Ethanol extract of antrodia camphorata for inducing apoptosis and preparation method thereof
a technology of antrodia camphorata and ethanol extract, which is applied in the field of extract of the fruiting body of antrodia camphorata, can solve the problems of inability to inhibit the growth of cancer cells of mycelia products, low apoptosis rate, and high price of ac, so as to achieve the effect of inhibiting the growth of leukemia, inducing apoptosis, and increasing polarities
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embodiment 1
Biological Experiments
[0043]Experiment 1: Ethanol Extract and Water Extract Prepared from Fruiting Body of Wild Antrodia campharata
[0044]Fifty-two grams (52 g) of the dried fruiting body of AC was ground as fine powder, which was boiled to flux with ethanol at 75° C. at a ratio of 1:10 (w / v) for 2 hours. The extract was cooled and then was allowed to precipitate overnight at 4° C. The supernatant of extract was further filtered by a filter paper, centrifuged at 3,000 rpm for 30 minutes to remove the precipitate, and then the extract was lyophilized and stored at −70° C. This extract is named as “the ethanol extract of the fruiting body of A. camphorata (EEAC)”. Further, the residue was further boiled to flux at a ratio of 1:10 (w / v) for 6 to 8 hours. The supernatants were filtered and then centrifuged at 3,000 rpm for 30 minutes to remove the precipitate. The product, named as “the water extract of the fruiting body of A. camphorata (WEAC)”, was lyophilized and stored at −70° C.
Ex...
experiment 4
, Cell Viability and Cytotoxicity
[0048]Human leukemia HL 60 cell line was purchased from American Type Culture Collection (ATCC, Manassas, Va.) and incubated in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin at 37° C. in a humidified atmosphere of 5% carbon dioxide.
[0049]Cell viability was determined by the trypan blue dye exclusion and cytotoxicity was assessed by MTT assay. First, 1×105 HL 60 cells were plated in 96-well plates and treated with different concentrations of EEAC dissolved in dimethyl sulfoxide (DMSO) at the concentrations of 0 to 150 μg / ml for 24 hours. Death cells then were stained with trypan blue, and the survival cells were calculated with hemocytometer. In MTT assay, 1.2 mg / ml MTT was added in each well, which was incubated for 2 hours to form crystal violet, formazan. Formazan was dissolved in DMSO, and absorbance was detected at a wavelength of 570 nm.
experiment 5
ry
[0050]HL 60 cells (1×106) were incubated in the 10-cm dish and treated with known concentrations of EEAC for 24 hours. The treated cells were washed with ice-cold phosphate-buffered saline (PBS) twice and harvested by centrifuging at 37° C. at 200×g for 5 minutes. Cells were fixed with 70% (v / v) ethanol at 37° C. for 30 minutes, and then were resuspended in 1 ml propidium iodium (PI) reagent (containing 0.1% Triton X-100, 100 μg / ml ribonuclease (RNase) A and 500 μg / ml PI) at 37° C. for 30 minutes. The cells were detected with the flow cytometer, and data were analyzed with FACScan and Cell Quest Program (Becton Dickinson).
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