiment 3
[0044] Example 3: Western Blot Analysis (Western Blot)
[0045] The Western Blot method was used to investigate the changes of the important protein PARP in the apoptosis pathway, so as to reflect the ability of the functional red yeast extract and DHA to induce apoptosis from the protein level.
[0046] experimental method:
[0047] 1) Inoculate tumor cells in a 6-well plate at 300,000-500,000 cells / well;
[0048] 2) After 24 hours, add corresponding concentrations of DHA and functional red yeast extract and the mixture;
[0049] 3) After 6, 12, and 24 hours of drug action, collect the cells, wash with PBS, and add 50 μL of pre-cooled cell lysate to lyse at 4°C for 40 min;
[0050] 4) After protein quantification, add 40 μg of the sample into an equal amount of loading buffer, and after 10% SDS-PAGE electrophoresis, transfer it to the PVDF membrane with an electrotransfer instrument;
[0051] 5) Add PARP and GAPDH specific antibodies and incubate overnight at 4°C. Fluores...
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