Water extract of antrodia camphorata for immunostimulatory effect and preparation method thereof
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experiment 2
action and Fractionation of ACW
[0046]ACW (216.4 mg) was extracted with solvents (sequentially as ethyl acetate, ethanol and water) of increasing polarities to obtain three different extracts, 8.9% (19.3 mg) of ethyl acetate extract (ACW-EA), 38.5% (83.3 mg) of ethanol extract (ACW-E) and 52.6% (113.8 mg) of a second water extract (ACW-W). Percentage is referred to the weight percentage of ACW, and weight is referred to the dry material extracted. The amount of carbohydrate in each extract was determined using phenol-sulfuric acid method (Chen et al., 2007), and the spectrum characteristics therein were analyzed with NMR.
experiment 3
[0047]The above-mentioned extracts were solved in deuterium solvents such as dideuterium monoxide (D2O), pyridine-D5 (C5D5N) and so on, and then 1H NMR spectra were recorded on a Varian Unity Inova-600 MHz NMR. Chemical shift was reported in parts per million (ppm, d).
experiment 4
of Human T Cells and DCs
[0048]The preparation method of DCs was mainly prepared and modified from Lin et al, 2006. First, peripheral blood mononuclear cells (PBMCs) were obtained from 12 healthy donors by centrifugation with the Ficoll-Hypaque (Amersham Bioscience, Uppsala, Sweden) density gradient centrifugation. PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1 mM L-glutamin, 100 μg / ml streptomycin and 100 U / ml penicillin for 2 hours. The nonadherent cells were removed, and the T cells were purified by nylon wool separation, as the source of mononuclear T lymphocytes. T cells were loaded into the column and the eluted nonadherent cell fraction was rich in T cells. As determined by FACS, the resulting cell population was 80% T cells with a high expression of CD3. The adherent cells were cultured for 6 days in RPMI 1640 medium with 50 ng / ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Half of the culture mediu...
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