Application of exocarpium citri rubrum expectoration extract in preparation of medicine for inhibiting human coronavirus infection
A technology of human coronavirus, orange-red phlegm cough, applied in the field of medicine
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[0027] The preparation method of the orange-red phlegm and cough extract is as follows: distill the orange-red and bitter almonds with water vapor, and collect the distillate; mix the dregs with Mibabu, Poria cocos, Shui Pinellia (manufactured), Baiqian, licorice and Schisandra chinensis Mix, respectively add (the total weight of the dregs and honey baebu, Poria cocos, Shui Pinellia (made), Baiqian, licorice and Schisandra chinensis) 6-8 times the amount of water, 4-6 times the amount of water to decoct twice, filter After that, combine the filtrate and distillate, concentrate to a relative density of 1.05-1.25 (80°C), add ethanol to make the alcohol content reach 75-80%, let it stand for 18-24 hours, take the supernatant, and concentrate to a relative density 1.05-1.25 (80°C), the orange red phlegm and cough extract is obtained.
[0028] HuH-7 cells were purchased from the Virus Laboratory of the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory...
Embodiment 1
[0030] Embodiment 1 cytotoxicity test (MTT method)
[0031] (1) Test drugs: as shown in Table 1;
[0032] Table 1 Drug name, experimental concentration and grouping
[0033]
[0034] (2) In a sterile 96-well culture plate, add 100 μL to each well with a concentration of 2×10 5 cells / mL Huh-7 cells, 37℃5%CO 2 Cultivate for 24 hours;
[0035] (3) The monolayer cells of the 96-well plate were washed once with PBS, and 100 μL of 2-fold serially diluted drug was added to each well. Add an equal volume of 2% serum medium to each well of the blank control group and normal cell group, and culture at 37°C for 4 days;
[0036] (4) Add 20 μL of MTT solution with a concentration of 5 mg / mL to each well and continue to incubate for 4 hours. Discard the supernatant, add 100 μL DMSO to each well, and shake at low speed for 5 minutes to fully melt the crystals;
[0037] (5) Select a wavelength of 490nm, measure the absorbance on an enzyme-linked immunosorbent analyzer, and calculate th...
Embodiment 2
[0040] Embodiment 2 test drug antiviral experiment
[0041] (1) Test drugs: as shown in Table 2
[0042] Table 2 Drug name, experimental concentration and grouping
[0043]
[0044] (2) In a sterile 96-well culture plate, add 100 μL to each well with a concentration of 2×10 5 cells / mL Huh-7 cells, 37℃5%CO 2 Cultivate for 24 hours;
[0045] (3) Add 100TCID to the culture plate experimental group and the virus control group 50 Virus solution 100 μL / well, 37°C 5% CO 2 Adsorption in the incubator for 2h;
[0046] (4) After 2 hours, the cell culture solution in the 96-well culture plate was discarded; the test drug was diluted to each concentration in Table 2, and each concentration was repeated in 3 wells, and 100 μl / well of the above drug solution was added;
[0047] (5) Set up cell control and virus control (negative control) at the same time;
[0048] (6) cells at 37°C, 5% CO 2 Incubate in the incubator for 3-4 days;
[0049] (7) Observing cytopathic changes (CPE) u...
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