Method for extracting medicament for killing hepatitis C virus from garlic
A technology for extracting hepatitis C virus and garlic, which is applied in antiviral agents, drug combinations, and pharmaceutical formulas, can solve the problems of no hepatitis C virus drugs, and achieve the effects of low cost, simple method, and cheap raw materials
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Embodiment 1
[0026] Determination of the cytotoxicity of the extract to Huh-7.5.1
[0027] Digest Huh-7.5.1 cells with trypsin, press 2×10 4 Inoculated into 96-well cell culture plates at a density of 5% CO 2 Incubate in an incubator at 37°C for 6 hours. After the cells adhere to the wall, add 10 μl of various extracts of different concentrations to each well, culture overnight at 37°C, add 10 μl of CCK-8 reagent to each well, and continue to incubate for 4 hours. At the same time, set up a negative control, add CCK-8 to the cell wells without extracting solution; add CCK-8 to the blank control, without cells, add DMEM culture solution. The 450nm absorbance value (OD value) of each well was measured with a microplate reader.
Embodiment 2
[0029] Antiviral activity of garlic extract:
[0030] In order to screen whether the extract has anti-HCV activity, as well as the pathway of antiviral effect, the following three groups of parallel experiments were designed:
[0031] The first group: kill the virus:
[0032] Take 3 concentrations: 10 μl each of garlic extracts of 20mg / ml, 5mg / ml, and 1mg / ml, and first mix them with 10μl HCVcc in equal amounts, and then inoculate them into 96-well plates after acting at 37°C for 4 hours. The cell density was 3 x 10 4 / hole, the same below. After culturing for 72 hours, the IFA experiment was carried out to detect the expression of HCV protein in the cells. A well directly infected with HCVcc was used as a positive control, and a well without extract and virus was used as a negative control.
[0033] The second group: blocking virus infection:
[0034] First add 10 μl of different concentrations of extracts to a 96-well plate containing Huh-7.5.1 cells, culture overnight...
Embodiment 3
[0038] IFA detection: Aspirate the culture supernatant in the 96-well plate, add 100 μl of methanol to each well, fix at -20°C for 20 minutes, wash with PBS 3 times for 3 minutes each time. Add 100 μl of 0.1% TritonX-100( ) prepared in PBS to each well, permeabilize at room temperature for 30 min, and wash with PBS (3 min×3 times). Add 100 μl of blocking solution (PBS containing 3% BSA) to each well, and block for 1 hour at room temperature. The blocking solution was sucked off; 50 μl of hepatitis C patient serum was added to each well (the serum diluted 1:50 with the blocking solution was used as the primary antibody), incubated at room temperature for 2 hours, and washed 3 times with PBS, 10 minutes each time. Fluorescein FITC-labeled anti-human IgG was added to each well as the secondary antibody, incubated in the dark for 1 hour, and washed with PBS (3 min×3 times).
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